CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit of priority from the U. S. Provisional application Serial No. 61 /896,601 filed 28 October 2013 which is herewith incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[002] In fuel-ethanol production, a substantial percentage, approximately 50%, of the 'DP2 peak' (DP - degree of polymerization - equivalent to a disaccharide), at the end-of-fermentation (EOF) is trehalose, thought to be internally synthesized by yeast for osmolarity stabilization, (for example, for protection from desiccation from cellular environments of high ethanol, high glucose, etc.). Furthermore, these trehalose levels can increase with fermentation temperature, measured to be as high as 60-70% of the EOF DP2 levels. Since formation of trehalose is in direct competition with glucose utilization, this lost carbon will result in less than theoretical yields of ethanol production. Nonetheless, the removal of trehalose for enhanced fermentation benefits could result in negative fermentation performance since trehalose is known to protect against stresses. However, there are literature references that suggest the hydrolysis of extracellular trehalose can be used for direct ethanol production as well as yeast growth (Basu et al., Biochem Boiphys. Acta 1760, 134-160 (2006)). The potential use of trehalase to lower EOF 'residual sugar' levels, while increasing ethanol yields, has industry interest.

[003] One molecule of trehalose is hydrolyzed to two molecules of glucose by the enzyme trehalase. Trehalase (a, a-trehalose-1 -C-glucohydrolase, EC 3.2.1 .28) has been reported from many organisms including prokaryotes, plants and animals. Though trehalose is not known to be present in mammals, trehalase enzyme is found to be present in the kidney brush border membrane and the intestinal villi membranes. Trehalose hydrolysis by trehalase enzyme is an important physiological process for various organisms, such as fungal spore germination, insect flight, and the resumption of growth in resting cells. At least two-types of trehalases, based on their pH optima, have been characterized; acid trehalases and neutral trehalases. It is also suggested that the neutral trehalase is mostly cytosolic while the acid trehalase is normally extracellularly secreted (Parrou JL et al., FEMS Yeast Res. (2005)5:503-1 1 ).

[004] Thus, there is a need in the industry to develop methods and compositions comprising trehalases that, among other things, improve ethanol yields, reduce DP2 peaks, and improve fermentation process. SUMMARY OF THE INVENTION [005] The present invention provides methods of increasing the production of ethanol from a liquefact, comprising: fermenting the liquefact with a glucoamylase, a fermenting organism and a trehalase; and recovering ethanol and other desired fermentation products at the end of the fermentation, wherein the trehalase-treated fermentation yields increased ethanol compared to the fermentation that is not treated with a trehalase. In another aspect, the invention provides methods of decreasing the final concentrations of DP2 in a fermentation product, comprising: fermenting a liquefact with a glucoamylase, a fermenting organism and a trehalase; and recovering the desired fermentation products at the end of the fermentation wherein the DP2 concentrations are reduced compared to a fermentation not treated with a trehalase. In a further aspect, the glucoamylase is DISTILLASE SSF. In another aspect, the trehalase is from a eukaryote or a filamentous fungi, such as Trichoderma. In another aspect, the trehalase is an acidic trehalase or a neutral trehalase. In another aspect, the trehalase is added at a concentration of about 0.1 μg/g DS to about 1000 μg/g DS of liquefact, preferably at a concentration of about 0.25 μg/g DS to about 100 μg/g DS of liquefact, more preferably at a concentration of about 0.5 μg/g DS to about 10 μg/g DS of liquefact.

[006] The invention also provides methods of increasing the production of ethanol from a liquefact, comprising fermenting the liquefact with a glucoamylase, a fermenting organism and a fermentation-stable trehalase; and recovering ethanol and other desired fermentation products at the end of the fermentation, wherein the trehalase-treated fermentation yields increased ethanol compared to the fermentation that is not treated with a trehalase.

[007] The invention also provides methods of improving a fermentation reaction comprising adding a trehalase, wherein the trehalase provides improved characteristics, such as healthier yeast cells during fermentation, improved ethanol yield or extended ethanol production. The trehalase may be added at the start of the fermentation or during fermentation.

[008] The invention also provides methods of improving ethanol production in a fermentation reaction comprising maintaining trehalose levels below a threshold level throughout the fermentation reaction, wherein the threshold level is below twice the trehalose concentration at the start of the fermentation (%wt/v). The invention also provides methods of improving ethanol production in a fermentation reaction comprising adding trehalase to the fermentation reaction, thereby maintaining trehalose levels below a threshold level throughout the fermentation reaction, wherein the threshold level is below half the trehalose concentration that would have been present during fermentation if no trehalase was added to the process (%wt/v). [009] The invention also provides methods as disclosed above, further comprising additional enzymes, selected from the group consisting of acyl transferases, alpha-amylases, β-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1 , 4- glucanases, endo-3-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, β- glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, and xylosidases.

[0010] The invention also provides method for improving ethanol production in a fermentation reaction comprising liquefact and backset by removing trehalose, comprising use of a trehalase, wherein trehalose levels are lower than 0.01 %w/v of the fermentation reaction. The invention also provides a method for improving ethanol production in a fermentation reaction by removing trehalose from the liquefaction comprising backset, comprising removal of trehalose in backset by treatment with a trehalase, wherein trehalose levels are lower than 0.05%w/v of the backset.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1 -3 show concentrations of trehalose during fermentation for various trehalase concentrations and controls.

[0012] Figure 4 shows trehalose profile at 36^ with and without trehalase.

[0013] Figure 5 shows DP2 profile at 36 ^< € with and without trehalase.

[0014] Figure 6 shows final ethanol production profile at 36^ with and without trehalase. [0015] Figures 7 and 8 show ethanol production profile for various fermentations with or without trehalase.

[0016] Figure 9 shows levels of trehalose in subsequent fermentations.

DETAILED DESCRIPTION

[0017] Described are compositions and methods comprising trehalases. Trehalases of the invention may be from a prokaryote or a eukaryote, such as from fungi, particularly from a Trichoderma fungus. Exemplary applications for the trehalase enzymes are for starch-based saccharification and fermentation. Saccharification and fermentation may be carried out simultaneously (SSF). These and other aspects of the compositions and methods are described in detail, below.

[0018] Prior to describing the various aspects and embodiments of the present compositions and methods, the following definitions and abbreviations are described.

1. Definitions and Abbreviations

[0019] In accordance with this detailed description, the following abbreviations and definitions apply. Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "the dosage" includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0020] The present document is organized into a number of sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the headings used for different sections of the disclosure should not be construed as limiting.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following terms are provided below.

1.1. Abbreviations and Acronyms

[0022] The following abbreviations/acronyms have the following meanings unless otherwise specified:

ABTS 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid

AE or AEO alcohol ethoxylate

AES or AEOS alcohol ethoxysulfate

AkAA Aspergillus kawcichii a-amylase

AnGA Aspergillus niger glucoamylase

AOS a-olefinsulfonate

AS alkyl sulfate cDNA complementary DNA

CMC carboxymethylcellulose

DE dextrose equivalent

DNA deoxyribonucleic acid

DPn degree of saccharide polymerization having n subunits ds or DS dry solids

DTMPA diethylenetriaminepentaacetic acid

EC Enzyme Commission

EDTA ethylenediaminetetraacetic acid

EO ethylene oxide (polymer fragment)

EOF End of Fermentation

GA glucoamylase

GAU/g ds glucoamylase activity unit/gram dry solids

HFCS high fructose corn syrup

HgGA Humicola grisea glucoamylase

IPTG isopropyl β-D-thiogalactoside

IRS insoluble residual starch

kDa kiloDalton

LAS linear alkylbenzenesulfonate

LAT, BLA B. licheniformis amylase

MW molecular weight

MWU modified Wohlgemuth unit; 1 .6x10 ^"5 mg/MWU = unit of activity

NCBI National Center for Biotechnology Information

NOBS nonanoyloxybenzenesulfonate

NTA nitriloacetic acid

OxAm Purastar HPAM 5000L (Danisco US Inc.)

PAHBAH p-hydroxybenzoic acid hydrazide

PEG polyethyleneglycol

pl isoelectric point

PI performance index

ppm parts per million, e.g., μg protein per gram dry solid

PVA polyvinyl alcohol)

PVP poly(vinylpyrrolidone)

RCF relative centrifugal/centripetal force (i.e., x gravity)

RNA ribonucleic acid

SAS alkanesulfonate

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SSF simultaneous saccharification and fermentation

SSU/g solid soluble starch unit/gram dry solids

sp. species TAED tetraacetylethylenediamine

Tm melting temperature

TrGA Trichoderma reesei glucoamylase

w/v weight/volume

w/w weight/weight

v/v volume/volume

wt% weight percent

°C degrees Centigrade

H ₂ 0 water

dH ₂ 0 or Dl deionized water

dlH ₂ 0 deionized water, Milli-Q filtration

g or gm grams

micrograms

mg milligrams

kg kilograms

μΙ_ and μΙ microliters

mL and ml milliliters

mm millimeters

μηι micrometer

M molar

mM millimolar

μΜ micromolar

U units

sec seconds

min(s) minute/minutes

hr(s) hour/hours

DO dissolved oxygen

Ncm Newton centimeter

ETOH ethanol

eq. equivalents

N normal

MWCO molecular weight cut-off

SSRL Stanford Synchrotron Radiation Lightsource

PDB Protein Database

CAZy Carbohydrate-Active Enzymes database

Tris-HCI tris(hydroxymethyl)aminomethane hydrochloride

HEPES 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid mS/cm milli-Siemens/cm CV column volumes

DP2 disaccharide

HPLC high-performance liquid chromatography

HPAEC-PAD high-performance anion exchange chromatography with pulsed amperometric detection

IC ion chromatography

RID differential refractive index detector

Da Dalton (molecular weight)... since you mention kDa as kiloDalton

1.2. Definitions

[0023] The term "trehalase" refers to enzymes that catalyze generation of two glucose molecules (oc-D glucose and β-D glucose) from a trehalose molecule. Trehalase (a, a-trehalose- 1 -C-glucohydrolase, EC 3.2.1 .28).

[0024] The terms "amylase" or "amylolytic enzyme" refer to an enzyme that is, among other things, capable of catalyzing the degradation of starch, a-amylases are hydrolases that cleave the a-D-(1→4) O-glycosidic linkages in starch. Generally, a-amylases (EC 3.2.1 .1 ; a-D-(1→4)- glucan glucanohydrolase) are defined as endo-acting enzymes cleaving a-D-(1→4) O- glycosidic linkages within the starch molecule in a random fashion yielding polysaccharides containing three or more (1 -4)-a-linked D-glucose units. In contrast, the exo-acting amylolytic enzymes, such as β-amylases (EC 3.2.1 .2; a-D-(1→4)-glucan maltohydrolase) and some product-specific amylases like maltogenic a-amylase (EC 3.2.1 .133) cleave the polysaccharide molecule from the non-reducing end of the substrate, β-amylases, a-glucosidases (EC

3.2.1 .20; a-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1 .3; a-D-(1→4)-glucan glucohydrolase), and product-specific amylases like the maltotetraosidases (EC 3.2.1 .60) and the maltohexaosidases (EC 3.2.1 .98) can produce malto-oligosaccharides of a specific length or enriched syrups of specific maltooligosaccharides. A preferred glucoamylase is TrGA variant of amino acid seqeunce:

SVDDFISTETPIALNNLLCNVGPDGCRAFGTSAGAVIASPSTIDPDYYYMWTRDSALVFK NLIDR FTETYDAGLQRRIEQYITAQVTLQGLSNPSGSLADGSGLGEPKFELTLKPFTGNWGRPQR DGP ALRAIALIGYSKWLINNNYQSTVSNVIWPIVRNDLNYVAQYWNQTGFDLWEEVNGSSFFT VAN QHRALVEGATLAATLGQSGSAYSSVAPQVLCFLQRFWVSSGGYVDSNINTNEGRTGKDVN SV LTSIHTFDPNLGCDAGTFQPCSDKALSNLKVVVDSFRSIYGVNKGIPAGAAVAIGRYAED VYYN GNPWYLATFAAAEQLYDAIYVWKKTGSITVTATSLAFFQELVPGVTAGTYSSSSSTFTNI INAVS TYADGFLSEAAKYVPADGSLAEQFDRNSGTPLSAVHLTWSYASFLTAAARRAGIVPPSWA NSS ASTIPSTCSGASVVGSYSRPTATSFPPSQTPKPGVPSGTPYTPLPCATPTSVAVTFHELV STQF GHTVKVAGNAAALGNWSTSAAVALDAVNYRDNHPLWIGTVNLEAGDVVEYKYIIVGQDGS VT WESDPNHTYTVPAVACVTQVVKEDTWQS (SEQ ID NO: 3); as disclosed in USPN 8058033. [0025] "Enzyme units" herein refer to the amount of product formed per time under the specified conditions of the assay. For example, a "glucoamylase activity unit" (GAU) is defined as the amount of enzyme that produces 1 g of glucose per hour from soluble starch substrate (4% DS) at 60 °C, pH 4.2. A "soluble starch unit" (SSU) is the amount of enzyme that produces 1 mg of glucose per minute from soluble starch substrate (4% DS) at pH 4.5, 50 'Ό. DS refers to "dry solids."

[0026] The term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C ₆ H _{1 0} O5) _x , wherein X can be any number. The term includes plant-based materials such as grains, cereal, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, milo, potato, sweet potato, and tapioca. The term "starch" includes granular starch. The term "granular starch" refers to raw, i.e., uncooked starch, e.g., starch that has not been subject to gelatinization.

[0027] The term 'liquefact' refers to a starch that has undergone liquefaction. [0028] The terms, "wild-type," "parental," or "reference," with respect to a polypeptide, refer to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more amino acid positions. Similarly, the terms "wild-type," "parental," or "reference," with respect to a polynucleotide, refer to a naturally-occurring polynucleotide that does not include a man-made nucleoside change. However, note that a polynucleotide encoding a wild-type, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type, parental, or reference polypeptide.

[0029] Reference to the wild-type polypeptide is understood to include the mature form of the polypeptide. A "mature" polypeptide or variant, thereof, is one in which a signal sequence is absent, for example, cleaved from an immature form of the polypeptide during or following expression of the polypeptide.

[0030] The term "variant," with respect to a polypeptide, refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes one or more naturally- occurring or man-made substitutions, insertions, or deletions of an amino acid. Similarly, the term "variant," with respect to a polynucleotide, refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context.

[0031] The term "recombinant," when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g., a heterologous promoter in an expression vector. Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences. A vector comprising a nucleic acid encoding an amylase is a recombinant vector.

[0032] The terms "recovered," "isolated," and "separated," refer to a compound, protein (polypeptides), cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component with which it is naturally associated as found in nature. An "isolated" polypeptides, thereof, includes, but is not limited to, a culture broth containing secreted polypeptide expressed in a heterologous host cell.

[0033] The term "purified" refers to material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

[0034] The term "enriched" refers to material (e.g., an isolated polypeptide or polynucleotide) that is in about 50% pure, at least about 60% pure, at least about 70% pure, or at least about 90% pure or more.

[0035] The terms "thermostable" and "thermostability," with reference to an enzyme, such as an amylase or a trehalase enzyme, refer to the ability of the enzyme to retain activity after exposure to an elevated temperature. The thermostability of an enzyme, such as an amylase enzyme, is measured by its half-life (ti ₂ ) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions. The half-life may be calculated by measuring residual a-amylase activity following exposure to (i.e., challenge by) an elevated temperature. [0036] A "pH range," with reference to an enzyme, refers to the range of pH values under which the enzyme exhibits catalytic activity.

[0037] The terms "pH stable" and "pH stability," with reference to an enzyme, relate to the ability of the enzyme to retain activity over a wide range of pH values for a predetermined period of time (e.g., 15 min., 30 min., 1 hour).

[0038] The term "amino acid sequence" is synonymous with the terms "polypeptide," "protein," and "peptide," and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an "enzyme." The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N→C). [0039] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0040] "Hybridization" refers to the process by which one strand of nucleic acid forms a duplex with, i.e., base pairs with, a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65 ^< € and 0.1 X SSC (where 1 X SSC = 0.15 M NaCI, 0.015 M Na ₃ citrate, pH 7.0). Hybridized, duplex nucleic acids are characterized by a melting temperature (T _m ), where one half of the hybridized nucleic acids are unpaired with the complementary strand. Mismatched nucleotides within the duplex lower the T _m . [0041] A "synthetic" molecule is produced by in vitro chemical or enzymatic synthesis rather than by an organism.

[0042] The terms "transformed," "stably transformed," and "transgenic," used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations. A non-native sequence may be an endogenous sequence.

[0043] The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

[0044] A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., an amylase) has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

[0045] The term "heterologous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

[0046] The term "endogenous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

[0047] The term "expression" refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation. [0048] A "selective marker" or "selectable marker" refers to a gene capable of being expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include but are not limited to antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.

[0049] A "vector" refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

[0050] An "expression vector" refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation. [0051] The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequences.

[0052] A "signal sequence" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0053] "Biologically active" refer to a sequence having a specified biological activity, such an enzymatic activity. [0054] The term "specific activity" refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed as units (U)/mg of protein.

[0055] As used herein, "water hardness" is a measure of the minerals (e.g., calcium and magnesium) present in water.

[0056] As used herein, an "effective amount of amylase," or similar expressions, refers to an amount of amylase sufficient to produce a visible, or otherwise measurable amount of starch hydrolysis in an particular application. Starch hydrolysis may result in, e.g., a visible cleaning of fabrics or dishware, reduced viscosity of a starch slurry or mash, and the like.

[0057] A "swatch" is a piece of material such as a fabric that has a stain applied thereto. The material can be, for example, fabrics made of cotton, polyester or mixtures of natural and synthetic fibers. The swatch can further be paper, such as filter paper or nitrocellulose, or a piece of a hard material such as ceramic, metal, or glass. For amylases, the stain is starch based, but can include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigment, oil, or mixtures of these compounds.

[0058] A "smaller swatch" is a section of the swatch that has been cut with a single hole punch device, or has been cut with a custom manufactured 96-hole punch device, where the pattern of the multi-hole punch is matched to standard 96-well microtiter plates, or the section has been otherwise removed from the swatch. The swatch can be of textile, paper, metal, or other suitable material. The smaller swatch can have the stain affixed either before or after it is placed into the well of a 24-, 48- or 96-well microtiter plate. The smaller swatch can also be made by applying a stain to a small piece of material. For example, the smaller swatch can be a stained piece of fabric 5/8" or 0.25" in diameter. The custom manufactured punch is designed in such a manner that it delivers 96 swatches simultaneously to all wells of a 96-well plate. The device allows delivery of more than one swatch per well by simply loading the same 96-well plate multiple times. Multi-hole punch devices can be conceived of to deliver simultaneously swatches to any format plate, including but not limited to 24-well, 48-well, and 96-well plates. In another conceivable method, the soiled test platform can be a bead made of metal, plastic, glass, ceramic, or another suitable material that is coated with the soil substrate. The one or more coated beads are then placed into wells of 96-, 48-, or 24-well plates or larger formats, containing suitable buffer and enzyme.

[0059] "A cultured cell material comprising an enzyme" or similar language, refers to a cell lysate or supernatant (including media) that includes an enzyme as a component. The cell material may be from a heterologous host that is grown in culture for the purpose of producing the enzyme.

[0060] "Percent sequence identity" means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:

Gap opening penalty: 10.0

Gap extension penalty: 0.05

Protein weight matrix: BLOSUM series

DNA weight matrix: IUB

Delay divergent sequences %: 40

Gap separation distance: 8

DNA transitions weight: 0.50

List hydrophilic residues: GPSNDQEKR Use negative matrix: OFF

Toggle Residue specific penalties: ON

Toggle hydrophilic penalties: ON

Toggle end gap separation penalty OFF. [0061] Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either termini are included. For example, a variant 500-amino acid residue polypeptide with a deletion of five amino acid residues from the C-terminus would have a percent sequence identity of 99% (495/500 identical residues ^χ 100) relative to the parent polypeptide. Such a variant would be encompassed by the language, "a variant having at least 99% sequence identity to the parent."

[0062] "Fused" polypeptide sequences are connected, i.e., operably linked, via a peptide bond between two subject polypeptide sequences.

[0063] The term "filamentous fungi" refers to all filamentous forms of the subdivision

Eumycotina, particularly Pezizomycotina species.

[0064] The term "degree of polymerization" (DP) refers to the number (n) of anhydro- glucopyranose units in a given saccharide. Examples of DP1 are the monosaccharides glucose and fructose. Examples of DP2 are the disaccharides maltose and sucrose. The term "DE," or "dextrose equivalent," is defined as the percentage of reducing sugar, i.e., D-glucose, as a fraction of total carbohydrate in a syrup.

[0065] The term "dry solids content" (ds) refers to the total solids of a slurry in a dry weight percent basis. The term "slurry" refers to an aqueous mixture containing insoluble solids.

[0066] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process in the production of biochemicals in which a microbial organism, such as an ethanologenic microorganism, and at least one enzyme, such as an amylase, are present during the same process step. SSF includes the contemporaneous hydrolysis of starch substrates (granular, liquefied, or solubilized) to saccharides, including glucose, and the fermentation of the saccharides into alcohol or other biochemical or biomaterial in the same reactor vessel.

[0067] An "ethanologenic microorganism" refers to a microorganism with the ability to convert a sugar or oligosaccharide to ethanol.

[0068] The term "fermented beverage" refers to any beverage produced by a method comprising a fermentation process, such as a microbial fermentation, e.g., a bacterial and/or fungal fermentation. "Beer" is an example of such a fermented beverage, and the term "beer" is meant to comprise any fermented wort produced by fermentation/brewing of a starch-containing plant material. Often, beer is produced exclusively from malt or adjunct, or any combination of malt and adjunct.

[0069] The term "malt" refers to any malted cereal grain, such as malted barley or wheat.

[0070] The term "adjunct" refers to any starch and/or sugar containing plant material that is not malt, such as barley or wheat malt. Examples of adjuncts include common corn grits, refined corn grits, brewer's milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes, rye, oats, potato, tapioca, cassava and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like.

[0071] The term "mash" refers to an aqueous slurry of any starch and/or sugar containing plant material, such as grist, e.g., comprising crushed barley malt, crushed barley, and/or other adjunct or a combination thereof, mixed with water later to be separated into wort and spent grains.

[0072] The term "wort" refers to the unfermented liquor run-off following extracting the grist during mashing.

[0073] "Iodine-positive starch" or "IPS" refers to (1 ) amylose that is not hydrolyzed after liquefaction and saccharification, or (2) a retrograded starch polymer. When saccharified starch or saccharide liquor is tested with iodine, the high DPn amylose or the retrograded starch polymer binds iodine and produces a characteristic blue color. The saccharide liquor is thus termed "iodine-positive saccharide," "blue saccharide," or "blue sac."

[0074] The terms "retrograded starch" or "starch retrogradation" refer to changes that occur spontaneously in a starch paste or gel on ageing.

[0075] The term "about" refers to ± 15% to the referenced value. 2. Enzymes of the Invention

[0076] Trehalases

[0077] Trehalases are enzymes that catalyze generation of two glucose molecules (oc-D glucose and β-D glucose) from a trehalose molecule. Trehalase (a, a-trehalose-1 -C- glucohydrolase, EC 3.2.1 .28) has been reported from many organisms including prokaryotes, plants and animals. Though trehalose is not known to be present in mammals, trehalase enzyme is found to be present in the kidney brush border membrane and the intestinal villi membranes. Trehalose hydrolysis by trehalase enzyme is an important physiological process for various organisms, such as fungal spore germination, insect flight, and the resumption of growth in resting cells. At least two-types of trehalases, based on their pH optima, are known; acid (extracellular) trehalases and neutral (cytosolic) trehalases. A trehalase of the invention may be isolated from an organism of interest using molecular biology techniques known in the art. Trehalase may be derived from a prokaryote or a eukaryote. For example, Trichoderma fungus is known to have at least two trehalases, acidic (extracellular) trehalases and neutral (cytosolic) trehalases. A prokaryotic trehalase is also available: MEGAZYMES (Megazymes International, Ireland). Trehalases may be a variant trehalase that may show improved properties over its parent molecule.

[0078] In some embodiments, the present enzymes have a defined degree of amino acid sequence identity to other enzymes, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, amino acid sequence identity. In some embodiments, the present enzymes are derived from a parental enzyme having a defined degree of amino acid sequence identity, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, amino acid sequence identity.

[0079] In some embodiments, the present enzymes comprise conservative substitution of one or several amino acid residues relative to the amino acid sequence of a parent enzyme.

Exemplary conservative amino acid substitutions are listed in the Table 1 Some conservative mutations can be produced by genetic manipulation, while others are produced by introducing synthetic amino acids into a polypeptide or other means.Table 1. Conservative amino acid substitutions

For Amino Cod Replace with any of

Acid e

Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His

Valine V D-Val, Leu, D-Leu, lie, D-lle, Met, D-Met

[0080] In some embodiments, the present enzymes comprises a deletion, substitution, insertion, or addition of one or a few amino acid residues. In some embodiments, the present enzymes are derived from the amino acid sequence of the parent enzyme by conservative substitution of one or several amino acid residues. In some embodiments, the present enzymes are derived from the amino acid sequence of the parent enzyme by deletion, substitution, insertion, or addition of one or a few amino acid residues. In all cases, the expression "one or a few amino acid residues" refers to 10 or less, i.e., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acid residues.

[0081] In some embodiments, the present enzymes are encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid sequence that is complementary to a nucleic acid that encodes the parent enzyme.

[0082] The present enzymes may be "precursor," "immature," or "full-length," in which case they may include a signal sequence, or "mature," in which case they may lack a signal sequence. Mature forms of the polypeptides are generally the most useful. Unless otherwise noted, the amino acid residue numbering used herein refers to the mature forms of the respective polypeptides. The present enzyme polypeptides may also be truncated to remove the N or C-termini, so long as the resulting polypeptides retain enzyme activity.

[0083] The present enzyme may be a "chimeric" or "hybrid" polypeptide, in that it includes at least a portion of a first enzyme polypeptide, and at least a portion of a second enzyme polypeptide (such chimeric enzymes have recently been "rediscovered" as domain-swap enzymes). The present enzymes may further include heterologous signal sequence or an epitope, foe example, to allow tracking or purification. Exemplary heterologous signal sequences are from B. licheniformis amylase (LAT), B. subtilis (AmyE or AprE), and

Streptomyces CelA.

[0084] In another aspect, nucleic acids encoding an enzyme polypeptide is provided. The nucleic acid may encode the enzyme having the amino acid sequence having a specified degree of amino acid sequence identity. In some embodiments, the nucleic acid encodes an enzyme having at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, amino acid sequence identity to a parental enzyme. In some embodiments, the nucleic acid has at least 80%, at least 85%, at least 90%, at least 95%, or even at least 98% nucleotide sequence identity to parental enzyme. [0085] In some embodiments, the present compositions and methods include nucleic acids that encode an enzyme having deletions, insertions, or substitutions, such as those mentioned, above. It will be appreciated that due to the degeneracy of the genetic code, a plurality of nucleic acids may encode the same polypeptide.

[0086] In another example, the nucleic acid hybridizes under stringent or very stringent conditions to a nucleic acid complementary to a nucleic acid encoding an enzyme having at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, amino acid sequence identity to a parent enzyme. In some embodiments, the nucleic acid hybridizes under stringent or very stringent conditions to a nucleic acid complementary to a nucleic acid having the sequence of a parent enzyme. Such hybridization conditions are described herein but are also well known in the art.

[0087] Nucleic acids may encode a "full-length" ("fl" or "FL") enzyme, which includes a signal sequence, only the mature form of an enzyme, which lacks the signal sequence, or a truncated form of an enzyme, which lacks the N or C-terminus of the mature form. Preferrably, the nucleic acids are of sufficient length to encode an active enzyme.

[0088] A nucleic acid that encodes an enzyme can be operably linked to various promoters and regulators in a vector suitable for expressing the enzyme in host cells. Exemplary promoters are from B. licheniformis amylase (LAT), B. subtilis (AmyE or AprE), and Streptomyces CelA. Such a nucleic acid can also be linked to other coding sequences, e.g., to encode a chimeric polypeptide.

3. Production of Enzymes of the Invention

[0089] The present enzymes can be produced in host cells, for example, by secretion or intracellular expression. A cultured cell material (e.g., a whole-cell broth) comprising an enzyme can be obtained following secretion of the enzyme into the cell medium. Optionally, the enzyme can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final enzyme. A gene encoding an enzyme can be cloned and expressed according to methods well known in the art. Suitable host cells include bacterial, fungal (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae or Trichoderma reesei. Other host cells include bacterial cells, e.g., Bacillus subtilis or B. licheniformis, as well as

Streptomyces.

[0090] The host cell further may express a nucleic acid encoding a homologous or

heterologous glucoamylase, i.e., a glucoamylase that is not the same species as the host cell, or one or more other enzymes. The glucoamylase may be a variant glucoamylase, such as one of the glucoamylase variants disclosed in U.S. Patent No. 8,058,033 (Danisco US Inc.), for example. Additionally, the host may express one or more accessory enzymes, proteins or peptides. These may benefit liquefaction, saccharification, fermentation, SSF processes.

Furthermore, the host cell may produce biochemicals in addition to enzymes used to digest the various feedstock(s). Such host cells may be useful for fermentation or simultaneous saccharification and fermentation processes to reduce or eliminate the need to add enzymes.

3.1. Vectors

[0091] A DNA construct comprising a nucleic acid encoding an enzyme can be constructed to be expressed in a host cell. Because of the well-known degeneracy in the genetic code, different polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding enzymes can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below. [0092] The vector may be any vector that can be transformed into and replicated within a host cell. For example, a vector comprising a nucleic acid encoding an enzyme can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector also may be transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional enzyme. Host cells that serve as expression hosts can include filamentous fungi, for example. The Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. See FGSC, Catalogue of Strains, University of Missouri, aiwww.fgsc.net (last modified January 17, 2007). A

representative vector is pJG153, a promoterless Cre expression vector that can be replicated in a bacterial host. See Harrison et at. (201 1 ) Applied Environ. Microbiol. 77:3916-22.

pJG153can be modified with routine skill to comprise and express a nucleic acid encoding an enzyme variant.

[0093] A nucleic acid encoding an enzyme can be operably linked to a suitable promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Exemplary promoters for directing the transcription of the DNA sequence encoding an enzyme, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or eel A promoters, the promoters of the Bacillus licheniformis a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens a-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae T AKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, A. niger acid stable a-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae those phosphate isomerase, or A. nidulans acetamidase. When a gene encoding an enzyme is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters, cbh l is an endogenous, inducible promoter from T. reesei. See Liu et at. (2008) "Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl ) promoter optimization," Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0094] The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence may be the DNA sequence naturally associated with the enzyme gene to be expressed or from a different Genus or species. A signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence is the cbh l signal sequence that is operably linked to a cbh l promoter.

[0095] An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding an enzyme. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

[0096] The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYCI 77, pUB1 10, pE194, pAMB1 , and plJ702.

[0097] The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the da/ genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xxsC, hygromycin resistance, or the selection may be accomplished by co-transformation.

[0098] Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of enzyme for subsequent enrichment or purification. Extracellular secretion of enzyme into the culture medium can also be used to make a cultured cell material comprising the isolated enzyme. [0099] The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the enzyme to a host cell organelle such as a peroxisome, or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence, SKL. For expression under the direction of control sequences, the nucleic acid sequence of the enzyme is operably linked to the control sequences in proper manner with respect to expression.

[00100] The procedures used to ligate the DNA construct encoding an enzyme, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art {see, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2 ^nd ed., Cold Spring Harbor, 1989, and 3 ^rd ed., 2001 ).

3.2. Transformation and Culture of Host Cells

[00101] An isolated cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of an enzyme. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination.

Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

[00102] Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streptomyces murinus; lactic acid bacterial species including Lactococcus sp. such as Lactococcus lactis; Lactobacillus sp.

including Lactobacillus reuteri; Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism. [00103] A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of

Saccharomyces, including Saccharomyces cerevisiae or a species belonging to

Schizosaccharomyces such as, for example, S. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma sp. such as Trichoderma reesei can be used as a host. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023. An enzyme expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type enzyme. The type and/or degree of glycosylation may impart changes in enzymatic and/or biochemical properties.

[00104] It may be advantageous to delete genes from expression hosts, where the gene deficiency may be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh2, egl1, and egl2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

[00105] Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, e.g., lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, e.g.,

Sambrook et al. (2001 ), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991 -1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding an enzyme is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques. [00106] The preparation of Trichoderma sp. for transformation, for example, may involve the preparation of protoplasts from fungal mycelia. See Campbell et al. (1989) Curr. Genet. 16: 53-56. The mycelia can be obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall, resulting in protoplasts. The protoplasts are protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1 .2 M, e.g., a 1 .2 M solution of sorbitol can be used in the suspension medium.

[00107] Uptake of DNA into the host Trichoderma sp. strain depends upon the calcium ion concentration. Generally, between about 10-50 mM CaCI ₂ is used in an uptake solution. Additional suitable compounds include a buffering system, such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 and polyethylene glycol. The polyethylene glycol is believed to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.

[00108] Usually transformation of Trichoderma sp. uses protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 10 ⁵ to 10 ⁷ /ml_, particularly 2x10 ⁶ /ml_. A volume of 100 μΙ_ of these protoplasts or cells in an appropriate solution (e.g., 1 .2 M sorbitol and 50 mM CaCI ₂ ) may be mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, it is useful to add about 0.25 volumes to the protoplast suspension. Additives, such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells. See, e.g., U.S. Patent No. 6,022,725.

3.3. Expression

[00109] A method of producing an enzyme may comprise cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and/or culture medium.

[00110] The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of an enzyme. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection). [00111] An enzyme secreted from the host cells can be used in a whole broth preparation. In the present methods, the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of an enzyme. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the enzyme to be expressed or isolated. The term "spent whole fermentation broth" is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term "spent whole fermentation broth" also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.

[00112] An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

[00113] The polynucleotide encoding an enzyme in a vector can be operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. The control sequences may in particular comprise promoters.

[00114] Host cells may be cultured under suitable conditions that allow expression of an enzyme. Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose. Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNT™ (Promega) rabbit reticulocyte system.

[00115] An expression host also can be cultured in the appropriate medium for the host, under aerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that host, e.g., from about 25^ to about 75°C (e.g., 30 °C to 45 °C), depending on the needs of the host and production of the desired enzyme. Culturing can occur from about 12 to about 100 hours or greater (and any hour value there between, e.g., from 24 to 72 hours). Typically, the culture broth is at a pH of about 4.0 to about 8.0, again depending on the culture conditions needed for the host relative to production of an enzyme.

3.4. Identification of Enzyme Activity

[00116] To evaluate the expression of an enzyme in a host cell, assays can measure the expressed protein, corresponding mRNA, or enzyme activity. For example, suitable assays include Northern blotting, reverse transcriptase polymerase chain reaction, and in situ hybridization, using an appropriately labeled hybridizing probe. Suitable assays also include measuring enzyme activity in a sample, for example, by assays directly measuring products in the culture media. For example, glucose concentration may be determined using glucose reagent kit No. 15-UV (Sigma Chemical Co.) or an instrument, such as Technicon

Autoanalyzer. a-amylase activity also may be measured by any known method, such as the PAHBAH or ABTS assays. Trehalase activity may be measured as described in the Examples section. Assays are also known in the art to measure other enzyme activities.

3.5. Methods for Enriching and Purifying Enzymes of the Invention [00117] Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare a concentrated enzyme polypeptide-containing solution.

[00118] After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.

[00119] It is desirable to concentrate an enzyme polypeptide-containing solution in order to optimize recovery. Use of unconcentrated solutions requires increased incubation time in order to collect the enriched or purified enzyme precipitate.

[00120] The enzyme containing solution is concentrated using conventional

concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein.

Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and/or ultrafiltration.

[00121] The enzyme solution is concentrated into a concentrated enzyme solution until the enzyme activity of the concentrated enzyme polypeptide-containing solution is at a desired level. [00122] During fermentation, an enzyme polypeptide accumulates in the culture broth. For the isolation, enrichment, or purification of the desired enzyme, the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme enrichment or purification. In one embodiment, the cell-free broth is subjected to salting out using ammonium sulfate at about 70% saturation; the 70% saturation-precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column, and eluted to recover the enzyme-active fraction. For further enrichment or purification, a conventional procedure such as ion exchange or affinity chromatography may be used.

[00123] For production scale recovery, enzyme polypeptides can be enriched or partially purified as generally described above by removing cells via flocculation with polymers.

Alternatively, the enzyme can be enriched or purified by microfiltration followed by

concentration by ultrafiltration using available membranes and equipment. However, for some applications, the enzyme does not need to be enriched or purified, and whole broth culture can be lysed and used without further treatment. The enzyme can then be processed, for example, into granules.

4. Compositions and Uses of Trehalases

[00124] The present trehalases are useful for a variety of industrial applications. For example, trehalases are useful in a starch conversion process, particularly in a saccharification and fermentation process of a starch that has undergone liquefaction. The desired end-product may be any product that may be produced by the enzymatic conversion of the starch substrate. For example, the desired product may be a syrup rich in glucose and maltose, which can be used in other processes, such as the preparation of HFCS, or which can be converted into a number of other useful products, such as ascorbic acid intermediates (e.g., gluconate; 2-keto-L- gulonic acid; 5-keto-gluconate; and 2,5-diketogluconate); 1 ,3-propanediol; aromatic amino acids (e.g., tyrosine, phenylalanine and tryptophan); organic acids (e.g., lactate, pyruvate, succinate, isocitrate, gluconic acid, and oxaloacetate); amino acids (e.g., serine, lysine, glutamic acid, and glycine); antibiotics; antimicrobials; enzymes; vitamins; and hormones.

[00125] The starch conversion process may be a precursor to, or simultaneous with, a fermentation process designed to produce alcohol for fuel or drinking (i.e., potable alcohol). One skilled in the art is aware of various fermentation conditions that may be used in the production of these end-products. Trehalases may also be useful in compositions and methods of food preparation. These various uses of trehalases are described in more detail below.

4.1. Preparation of Starch Substrates

[00126] Those of general skill in the art are well aware of available methods that may be used to prepare starch substrates for use in the processes disclosed herein. For example, a useful starch substrate may be obtained from tubers, roots, stems, legumes, cereals or whole grain. More specifically, the granular starch may be obtained from corn, cobs, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, rice, peas, bean, banana, or potatoes. Corn contains about 60-68% starch; barley contains about 55-65% starch; millet contains about 75-80% starch; wheat contains about 60-65% starch; and polished rice contains 70-72% starch. Specifically contemplated starch substrates are corn starch and wheat starch. The starch from a grain may be ground or whole and includes corn solids, such as kernels, bran and/or cobs. The starch may also be highly refined raw starch or feedstock from starch refinery processes. Various starches also are commercially available. For example, corn starch is available from Cerestar, Sigma, and Katayama Chemical Industry Co. (Japan); wheat starch is available from Sigma; sweet potato starch is available from Wako Pure Chemical Industry Co. (Japan); and potato starch is available from Nakaari Chemical Pharmaceutical Co. (Japan).

[00127] The starch substrate can be a crude starch from milled whole grain, which contains non-starch fractions, e.g., germ residues and fibers. Milling may comprise either wet milling or dry milling or grinding. In wet milling, whole grain is soaked in water or dilute acid to separate the grain into its component parts, e.g., starch, protein, germ, oil, kernel fibers. Wet milling efficiently separates the germ and meal (i.e., starch granules and protein) and is especially suitable for production of syrups. In dry milling or grinding, whole kernels are ground into a fine powder and often processed without fractionating the grain into its component parts. In some cases, oils from the kernels are recovered. Dry ground grain thus will comprise significant amounts of non-starch carbohydrate compounds, in addition to starch. Dry grinding of the starch substrate can be used for production of ethanol and other biochemicals. The starch to be processed may be a highly refined starch quality, for example, at least 90%, at least 95%, at least 97%, or at least 99.5% pure.

4.2. Gelatinization and Liquefaction of Starch

[00128] As used herein, the term "liquefaction" or "liquefy" means a process by which starch is converted to less viscous and shorter chain dextrins. Generally, this process involves gelatinization of starch simultaneously with or followed by the addition of an a-amylase, although additional liquefaction-inducing enzymes optionally may be added. In some embodiments, the starch substrate prepared as described above is slurried with water. The starch slurry may contain starch as a weight percent of dry solids of about 10-55%, about 20- 45%, about 30-45%, about 30-40%, or about 30-35%. a-Amylase (EC 3.2.1 .1 ) may be added to the slurry, with a metering pump, for example. The α-amylase typically used for this application is a thermally stable, bacterial a-amylase, such as a Geobacillus stearothermophilus a-amylase. The α-amylase is usually supplied, for example, at about 1500 units per kg dry matter of starch. To optimize a-amylase stability and activity, the pH of the slurry typically is adjusted to about pH 5.5-6.5 and about 1 mM of calcium (about 40 ppm free calcium ions) can also be added. Geobacillus stearothermophilus variants or other trehalases may require different conditions. Bacterial a-amylase remaining in the slurry following liquefaction may be deactivated via a number of methods, including lowering the pH in a subsequent reaction step or by removing calcium from the slurry in cases where the enzyme is dependent upon calcium.

[00129] The slurry of starch plus the α-amylase may be pumped continuously through a jet cooker, which is steam heated to 105°C. Gelatinization occurs rapidly under these conditions, and the enzymatic activity, combined with the significant shear forces, begins the hydrolysis of the starch substrate. The residence time in the jet cooker is brief. The partly gelatinized starch may be passed into a series of holding tubes maintained at 105-1 10 °C and held for 5-8 min. to complete the gelatinization process ("primary liquefaction"). Hydrolysis to the required DE is completed in holding tanks at 85-95 ^ or higher temperatures for about 1 to 2 hours ("secondary liquefaction"). These tanks may contain baffles to discourage back mixing. As used herein, the term "minutes of secondary liquefaction" refers to the time that has elapsed from the start of secondary liquefaction to the time that the Dextrose Equivalent (DE) is measured. The slurry is then allowed to cool to room temperature. This cooling step can be 30 minutes to 180 minutes, or more. The liquefied starch typically is in the form of a slurry having a dry solids content (w/w) of about 10-50%; about 10-45%; about 15-40%; about 20-40%; about 25-40%; or about 25-35%.

[00130] In particular embodiments, starch liquifaction is performed at a temperature range of 90-1 15 ^Q C, for the purpose of producing high-purity glucose syrups, HFCS,

maltodextrins, etc.

4.3. Saccharification [00131] The liquefied starch can be saccharified into a syrup rich in lower DP (e.g., DP1 + DP2) saccharides, using a-amylases, optionally in the presence of other enzyme(s). The exact composition of the products of saccharification depends on the combination of enzymes used, as well as the type of granular starch processed. Advantageously, the syrup obtainable using the provided trehalases may contain a weight percent of DP2 of the total

oligosaccharides in the saccharified starch exceeding 30%, e.g., 45% - 65% or 55% - 65%. The weight percent of (DP1 + DP2) in the saccharified starch may exceed about 70%, e.g., 75% - 85% or 80% - 85%. The amylases also produce a relatively high yield of glucose, e.g., DP1 > 20%, in the syrup product.

[00132] Whereas liquefaction is generally run as a continuous process, saccharification is often conducted as a batch process. Saccharification typically is most effective at

temperatures of about 60-65 'Ό and a pH of about 4.0-4.5, e.g., pH 4.3, necessitating cooling and adjusting the pH of the liquefied starch. Saccharification may be performed, for example, at a temperature between about 40 °C, about 50 ^< €, or about 55 °C to about 60 ^< € or about 65 °C. Saccharification is normally conducted in stirred tanks, which may take several hours to fill or empty. Enzymes typically are added either at a fixed ratio to dried solids as the tanks are filled or added as a single dose at the commencement of the filling stage. A saccharification reaction to make a syrup typically is run over about 24-72 hours, for example, 24-48 hours. When a maximum or desired DE has been attained, the reaction is stopped by heating to 85 'Ό for 5 min., for example. Further incubation will result in a lower DE, eventually to about 90 DE, as accumulated glucose re-polymerizes to isomaltose and/or other reversion products via an enzymatic reversion reaction and/or with the approach of thermodynamic equilibrium. When using an amylase, saccharification optimally is conducted at a temperature range of about 30 'Ό to about 75 ^q C, e.g., 45 ^q C - 75°C or 47 ^< Ό - 74 °C. The saccharifying may be conducted over a pH range of about pH 3 to about pH 7, e.g., pH 3.0 - pH 7.5, pH 3.5 - pH 5.5, pH 3.5, pH 3.8, or pH 4.5.

[00133] An amylase may be added to the slurry in the form of a composition. Amylase can be added to a slurry of a granular starch substrate in an amount of about 0.6 - 10 ppm ds, e.g., 2 ppm ds. An amylase can be added as a whole broth, clarified, enriched, partially purified, or purified enzyme. The specific activity of the amylase may be about 300 U/mg of enzyme, for example, measured with the PAHBAH assay. The amylase also can be added as a whole broth product.

[00134] An amylase may be added to the slurry as an isolated enzyme solution. For example, an amylase can be added in the form of a cultured cell material produced by host cells expressing an amylase. An amylase may also be secreted by a host cell into the reaction medium during the fermentation or SSF process, such that the enzyme is provided continuously into the reaction. The host cell producing and secreting amylase may also express an additional enzyme, such as a glucoamylase. For example, U.S. Patent No. 5,422,267 discloses the use of a glucoamylase in yeast for production of alcoholic beverages. For example, a host cell, e.g., Trichoderma reesei or Aspergillus niger, may be engineered to co-express an amylase and a glucoamylase, e.g., HgGA, TrGA, or a TrGA variant, during saccharification. The host cell can be genetically modified so as not to express its endogenous glucoamylase and/or other enzymes, proteins or other materials. The host cell can be engineered to express a broad spectrum of various saccharolytic enzymes. For example, the recombinant yeast host cell can comprise nucleic acids encoding a glucoamylase, an alpha-glucosidase, an enzyme that utilizes pentose sugar, an a-amylase, a pullulanse, an isoamylase, and/or an

isopullulanase. See, e.g., WO 201 1/153516 A2. 4.4. Isomerization

[00135] The soluble starch hydrolysate produced by treatment with amylase can be converted into high fructose starch-based syrup (HFSS), such as high fructose corn syrup (HFCS). This conversion can be achieved using a glucose isomerase, particularly a glucose isomerase immobilized on a solid support. The pH is increased to about 6.0 to about 8.0, e.g., pH 7.5 (depending on the isomerase), and Ca ²⁺ is removed by ion exchange. Suitable isomerases include SWEETZYME®, IT (Novozymes A/S); G-ZYME® IMGI, and G-ZYME® G993, KETOMAX®, G-ZYME® G993, G-ZYME® G993 liquid, and GENSWEET® IGI.

Following isomerization, the mixture typically contains about 40-45% fructose, e.g., 42% fructose.

4.5. Fermentation

[00136] The soluble starch hydrolysate, particularly a glucose rich syrup, can be fermented by contacting the starch hydrolysate with a fermenting organism typically at a temperature around 32 ^< Ό, such as from 30 'Ό to 35^ for alcohol-producing yeast. The temperature and pH of the fermentation will depend upon the fermenting organism. EOF products include metabolites, such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono delta-lactone, sodium erythorbate, lysine and other amino acids, omega 3 fatty acid, butanol, isoprene, 1 ,3-propanediol, pyruvate, 2,3-butanediol and other biomaterials.

[00137] Ethanologenic microorganisms include yeast, such as Saccharomyces cerevisiae and bacteria, e.g., Zymomonas moblis, expressing alcohol dehydrogenase and pyruvate decarboxylase. The ethanologenic microorganism can express xylose reductase and xylitol dehydrogenase, which convert xylose to xylulose. Improved strains of ethanologenic microorganisms, which can withstand higher temperatures, for example, are known in the art and can be used. See Liu et al. (201 1 ) Sheng Wu Gong Cheng Xue Bao 27:1049-56.

Commercial sources of yeast include ETHANOL RED® (LeSaffre); THERMOSACC®

(Lallemand); RED STAR® (Red Star); FERMIOL® (DSM Specialties); and SUPERSTART® (Alltech). Microorganisms that produce other metabolites, such as citric acid and lactic acid, by fermentation are also known in the art. See, e.g., Papagianni (2007) Biotechnol. Adv. 25:244- 63; John et al. (2009) Biotechnol. Adv. 27:145-52.

[00138] The saccharification and fermentation processes may be carried out as an SSF process. Fermentation may comprise subsequent enrichment, purification, and recovery of ethanol, for example. During the fermentation, the ethanol content of the broth or "beer" may reach about 8-18% v/v, e.g., 14-15% v/v. The broth may be distilled to produce enriched, e.g., 96% pure, solutions of ethanol. Further, C0 ₂ generated by fermentation may be collected with a C0 ₂ scrubber, compressed, and marketed for other uses, e.g., carbonating beverage or dry ice production. Solid waste from the fermentation process may be used as protein-rich products, e.g., livestock feed. [00139] As mentioned above, an SSF process can be conducted with fungal cells that express and secrete amylase continuously throughout SSF. The fungal cells expressing amylase also can be the fermenting microorganism, e.g., an ethanologenic microorganism. Ethanol production thus can be carried out using a fungal cell that expresses sufficient amylase or trehalase so that less or no enzyme has to be added exogenously. The fungal host cell can be from an appropriately engineered fungal strain. Fungal host cells that express and secrete other enzymes, in addition to amylase, or trehalase also can be used. Such cells may express glucoamylase and/or a pullulanase, phytase, a/p ?a-glucosidase, isoamylase, beta-amylase cellulase, xylanase, other hemicellulases, protease, befa-glucosidase, pectinase, esterase, redox enzymes, transferase, or other enzyme.

[00140] A variation on this process is a "fed-batch fermentation" system, where the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression may inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. The actual substrate

concentration in fed-batch systems is estimated by the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases, such as C0 ₂ . Batch and fed- batch fermentations are common and well known in the art.

[00141] Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation permits modulation of cell growth and/or product concentration. For example, a limiting nutrient such as the carbon source or nitrogen source is maintained at a fixed rate and all other parameters are allowed to moderate. Because growth is maintained at a steady state, cell loss due to medium being drawn off should be balanced against the cell growth rate in the fermentation. Methods of optimizing continuous fermentation processes and maximizing the rate of product formation are well known in the art of industrial microbiology.

4.6. Compositions

[00142] Trehalases of the invention are used in fermentation of various starch substrates. Trehalases may be used in fermentation of a liquefact. Trehalase may also be used in simultaneous saccharification and fermentation (SSF). Trehalases may be used at a suitable pH, temperature and time. Use of a trehalase may improve the health of the yeast in fermentation media as evidenced by increased ethanol production and/or continued

degradation of trehalose as evidenced by lower DP2 levels or trehalose levels. Certain fungal trehalases may be more effective for such functionalities. Trehalases may be used at any desired concentrations, e.g., from about 0.1 μg/g to 1 mg/g of DS in the fermentation. Preferred concentrations include 0.1 , 0.2, 0.25, 0.5, 1 .0, 2, 4, 6, 8, 10, 20, 50, 100 μg/g of DS in the fermentation.

[00143] Trehalases may be combined with a glucoamylase (EC 3.2.1 .3), e.g., a

Trichoderma glucoamylase or variant thereof. An exemplary glucoamylase is Trichoderma reesei glucoamylase (TrGA) and variants thereof that possess superior specific activity and thermal stability. See U.S. Published Applications Nos. 2006/0094080, 2007/0004018, and 2007/0015266 (Danisco US Inc.). Suitable variants of TrGA include those with glucoamylase activity and at least 80%, at least 90%, or at least 95% sequence identity to wild-type TrGA. a- amylases advantageously increase the yield of glucose produced in a saccharification process catalyzed by TrGA.

[00144] Alternatively, the glucoamylase may be another glucoamylase derived from plants (including algae), fungi, or bacteria. For example, the glucoamylases may be Aspergillus niger G1 or G2 glucoamylase or its variants {e.g., Boel et al. (1984) EMBO J. 3:1097-1 102; WO 92/00381 ; WO 00/04136 (Novo Nordisk A S)); and A. awamori glucoamylase {e.g., WO

84/02921 (Cetus Corp.)). Other contemplated Aspergillus glucoamylase include variants with enhanced thermal stability, e.g., G137A and G139A (Chen et al. (1996) Prot. Eng. 9:499-505); D257E and D293E/Q (Chen et al. (1995) Prot. Eng. 8:575-582); N182 (Chen et al. (1994) Biochem. J. 301 :275-281 ); A246C (Fierobe et al. (1996) Biochemistry, 35: 8698-8704); and variants with Pro residues in positions A435 and S436 (Li et al. (1997) Protein Eng. 10:1 199- 1204). Other contemplated glucoamylases include Talaromyces glucoamylases, in particular derived from T. emersonii {e.g., WO 99/28448 (Novo Nordisk A S), T. leycettanus {e.g., U.S. Patent No. RE 32,153 (CPC International, Inc.)), T. duponti, or T. thermophilus {e.g., U.S. Patent No. 4,587,215). Contemplated bacterial glucoamylases include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum {e.g., EP 135138 (CPC International, Inc.) and C. thermohydrosulfuricum {e.g., WO 86/01831 (Michigan Biotechnology Institute)). Suitable glucoamylases include the glucoamylases derived from Aspergillus oryzae, such as a glucoamylase shown in SEQ ID NO:2 in WO 00/04136 (Novo Nordisk A S). Also suitable are commercial glucoamylases, such as AMG 200L; AMG 300 L; SAN™ SUPER and AMG™ E (Novozymes); OPTIDEX® 300 and OPTIDEX L-400 (Danisco US Inc.); AMIGASE™and AMIGASE™PLUS (DSM); G-ZYME® G900 (Enzyme Bio-Systems); and G-ZYME® G990 ZR {A. niger glucoamylase with a low protease content). Still other suitable glucoamylases include Aspergillus fumigatus glucoamylase, Talaromyces glucoamylase, Thielavia glucoamylase, Trametes glucoamylase, Thermomyces glucoamylase, Athelia glucoamylase, or Humicola glucoamylase {e.g., HgGA). Glucoamylases typically are added in an amount of about 0.1 - 2 glucoamylase units (GAU)/g ds, e.g., about 0.16 GAU/g ds, 0.23 GAU/g ds, or 0.33 GAU/g ds.

[00145] Other suitable enzymes that can be used with trehalases include a phytase, protease, pullulanase, β-amylase, isoamylase, a different a-amylase, alpha-glucosidase, cellulase, xylanase, other hemicellulases, beta-glucosidase, transferase, pectinase, lipase, cutinase, esterase, redox enzymes, or a combination thereof. For example, a debranching enzyme, such as an isoamylase (EC 3.2.1 .68), may be added in effective amounts well known to the person skilled in the art. A pullulanase (EC 3.2.1 .41 ), e.g., PROMOZYME®, is also suitable. Pullulanase typically is added at 100 U/kg ds. Further suitable enzymes include proteases, such as fungal and bacterial proteases. Fungal proteases include those obtained from Aspergillus, such as A. niger, A. awamori, A. oryzae; Mucor (e.g., M. miehei); Rhizopus; and Trichoderma.

[00146] β-Amylases (EC 3.2.1 .2) are exo-acting maltogenic amylases, which catalyze the hydrolysis of 1 ,4-a-glucosidic linkages into amylopectin and related glucose polymers, thereby releasing maltose. β-Amylases have been isolated from various plants and

microorganisms. See Fogarty et al. (1979) in PROGRESS IN INDUSTRIAL MICROBIOLOGY, Vol. 15, pp. 1 12-1 15. These β-Amylases have optimum temperatures in the range from 40 ^ to 65^ and optimum pH in the range from about 4.5 to about 7.0. Contemplated β-amylases include, but are not limited to, β-amylases from barley SPEZYME® BBA 1500, SPEZYME® DBA, OPTIMALT™ ME, OPTIMALT™ BBA (Danisco US Inc.); and NOVOZYM™WBA (Novozymes A/S).

[00147] Compositions comprising the present trehalases may be aqueous or nonaqueous formulations, granules, powders, gels, slurries, pastes, etc., which may further comprise any one or more of the additional enzymes listed, herein, along with buffers, salts, preservatives, water, co-solvents, surfactants, and the like. Such compositions may work in combination with endogenous enzymes or other ingredients already present in a slurry, water bath, washing machine, food or drink product, etc., for example, endogenous plant (including algal) enzymes, residual enzymes from a prior processing step, and the like. EXAMPLES

Example 1 : Materials and Methods

[00148] Liquefactions. All of the following lab-based fermentations were completed with liquefact samples collected from industrial fuel ethanol plants. Liquefacts were collected at the plant and brought back to labs for immediate freezing and storage. On the day of fermentation use, liquefact is brought up to fermentation temperature in a water-bath. Prior to fermentation, the liquefact pH was adjusted to the starting pH (4.8) of fermentation using sulfuric acid. Because these liquefacts were from fuel ethanol plants, a significant portion of backset (also known as thin stillage) had been used to generate these liquefact samples. This addition of backset may have resulted in industry-wide fermentations starting off with measureable trehalose levels.

[00149] Fermentations:

[00150] Simultaneous saccharification and fermentation (SSF), or fermentation experiments were conducted by long-standing internal protocols using 100g-scale

fermentations. . All fermentation comparisons were done with triplicate (n=3) 100g-scale flask conditions and data displayed herein are averages of those triplicate fermentations.

Fermentations were conducted within temperature controlled (either 32 °C isotemp or 36°C ramp temperature profiles) shaker incubators (150rpm shaking). Before enzyme dosing, 600ppm urea and 0.1 % yeast (typically dry-pitched) were also added to the liquefact. All trehalase dosing was carried out at the beginning of the fermentation along with the

DISTILLASE® SSF (an enzyme blend comprising glucoamylase from GENENCOR) dosing. For Example 2 only, for equal dilution comparisons, trehalase dosing included the enzyme of interest plus a volume of water necessary to equalize the total dosing volume to 670μΙ_ (the largest volume needed to obtain 8ug/gDS TrnT (EH)).

[00151] Sample preparation:

[00152] Fermentation broths were sampled into 2ml_ tubes for subsequent 3min

13000rpm centrifugations. A 0.500ml_ supernatant aliquot was pipetted into glass tube followed by a 50μΙ_ 1 M sulfuric acid addition to deactivate GA activity. After 5 minute incubations, the fermentation sample was diluted to 10x dilution with Dl water. This procedure is an internal standard protocol of sampling for organic acid HPLC analysis that not only deactivates the enzymes before analysis, but also purposefully provides a background sulfuric acid comparable to the HPLC mobile phase level. An approximate 1 ml_ aliquot of this sample preparation was filtered using 0.22μηι nylon syringe filter and placed into an HPLC sample vial.

[00153] For trehalose determination by the ion chromatography method of high- performance anion exchange with pulsed amperometric detection (HPAEC-PAD), a separate 0.500mL aliquot from the above 10x diluted sample preparation was diluted an additional 10 fold in 9mM NaOH (to neutralize the acid added earlier to kill enzyme activity). This 100x dilution sample was filtered using a 0.22um nylon syringe filter into a 2mL IC vial for HPAEC- PAD analysis.

[00154] Organic Acid HPLC Separations

Column: Phenomenex® Rezex™ Organic Acid (ROA), 300 x 7.80 mm

Guard column: Phenomenex® SecurityGuard™ Carbo-H cartridges.

Column temperature: 65^

Mobile phase: 0.01 N sulfuric acid. Flow rate: 0.6 or 0.7 mL/min

Injection volume: 20 μΙ_

Detection: differential refractive index held at 40^

[00155] Advanced Carbohydrate Separations using HPAE-PAD:

Instrument: Dionex® ICS-5000

Column: Dionex® CarboPac PA20, 250 x 4 mm.

Guard column: Dionex® CarboPac PA20G, 50 x 4 mm.

Column temperature: 35°C

Mobile phase: gradient elution with 10mM and 200mM NaOH MP concentrations (made from 50% soln)

Gradient: initial 1 mM NaOH isocratic for 5min followed by a single-stage gradient to 33mM with subsequent regeneration and equilibration

Flow rate: 0.5 mL/min

Injection volume: 10 μ\- Detection: post-column gold electrochemical cell (4-step pulsed amperometric mode)

[00156] Trehalase dosing levels were based on total protein levels by Bradford assay. TraT #9, shake-flask, 772 μg/mL total protein, Endo-H treated; TrnT #22, shake-flask, 369 μg/mL total protein, Endo-H treated; TaaT #24, shake-flask, 9^g/mL total protein, Endo-H treated; Megazyme trehalase Specific activity: 150 U/mg(40 ^q C, pH 5.5), 2100 U/mL.

Example 2: Trehalase activity from Trichoderma cellulase enzyme preparation

[00157] Fermentations were performed as described above for 55 hours (hr). Samples tested were as follows: 1 . SSF (control); 2. SSF with added benchmark trehalase (prokaryotic trehalase from MEGAZYMES; cat. E-TREH); 3. SSF with 250μΙ cellulosic enzyme mixture from T. reesei strain (TRIO). Levels of trehalose, DP1 , DP2 and ethanol (EtOH) were quantitiated at the end of fermentation (EOF) time point of 55 hr and are shown in Table 1 .

TABLE 1

[00158] A greater than 0.3% wt/v increase in ethanol production was seen from T. reesei cellulosic enzyme mixture (TRIO) dosed fermentations as compared to the control (16.76% wt/v versus 16.41 % wt/v). Further, a greater than 0.2% wt/v increase in ethanol production was seen from T. reesei cellulosic enzyme mixture (TRIO) dosed fermentations as compared to the benchmark trehalase (16.76% wt/v versus 16.55% wt/v). This improved yield appears to be attributable to a decrease in DP2 levels, of which trehalose is a constituent. This indicates that the T. reesei cellulosic enzyme mixture contains trehalase activity (data not shown). This result further indicates that the trehalase enzyme(s) in the T. reesei cellulosic enzyme has improved stability and/or activity as compared to the benchmark trehalase.

Example 3: Production of T. reesei (extracellular) trehalase and neutral (cytosolic) trehalase (TraT and TrnT, respectively)

[00159] Sequences for each Trehalase candidate are available as Neutral (cytosolic) trehalase GH37 (tre2) = JGI PID 123226 = Genbank EGR46144.1 ; amino acid sequence:

mpplfslaal Igtailtihs nvahalying sviapcdspi ychgdilrei elahpfsdsktfvdmpakrp Iseiqtafan Ipkplrndss Iqtflasyfa daggeliqvp ranlttnptflskindtvie qfvtqvidiw pdltrryagd aavkncsscp nsfipvnrtf

vvaggrfrepyywdsywive gllrtggafv giarntidnf Idfierfgfv pngarlyyln rsqppllsrmvkvyidhtnd tailrralpl Ivkehefwtr nrtvdvrvnn ktyvlnqyav qntqprpesfredfqtannr syyaasgiiy patkplnesq ieelyanlas gaesgndyta rwladpsdamrdvyfplrsl nnkdivpvdl nsilygnela iaqfynqtgn ttaarewssl

aanrsasiqavfwnetlfsy fdynltsssq niyvpldkda valdrqtapp gkqvlfhvgq fypfwtgaapeylrnnpfav trifdrvksy Idtrpggipa snvntgqqwd qpnvwpphmh ilmeslnsvpatfseadpay qdvrnlslrl gqryldftfc twratggsts etpklqgltd qdvgimfekyndnstnaagg ggeyqvvegf gwtngvllwt adtfgsqlkr pqcgnimagh papskrsavqldmwdasrvk kfgrraegrm gtlhaw (SEQ ID NO: 1 )

[00160] Acid (extracellular) trehalase GH65 (trel ) = JGI PID 123456 = Genbank

EGR45658.1 (JGI— Joint Genome institute). Amino acid sequence:

mrstvtsaaa llsllqlvsp vhgttlvdrv tkclsrhdgs daeshfsknv yktdfagvtwdednwllstt qlkqgafear gsvangylgi nvasvgpffe vdteedgdvi sgwplfsrrqsfatvagfwd aqpqmngtnf pwlsqygsdt aisgiphwsg Ivldlgggty Idatvsnktishfrstydyk agvlswsykw tpkgnkgsfd isyrlfankl hvnqavvdmq

vtasknvqasivnvldgfaa vrtdfvesge dgsaifaavr pngvanvtay vyaditgsgg vnlssrkivhnkpyvhanas siaqavpvkf aagrtvrvtk fvgaassdaf knpkqvakka aaaglsngytkslkahveew atvmpessvd sfadpktgkl padshivdsa iiavtntyyl Iqntvgkngikavdgapvnv dsisvgglts dsyagqifwd adlwmqpglv aahpeaaeri tnyrlarygqakenvktaya gsqnetffsa saavfpwtsg rygnctatgp cwdyeyhlng digislvnqwvvngdtkdfe knlfpvydsv aqlygnllrp nktswtltnm tdpdeyanhv daggytmpliaetlqkansf rqqfgieqnk twndmasnvl vlrengvtle ftamngtavv kqadvimltyplsygtnysa qdalndldyy ankqspdgpa mtyaffsiva neispsgcsa ytyaqnafkpyvrapfyqis eqliddasvn ggthpaypfl tghggahqvv Ifgylglrlv pddvihiepnlppqipylry rtfywrgwpi sawsnythtt Israagvaal egadqrfark pitihagpeqdptayrlpvk gsvvipnkqi gsqqtyagnl vqchaasspn dyvpgqfpia avdgatstkwqpasadkvss itvsldkedv gslvsgfhfd waqappvnat vifhdealad patalasahkhnskyttvts Itnielsdpy vstkdlnaia ipignttnvt Ishpvaasry asllivgnqgldpvdvkakn gtgatvaewa ifghgkehsg kpsshskrrl nvrtaatlsn prsfmrrrl (SEQ ID NO: 2) [00161] Amplified from Genomic DNA, cloned into a standard Trichoderma expression vector in E.coli, sequenced, digested and gel purified out the bacterial DNA, PCR amplified the exempt expression cassette. Performed PEG transformation of the exempt trehalase into LVS- EndoT Delete strain and plated on VOGELs plates. Transformants appeared after 3-4 days. Picked approximately 100 transformants of each stable trehalase. Transferred each stable transformant to 24-well plate and allowed to grow 6 days at 28 ^Q C in Aachen media. Screened broth of transformants by protein expression (SDS-PAGE gels) and activity (MEGAZYMES trehalase activity assay). Top transformants were inoculated into 250ml_ shake flasks in duplicate in Aachen medium as well as in slow-release microtiter plates in NREL media.

Cultures were allowed to grow at 28 ^Q C for 6 days. Broth samples were checked by activity assay/SDS-Page for highest activity/expression. Top transformants were selected for 14L evaluation. Final top Trehalase candidate selected: LVS-ETD T.reesei acid trehalase #23.

Example 4: Comparison of TraT and TrnT stability/activity during fermentation to TaaT and benchmark trehalase

[00162] Comprehensive fermentation performance views for trehalose dynamics for each set of trehalase dosing sets are shown in Figures 1 (TraT), 2 (TrnT) and 3 (TaaT; Trichoderma atroviride trehalase). Each of these comparisons also included the control and benchmark (MEGAZYMES) fermentations. The trehalose time profile for the control was as expected, with a small initial decrease in trehalose followed by an almost equal increase latter in fermentation. These trehalose dynamics within this control could have been either due to natural fluxes in and out of the yeast cells and/or trace levels of background trehalases known to exist.

[00163] When comparing trehalase-dosed fermentations, complete trehalose hydrolysis was seen for the 22 hour sampling time. However, later time points in fermentation revealed significant differences in performance. Specifically, for both the Trichoderma reesei trehalases, the trehalose levels remained low throughout the entire course of the fermentation (Figures 1 and 2). However, the Trichoderma atroviride profiles showed significant increases in the extracellular trehalose levels at later time points. Since trehalose levels were initially reduced to below detectable limits in all samples, this significant rise in trehalose levels towards the end of fermentation was evidence of TaaT stability/activity issues. This trehalase application- stability issue was also apparent for the benchmark (Megazyme) trehalase (shown in each of Figures 1 , 2, and 3; dotted line; note the marked increase in trehalose at later time points in fermentation). Example 5: Effect of adding TraT in EtOH fermentation

[00164] Based on the results above, the addition of TraT to fermentations was assessed for its effectiveness in improving the production of ethanol in fermentations. Fermentations were set up as described above in Example 1 with (1 ) no added trehalase (control), (2) with 0^g/g DS TraT added at the beginning of fermentation, or (3) with 0.25μg/g DS TraT added at the beginning of fermentation. Fermentations were performed for 72 hours with samples taken at 0, 24, 32, 48, 56, and 72 hours. The fermentations were run at 36°C temperature ramp, not only to be more indicative of a fermentation plant conditions, but also to provide a more stressed environment for the yeast cells.

[00165] As shown in Figure 4, the addition of either 0^g/g DS or 0.25μg/g DS TraT at the beginning of fermentation results in reduced trehalose levels throughout the fermentation as compared to the control (no trehalase added). The peak trehalose level observed in the TraT samples (32 hr time point) was about 0.2%wt/v as compared to about 0.35%wt/v in the control (48 hr timepoint). DP2 levels were also measured for these time points and are shown in Figure 5. DP2 levels decreased in all the samples, however, the decrease was more pronounced for the fermentations treated with trehalase TraT.

[00166] Figure 6 shows that the EtOH levels (%v/v) at EOF (72 hr) for the TraT- containing fermentations were significantly higher than the control fermentation (no added trehalase). It is noted that the increase in EtOH observed is more than would be expected based solely on the increased consumption of glucose by the yeast that is derived from trehalose hydrolysis by trehalase. For example, if a 0.2% wt/v trehalose level in the

extracellular supernatant environment is completely hydrolyzed by the end of fermentation, a 0.14%v/v increase in ethanol would be expected. However, greater ethanol increases were seen in these experiments. Specifically, heat ramped fermentations showed a 0.22% wt/v reduction in trehalose over control (Figure 4, 0.5 μg/g DS TraT), and therefore a 0.12%v/v ethanol increase is expected. Instead, an approximate 50% larger than expected increase was measured (~0.28%v/v ethanol increase; Figure 6, 0.5 μg/g DS TraT). This data also shows that the EOF DP2 level is not the absolute determining factor for the amount of ethanol increase due to trehalase treatment. Even though both doses show a similar 72hr EOF trehalose level, the higher dose that kept trehalose levels lower throughout SSF showed the largest increase in ethanol production.

[00167] With regard to this finding, additional lab scale fermentation data show that addition of trehalase to SSF keeps the fermentative yeast active for longer during fermentation for extra ethanol conversion. Specifically, the addition of trehalase at the start of fermentation allows the yeast to reach higher EOF ethanol levels than control fermentations (see Figures 7 and 8).

Example 6

The effect of trehalase treatment on the levels of trehalose in subsequent fermentations [00168] The fermentations were carried out as described above. However, after the first fermentation with a trehalase treatment, the backsets from those fermentations were used in preparing the subsequent fermentation reactions. The amount of trehalose was measured at various timepoints. The data is shown in the Table below and Figure 9. As can be seen, the trehalose profiles take a few fermentations to come down to lower levels. Furthermore, the trehalose profiles after ending a trehalase trial show the longer-lasting effects of reducing trehalose. Not wishing to be bound by the theory, it is possible that both of these effects are due to the time required to re-equilibrate the backset trehalose levels, as supported by the size and rate of the particular fuel ethanol plant from which this data was generated.

[00169] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.