Novel Esterases and Their Use

Field of the Invention

The present invention relates to novel esterases, and more precisely to polyesterase proteins having cutinase and/or suberinase activity. Said enzymes may be obtained from the fungal genus Coprinus or Trichoderma. The invention also relates to isolated polynucleotides encoding said proteins, and to vectors and genetically modified microorganisms comprising the polynucleotides, as well as to a method for producing the proteins. Still further the invention relates to an enzyme preparation comprising the polyesterase protein, and to the use of the protein or preparation. Finally the invention relates to a method of hydrolysis of cutin and/or suberin or other polyesters using the polyesterases.

Technical Background

Cutinases and suberinases are polyesterases, which are able to degrade or partially depolymerise plant polyester waxes, i.e. cutin and suberin. Significant amounts of cutin/suberin are present in different agricultural and forest raw materials and by-products, such as birch bark and cork, berries, cereals, vegetables and their processing by-products. The presence of these waxes in plant raw materials may impair the industrial processing of plant ma- terials due to their hydrophobic character and recalcitrant structure.

Modification of the polyesters would improve the processing and exploitation of several natural materials, and would reduce disposal of process co-products or wastes. These waste fractions could be exploited as a source of more valuable compounds, e.g. suberin-based oligoesters could be potential raw materials in lubricants and binders. The use of polyesterases improve the processing and exploitation of several plant materials, such as cereals, fruits, vegetables and berries, and also improve release and recovery of valuable bioactive and functional components from these raw materials.

Sustainable use of natural resources and waste management con- tribute to minimise waste production. The use of enzymes in synergy with chemical and physical processes is an environmentally friendly means to add value to waste co-products. Cutinases/suberinases can also be utilised e.g. in laundry and dishwashing applications to remove fats as well as in cotton bio- scouring and surface modification of man-made polyester fibres.

Although lipids and waxes are abundant constituents of different industrial products and lignocellulosic residues, only a limited set of lipid modifying enzymes, other than conventional lipases, are commercially available. Cu- tinases and suberinases are regarded as potential enzymes for modification of natural lipids and waxes, which cannot be hydrolyzed by conventional lipases.

A cutinase from the plant/human pathogen fungus Fusarium solani sp. pisi is the most studied cutinase so far (Carvalho et a/., 1999), but cuti- nases have also been found in microorganisms such as Alternaria brassicicola (Trail and Kόller, 1993), Botrytis cinerea {Gindro and Pezet 1999), Venturia In- aequalis {Kόller and Parker, 1989), Aspergillus oryzae (Maeda et a/., 2005) and in certain Streptomyces species (Fett et a/., 1992). All of the biochemically well-characterized cutinases are serine esterases, containing the classical Ser- His-Asp triad common in serine proteases and in several lipases. The characterized cutinases possess a pH optimum from neutral to alkaline. Cutinases have been suggested for a number of uses of which only a few are mentioned herein. WO2004/029193 for example suggests the use of lipases including cutinases in fermentation processes, in particular in ethanol production processes. US 6,255,451 relates to degradation of biodegradable polymers with lipase and cutinase. A great number of potential lipolytic enzyme production organisms have been listed, including i.a. Coprinus cinerius and Trichoderma reesei. However, there is no disclosure of lipases from these organisms. Garcia-Lepe et al., 1997 screened for lipase activity in autolysed cultures of fifty-one fungi from different genera and strains. Fungi from the genus Fusarium were found to be the best producers of lipase activity and they also showed a low activity on cutin and suberin. Aspergillus was also found to have some activity, whereas Penicillium species had very low activity. Other species and strains from genus Trichoderma, order Mucorales and class Basidiomy- cetes did not show lipase activity.

Cutinases are frequently produced by phytopathogenic fungi, be- cause they are involved in the disruption of structural cutin polymer of higher plants. Cutinases are secreted proteins, which allow pathogenic fungi to penetrate through the cuticular barrier into the host plant during the initial stage of fungal infection. However, phytopathogenic fungi are undesirable sources of industrial enzymes due to negative user perceptions. In fact, food grade poly- esterases and suberin-processing enzymes are currently not commercially

available. Thus there is still a need for novel and more efficient polyesterases. The present invention meets this need.

Summary of the Invention

One object of the present invention is a polyesterase protein com- prising an amino acid sequence having at least 50 % sequence identity to SEQ ID NO: 2, 6, 11 , or 13, or a variant or fragment thereof having polyesterase activity.

Another object of the invention is an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a nucleotide sequence of SEQ ID

NO: 1 , 3, 5, 10 or 12, or a nucleotide sequence encoding a protein of claim 1 , b) a complementary strand of a), and c) a sequence that is degenerate as a result of the genetic code to any one of a) or b). One further object of the invention is a vector comprising said polynucleotide, and a genetically modified microorganism, which has been transformed with this vector.

Stiil one object of the invention is a method for producing said polyesterase protein, which method comprises transforming a microorganism with a vector comprising said polynucleotide, culturing the transformed microorganism under conditions allowing the expression of said polynucleotide, and recovering the expressed protein.

The invention also encompasses an enzyme preparation comprising said polyesterase protein. Further the invention encompasses a method of hydrolysis of cutin, suberin, or other polyester, said method comprising treating a cutin, suberin, or other polyester containing material with said polyesterase protein under conditions allowing partial or total hydrolysis of said polyester.

Still further the invention encompasses the use of said polyesterase protein or enzyme preparation in food industry, pulp and paper industry, textile industry, or in laundry and dishwashing applications, or in chemical synthesis. Specific embodiments of the invention are set forth in the dependent claims. Other objects, details and advantages of the present invention will become apparent from the following drawings, detailed description and examples.

Brief Description of the Drawings

Figure 1 shows the amino acid sequences of the cutinase-like proteins of Coprinus cinereus.

Figure 2 shows extracellular production of Coprinus cinereus cuti- nase 09668 (CcCUT) in a 20 L bioreactor cultivation.

Figure 3 shows extracellular production of Trichoderma reesei cuti- nase (TrCUT) and suberinase (TrSUB) in a 20 L bioreactor cultivation.

Figure 4 shows the effects of fatty acid chain length on esterolytic activity of Coprinus cinereus cutinase 09668 (CcCUT) and Trichoderma reesei cutinase (TrCUT) measured at pH 7 and 40 ⁰ C.

Detailed Description of the Invention

The invention provides novel enzyme proteins, which are capable of hydrolysing ester bonds in natural and man-made polyesters. At least some of them have substantial activity also at acidic pH, which is an advantage in cer- tain applications. The proteins are "esterases", which encompass enzymes classified in (EC 3.1.1 ), also called carboxylic ester hydrolases. In particular the proteins of the present invention are "polyesterases", which means that they have significant activity on various polyesters, such as e.g. plant polyester waxes, i.e. cutin and suberin or man-made polyesters. According to a preferred embodiment of the invention the protein has cutinase activity. "Cutinase" is an enzyme classified in (EC 3.1.1.74). A cutinase is a serine esterase containing the classical Ser, His, Asp triad of serine hydrolases. According to another embodiment of the invention the protein has suberinase activity. "Suberinase" is an enzyme capable of degrading suberin. The proteins may have more than one of said enzyme activities as measured by using model substrates or isolated cutin or suberin as substrate. The polymerase activity may thus be at least cutinase activity, or suberinase activity, or both. In addition the proteins may have other enzyme activities such as e.g. lipase activity, which is also classified in EC 3.1.1. The polyesterases comprise an amino acid sequence that has at least 50%, or preferably at least 60%, 70%, 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 2, 6, 11 or 13, or a variant, or fragment thereof having polyesterase activity. According to a preferred embodiment, the polyesterase comprises an amino acid sequence having at least 50% sequence identity to SEQ ID NO:2, or a variant or fragment thereof having cutinase activity. Such

polyesters are e.g. those comprising the amino acid sequence of SEQ ID NO: 4, 7, 8 or 9, or an enzymatically active variant or fragment thereof. Such a protein may have at least 50, 60, 70, 80, 90, 95 or 98% sequence identity to SEQ ID NO: 4. The term "identity" means here the sequence identity between two amino acid sequences compared to each other. The identity of sequences is here determined using Clustal w multiple alignment programme found on the webpages of European Molecular Biology Laboratory - European Bioinformat- ics Institute (EMBL-EBI; http://www.ebi.ac.uk/clustalw/) using default settings and Blosum62 as substitution matrix (Thompson et al., 1994).

It is well known that deletion, addition or substitution of one or a few amino acids does not necessarily change the catalytic properties of an enzyme protein. Therefore the invention also encompasses variants and fragments of the given amino acid sequences having polyesterase activity. The term "vari- ant" as used herein refers to a sequence having minor changes in the amino acid sequence as compared to a given sequence. Such a variant may occur naturally e.g. as an allelic variant within the same strain, species or genus, or it may be generated by mutagenesis or other gene modification. It may comprise amino acid substitutions, deletions or insertions, but it still functions in substan- tially the same manner as the given enzymes, in particular it retains its catalytic function as a polyesterase.

A "fragment" of a given protein sequence means part of that sequence, i.e. a sequence that has been truncated at the N- and/or C-terminal end. It may for example be the mature part of a protein comprising a signal se- quence, or it may be only an enzymatically active fragment of the mature protein.

The invention is also directed to isolated polynucleotides, which encode the polyesterases disclosed, including complementary strands and degenerate strands. A polynucleotide that is "degenerate as a result of the ge- netic code" to a given sequence, means that it contains one or more different codons, but encodes for the same amino acids. A "polynucleotide" as used herein may be a single or double stranded polynucleic acid. The term encompasses genome DNA, cDNA and RNA.

Genes from different organisms encoding enzymes with the same catalytic activity often have sequence similarities. These similarities can be ex-

ploited in many ways to clone other genes from other organisms with the same or similar catalytic activity.

Polynucleotides encoding the novel esterases may be identified e.g. in silico by comparing nucleotide sequences. If such sequences are not avail- able one can identify a conserved region in the nucleotide or amino acid sequence and clone a gene fragment using PCR techniques. Cloning means transfer of a DNA fragment of interest from one organism to a self-replicating genetic element and furthermore, possibly to a foreign host cell. After sequencing the fragment the complete gene can be obtained e.g. by using a cDNA Ii- brary in a manner known per se. Another way to identify a polyesterase gene is by conventional nucleic acid hybridization.

Specific probes for cloning can be prepared for example from corresponding mRNA or the probe can be prepared if part of the amino acid sequence of the protein encoded by the gene is known. Once candidate DNA sequences have been determined, algorithmic methods can be utilized to efficiently search a target genome for matches. BLAST (Basic Local Alignment Search Tool) is a widely used system designed for this purpose.

The proteins or polynucleotides of the invention may be derived from any suitable organism including bacterial, fungal, yeast, plant or mammal- ian cells containing them. Preferably the enzyme is derived from a fungus, and in particular from a filamentous fungus e.g. from the genus Coprinυs or Tricho- derma, and especially from C. cinereus, or T. reesei {Hypocrea jecorina).

Proteins or polynucleotides "derived from" a particular organism encompass products isolated from said organism, as well as modifications thereof. A protein derived from a particular organism may be a recombinantly produced product, which is identical to, or a modification of the naturally occurring protein. The protein may also be modified e.g. by glycosylation, phosphorylation or other chemical modification. The modification may also include attachment of a suitable peptide or protein fusion partner to the protein of in- terest. The fusion partner may have a beneficial role, such as it may enhance the hydrolysis or processing efficiency of the protein of interest, or the fusion partner may aid in purification of the protein of interest. Examples of such fusion partners are e.g. fungal hydrophobins. Products derived from the particular organism also encompass mutants and natural variants of the products, where one or more nucleic acid and/or amino acid is deleted, inserted and/or substituted.

As set forth above, the protein may be isolated from the organism, where it occurs naturally, or it may be produced recombinantly in a host cell, or produced synthetically e.g. by peptide synthesis. Preferably the protein is a recombinant protein. It may be prepared by first isolating a fragment comprising the protein encoding polynucleotide by amplification in a PCR reaction (Coen, 2001) or other recombinant DNA methods (Sambrook et al., 1989). The isolated polynucleotide is then inserted into a vector e.g. a plasmid vector, especially an expression vector, which comprises the following operably linked elements: a transcriptional promotor, a segment encoding the polyesterase, and a transcriptional terminator. The promotor is preferably a strong promotor, which enables overexpression of the protein. One suitable promotor is the cellobio- hydrolase I (cbh1) promotor of T. reesei. The promoter is chosen to be capable of driving expression of the gene of interest in the selected production host. The vector can be one that is integrated into the chromosome or an autono- mously replicating one.

The vector is then transformed into a heterologous or homologous host cell to generate a "genetically modified microorganism", which is cultivated under conditions enabling the expression of the protein. Methods for protein production by recombinant technology in different host systems are well known in the art (Gellissen, 2005). Alternatively only the strong promotor is operably linked to the polyesterase gene on the host's chromosome, whereby the expression of said gene is overexpressed. The host cell may be any suitable eukaryotic or prokaryotic cell. Preferably it is a fungus e.g. a filamentous fungus or yeast, and most preferably it belongs to the genus Tricho- derma, especially it is 7. reesei. It may also be a Saccharomyces or a Pichia strain, such as S. cerevisiae and P. stipitis, respectively. Further it may be an Aspergillus strain, such as A. nidulans, A. niger or A. oryzae or even a bacterial host.

The polyesterase protein is preferably produced extracellularily, whereby the secreted protein may be obtained from the culture medium. Alternatively the cefls may be disrupted to release the enzyme, which then may be obtained from the supernatant after removal of the cell debris. The enzyme may be further purified using various protein purification methods, if desired. Such purification may include e.g. concentration, precipitation, chroma- tograhpy, immunopurification, phase separation etc. to remove other proteins and especially other enzymes.

An "enzyme preparation" in the present context may be any composition comprising at least one of the polyesterases of the invention. It may further comprise one or more other enzymes. It may be in crude form, e.g. in the form of a spent culture or cell supernatant, or it may contain the polyesterase in a purified or substantially purified form.

The polyesterases are useful for hydrolysis of cutin, suberiπ or other polyester containing material. The cutin and/or suberin containing material is usually of plant origin, whereas the other polyester containing material may be plant-derived or man-made. An amount of the enzyme efficient to catalyze the desired reaction is added to the material to be treated under conditions allowing hydrolysis. The polyesterases may e.g. be used to degrade or partially de- polymerise plant polyester waxes, i.e. cutin and suberin. The polyesterases may thus be used e.g. for treating agricultural or food raw materials or byproducts obtained from vegetables, fruits, berries, and cereals. They may also be applied in non-food processes e.g. in methods comprising treating of wood raw materials, pulp and paper products, or process wastes or waters or byproducts, or modifying synthetic or other man-made polyester fibres or textiles, or removing stickies or fat from laundry and dishes.

The polyesterases may under appropriate conditions also be used for catalyzing a reverse reaction i.e. esterification, that is formation of ester bonds e.g. between fatty acids and alcohols.

The invention is illustrated by the following non-limiting examples. It should be understood, however, that the embodiments given in the description above and in the examples are for illustrative purposes only, and that various changes and modifications are possible within the scope of the claims.

Example 1. Measurement of polyesterase activities

Methods to model suberin degradation

The degradation of the aliphatic layer of suberin was imitated by model substrates, i.e. naphthol derivatives differing both in the bulkiness of the chromophore (1-naphthyl, 2-naphthyl, Naphthol AS, Naphthol AS-D) and the length of the ester-bonded carbon chain. Substrate solutions of naphthol derivatives (0.5-1 mM) were prepared in 1% acetone and 1% Triton X-100 in 50 mM Na-citrate (pH 5) or 50 mM NaP (pH 8). The reaction mixture containing 170 μl substrate solution and 10 μl enzyme sample was incubated at 4O ⁰ C for 20 minutes. After incubation, 20 μl of 1% Fast Blue BB salt dye was added,

and absorbance (1 NA substrates - 450 nm, 2NA substrates - 510 nm, NAS substrates - 595 nm, NASD substrates - 595 nm) was measured after additional incubation of 10 min. The enzyme activities were determined by reference to a standard curve prepared from various amounts of 1 NA, 2NA, NAS or NASD (the coloured reaction products).

The degradation of the layers of suberin containing aromatics was monitored with a model substrate 4-methylumbelliferyl 4-methyl ferulic acid ester (MUFE) containing p-coumaric acid derivatives (observed in native suberin) esterified with a fluorescent molecule (4-methylumbelliferσne, 4MU). MUFE assay was performed by incubating 190 μl of 0.1 mM substrate solution with 10 μl of enzyme solution at 4O ⁰ C. Fluorescence was measured (A _6x = 355 nm; λ _em = 465 nm) after 20 min incubation using 4-methylumbelliferone (4MU) as standard.

Degradation of suberin was also measured using radioactively Ia- belled suberin as a substrate. Suberin isolated from birch outer bark was labelled with [ ³ H]NaBH ₄ . The reaction mixture contained 10 mg suberin (5x10 ⁵ - 10 ⁶ dpm/mg), 1.9 ml buffer (0.1 % Triton X-100 in 50 mM Na-citrate buffer, pH 5 or in 50 mM Na-phosphate buffer, pH 7) and 0.1 ml enzyme solution. The reaction mixture was incubated at 37°C, and reaction samples of 0.1 ml were taken during 48 h incubation. The hydrolysis products ( ³ H labelled monomers) released by the enzymatic action were extracted from reaction samples with ethyl acetate, and the resulting radioactivity was measured by liquid scintillation counter. The degree (%) of enzymatic degradation was quantified by measuring the radioactivity released after a total hydrolysis of suberin by alkali.

Methods to model cutin degradation

Esterase activity modelling the cutinase activity was measured by a spectrophotometric assay (slightly modified from Davies et al., 2000) with 2.1 mM p-nitrophenyl butyrate (p-NPB) as substrate. The reaction was carried out in 0.1 M sodium phosphate buffer (pH 7.0) at 4O ⁰ C for 10 minutes and the amount of released p-nitrophenol was measured at 340 nm, using commercial p-nitrophenol as standard. This method enabled a convenient and rapid assay for non-specific esterase activity.

Cutinase activity was also measured using ³ H labelled apple cutin as substrate by an adaptation of the methodology presented in Kδller et al. (1982) and Davies et al. (2000). The reaction mixture contained 8 mg cutin (5x10 ⁶ dpm/mg), 1.9 ml master mix (containing 0.025 % Triton X-100 in 50 mM

Na-phosphate buffer, pH 7.0) and 0.1 ml enzyme solution. The reaction mixture was incubated at 37°C, and the reaction was followed for 24 h. The hydrolysis products ( ³ H labelled monomers) released by the action of cutinase were extracted from the reaction sample of 0.1 ml with ethyl acetate, and the resulting radioactivity was measured by liquid scintillation counter. The degree (%) of enzymatic degradation can be quantified by measuring the radioactivity released after a total hydrolysis of cutin by alkali.

Example 2. Screening of polyesterolytic activities

Altogether 55 microorganisms, mostly filamentous fungi, were screened for their ability to produce suberin modifying enzymes in suberin- induced conditions. The screening was based on the enzymatic assays of culture supernatants (hydrolysis of naphtol substrates and a fluorescently labelled aromatic compound and radiolabeled suberin, as described in Example 1 ) and GC/MS analysis of separated solids, whereby increased amounts of long fatty acids, such as hydroxy fatty acids and diols, confirmed that a microorganism was able to degrade suberin during its growth. Coprinus cinereus and Tricho- derma reesei were found to be potential producers of cutin/suberin degrading enzymes.

Example 3. Genome analysis of Coprinus cinereus for polyesterase en- coding genes

Coprinus cinereus was found to be able to produce polyesterases having activity on natural polyesters such as cutin and suberin (Example 2). The published genome of Coprinus cinereus

(http://www.broad.mit.edu/annotation/genome/coprinus_cine reus/Home.html) was exploited for similarity searches based on known polyesterases (cutinases and suberinases), and six different cutinase-like genes were found. Protein similarities were analysed with Clustal w multiple alignment program. Five of the genes (CC1G_09668.1 , CC1G_03922.1, CC1G_11503.1 , CC1G_07482.1 , and CC1G_09365.1 ) showed high sequence homology to cutinases and one (CC1G_05430.1) shared higher homology with acetyl xylan esterases (AXE) having e.g. a sequence identity of 30% with Trichoderma reesei AXE1. The results are shown in Figure 1 , where the serine active site and the aspartate and histidine active sites of the cutinases are indicated. Said genes and corresponding enzymes are hereinafter also simply called 09668, 03922, 11503, 07482, 09365 and 05430, respectively.

The sequence identities between the Coprinus cinereus cutinases analysed by Clustal w multiple alignment programme are shown in Table 1. Genes 09668, 03922, and 11503 had 199 amino acids, 07482 had 200 amino acids, 09365 had 216, and 05430 had 229 amino acids.

Table 1. Sequence identity between the Coprinus cinereus cutinases

Example 4. Genome analysis of Trichoderma reesei for polyesterase encoding genes

Trichoderma reesei was found to have activity against cutin and suberin (Example 2). The published genome of 7 ^" . reesei (http://genome.jgi- psf.org/Trire2/Trire2.home.html) was exploited for similarity searches based on known cutinases, and one cutinase (like) gene (v1.2: tre17732, v2.0: tre60489, scaffold 7) was found.

A suberinase like gene (v1.2: tre40871 , v2.0: tre31227, scaffold 37) was found through extensive blasting. Protein sequence of Steptomyces scabies suberinase was first used for blasting with BLAST program (blastp) at National Center for Biotechnology Information, NCBI using default parameters (Matrix: Blosum62, gap costs: existence 11 , extension 1). Subsequently, Trichoderma reesei genome was blasted with fungal sequences having similarity with S. scabies suberinase (containing SEST-like domains) using default parameters.

Enzymes containing this SEST-domain act as esterases and lipases, but have little sequence homology to true lipases. The tertiary fold of these enzymes is substantially different from that of the alpha/beta hydrolase family and unique among all known hydrolases. Proteins containing this type of esterase domain have been found in a variety of hydrolases. Those with structural information include an esterase from Steptomyces scabies (SEST), a

causal agent of the potato scab disease, which hyclrolyzes a specific ester bond in suberin. Some hypothetical or putative proteins have also been found to have similarity with S. scabies esterase.

Example 5. Cloning of novel polyesterases from Coprinus cinereus Three different types of polyesterases from Example 3 {09668,

07482, 05430) sharing lowest homology among themselves were selected for over-expression in Trichoderma reesei. The selected cutinases had optimal codon usage and suitable native signal sequences for the expression host.

For isolation of chromosomal DNA the Coprinus cinereus strain VTT-D-041011 was grown as mycelium in liquid cultures started from spores. The spores were inoculated in 50 ml of YP medium and grown for 2 days at 24°C shaking. The mycelia were harvested by filtration and the genomic DNA was isolated by the method of Raeder and Broda, 1985. The genomic DNA was used as a template for PCR amplifications of the two cutinase genes (CC1G_09668.1 , CC1G_07482.1) and the AXE-like gene (CC1G_05430.1) with primers which were designed to create a C-terminal His ₆ -tag and bearing phage lambda-based site specific recombination sequences. The native signal sequences of the genes were used. The primers used were the following; CC1G_09668.1 forward: SEQ ID NO: 14, CC1G_09668.1 reverse: SEQ ID NO: 15, CC1G_07482.1 forward: SEQ ID NO: 16, CC1G_07482.1 reverse: SEQ ID NO: 17, CC1G_05430.1 forward: SEQ ID NO: 18, CC1G_05430.1 reverse: SEQ ID NO: 19. The PCR reactions were done with the Phusion thermostable polymerase (Finnzymes, Finland) in a reaction mixture recommended by the manufacturer. The PCR program had an initial denaturation step of 30 seconds at 98 ⁰ C, followed by 25 cycles of 10 seconds at 98 ⁰ C, 30 seconds at 64 ⁰ C and 30 seconds at 72 ⁰ C, where the annealing temperature was decreased with 1 ⁰ C per cycle until 50 ⁰ C was reached. This was followed by a final elongation step of 10 minutes at 72 ⁰ C. The amplified PCR products were recombined into the Gateway donor vector pDONR221 (Invitrogen) by Gateway Recombination kit (Invitrogen) and sequenced. The sequences were as shown in Table 2.

Table 2. Cloned Coprinus cinereus cutinase sequences

Two clones 3.1 and 3.5 of 09668 were sequenced. There were a few differences between the nucleotide sequences of clone 3.5 and the ge- nome sequence, but all three nucleotide sequences encode the same amino acid sequence (SEQ ID NO: 2). The published genome sequence is derived from a haploid genome and is based on automated genome annotation. Therefore, the sequences of the cloned genes may differ from the published genome sequences. Differences may also have been introduced during PCR. SEQ ID NO: 4 and SEQ ID NO: 6, respectively, differ by one amino acid from the amino acid sequence deduced from the genome. This difference is indicated in Figure 1 , where the two differing amino acids are shaded. The sequences of the other three cutinase-like proteins CC1G_03922, CC1G_11503, and CC1G_09365 are given in the Sequence Listing as SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

The genes were transferred by LR recombination reactions from the pDONR221 vector to the Trichoderma reesei expression vector pMS186, giving rise to the plasmids pAWP26 (CC1G_09668.1 ), pAWP27 (CC1G_07482) and pAWP28 (CC1G_05430.1). The pMS186 vector contains the Gateway reading frame cassette C (RfC) inserted between the cbh1 (cellobiohydrolase 1) promoter and terminator, and a hygromycin resistance cassette. The LR recombination reaction was done with the Gateway Recombination kit (Invitro- gen) as instructed by the manufacturer.

Example 6. Cloning of novel polyesterases from Trichoderma reesei The cutinase (v1.2: tre17732, v2.0: tre60489, scaffold 7) and suberi- nase (v1.2: tre40871 , v2.0: tre31227, scaffold 37) cDNA from Trichoderma reesei were isolated by RT-PCR from a cDNA expression library of Tricho-

derma reesei RutC-30 (Margolles-Ciark E., et al., 1996) with primers which were designed to create a C-terminal Hise-tag and bearing phage lambda- based site specific recombination sequences; cutinase forward: (SEQ ID NO:20), cutinase reverse: (SEQ ID NO:21), suberinase forward: (SEQ ID NO:22, suberinase reverse: (SEQ ID NO:23). The native signal sequence of the cutinase was used, whereas the signal sequence of cbhl was used for the suberinase construct. The PCR reactions were done with the Phusion thermostable polymerase (Finnzymes, Finland) in a reaction mixture recommended by the manufacturer. The PCR program had an initial denaturation step of 30 seconds at 98 ⁰ C, followed by 25 cycles of 10 seconds at 98 ⁰ C, 30 seconds at 64 ⁰ C and 30 seconds at 72 ⁰ C, where the annealing temperature was decreased with 1 ⁰ C per cycle until 50 ⁰ C was reached. This was followed by a final elongation step of 10 minutes at 72 ⁰ C. The amplified PCR products were recombined into the Gateway donor vector pDONR221 (Invitrogen) by Gate- way Recombination kit (Invitrogen) and sequenced. The sequences are shown in Table 3.

Table 3. Cloned Trichoderma reesei cutinase and suberinase sequences

The cloned nucleotide sequence and deduced amino acid sequence of cutinase was longer at both 5' and 3' ends than predicted by the computational annotation of T. reesei genome.

The cutinase and suberinase genes were transferred by LR recombination reactions from the pDONR221 vector to the Trichoderma reesei ex- pression vector pMS186, giving rise to the plasmids pAWP24 (cutinase) and pAWP25 (suberinase). The pMS186 vector contains the Gateway reading frame cassette C (RfC) inserted between the cbhl (cellobiohydrolase 1 ) promoter and terminator, and a hygromycin resistance cassette. The LR recombination reaction was done with the Gateway Recombination kit (Invitrogen) as instructed by the manufacturer.

Example 7. Expression of novel polyesterases in Trichoderma reesei

The polyesterase genes were expressed in T. reesei under the strongly inducible promoter of the major cellufase gene cbh1. Circular expression vectors (5 μg) were transformed into the T. reesei cbh1 negative strain VTT-D-04966 by PEG-mediated transformation, essentially as described by Penttila M., et al, 1987, and transformants were selected for hygromycin resistance on plates containing 125 μg/ml of hygromycin B. The transformants were streaked on the selective medium for two successive rounds and tested by PCR for integration into the genome. Positive transformants were purified by single-spore cultures and were tested for cutinase activity in liquid cultures using p-nitrophenylbutyrate (p-NPB) as a model substrate (Example 1 ). 50 ml of culture medium (TrMM + 4% lactose, 2% spent grain, 100 mM PIPPS, pH 5.5) was inoculated with 1 x 10 ⁷ spores and grown for a maximum of 10 days at 28 ⁰ C shaking at 250 rpm. All three Trichoderma constructs i.e. those trans- formed with Coprinus gene 09668, 07482, and 05430, respectively, showed p- NPB activity. The six transformants showing highest activities of each gene were re-cultivated for more thorough analysis. C. cinereus 09668 seemed to be most promising candidate and it was cultivated in a faboratory-scale fer- menter. The most potential transformants (on the basis of the activity assay with p-NPB) carrying T. reesei cutinase or suberinase gene were also selected for cultivation in fermenter.

Example 8. Production of novel polyesterases in laboratory-scale fermenter

The transformant of 09668 producing cutinase (CcCUT) was cuiti- vated in a Braun Biostat C fermenter (B. Braun Biotech, Germany), working volume 20 litres. The medium contained (in g I ^"1 ): lactose (60), (NH-OaSO ₄ (5) and KH ₂ PO ₄ (5). The liquid phase of the medium was an aqueous extract of distiller's spent grain prepared by heating 60 g I ^"1 spent grain at 115 ⁰ C for 20 minutes in an autoclave, cooling and centrifuging to remove the solid compo- nents. The centrifugation supernatant containing both nitrogen source and inducers was used in the medium as the only liquid. Cultivation temperature was 28 ⁰ C and pH was 5.0-5.5 (controlled by addition of ammonium hydroxide and phosphoric acid). Dissolved oxygen was maintained at >30% by agitation at 300...700 rpm, with a constant aeration of 8 I min ^"1 . Foaming was controlled by automatic addition of Struktol J633 polyoieate antifoam agent (Schill & Seila-

cher, Germany). After the cultivation, cells were removed by centrifugation and the culture supernatant was concentrated by ultrafiltration using Millipore (France) BioMax 10 membranes, nominal cut-off 10 kDa.

The C. cinereus cutinase (CcCUT) was successfully produced in the fermenter. Cutinase production increased to a maximum of over 8000 nkat ml ^"1 after 96 h (Figure 2). A ten-fold culture filtrate had an esterase activity (with p- NPB) of 70 000 nkat ml ^"1 , a total protein content of 104 mg ml ^"1 , and an amount of cutinase of approx. 23 mg ml ^"1 . The presence of cutinolytic activity in the culture supernatant was also verified on isolated apple cutin before further studies (Table 4). Cutin was treated in the presence of 0.1% Triton X-100 with culture supernatant (45h sample, p-NPB -activity of 1780 nkat ml ^"1 ) using enzyme dosages of 1000, 5000 and 20000 nkat g ^"1 substrate (pH 7, 40 ⁰ C).

Table 4. Cutinolytic activity of CcCUT

* total amount, containing mono- and oligomers

The transformants producing Trichoderma reesei cutinase (TrCUT) and suberinase (TrSUB) were cultivated in a laboratory fermenter similarly as described earlier for CcCUT. Enzyme activities are shown as a function of time in Figure 3.

Example 9. Purification of recombinant enzymes

The presence of C-terminal His(6)-tag enabled a one-step purification of CcCUT and TrCUT using immobilized metal affinity chromatography (IMAC). The concentrated culture supernatant was applied to a chelating Sepharose FF column (Amersham Biosciences, Uppsala, Sweden) preloaded with Ni ²⁺ and equilibrated with 50 mM sodium phosphate containing 500 mM NaCI and 5 mM imidazole, pH 7.2. The column was washed with equilibrating buffer supplemented with 50 mM (for CcCUT) or 20 mM imidazole (for TrCUT) in order to remove the unbound material. The recombinant protein was eluted

with equilibrating buffer supplemented with 200 mM imidazole and fractions were collected and screened for the activity on p-NPB and the presence of the protein by SDS-PAGE. SDS-PAGE (12% Tris-HCI Ready Gel, Bio-Rad) was performed according to Laemmli (1970), using Pre-stained SDS-PAGE Stan- dards (Broad Range Cat. no. 161-0318, Bio-Rad or LMW, Cat. No 17-0446-01, GE Healthcare) and Coomassie Brilliant Blue (R350; Pharmacia) for staining the proteins.

The purified CcCUT showed homogeneity on SDS-PAGE and approx. 10 grams of purified enzyme was prepared for further characterization and hydrolysis studies. 3 grams of TrCUT was purified, the preparation having a purity around 95% (based on SDS-PAGE analysis). TrSUB is purified similar to CcCUT and TrCUT for characterization.

Example 10. Characterization of the novel polyesterases

The purified Coprinus cinereus (CcCUT) and Trichoderma reesei (TrCUT) cutinases were biochemically characterized with respect to size, activity, substrate specificity, pH and temperature characteristics.

Substrate specificity

The substrate specificity was determined using p-nitrophenols es- terified with acetate (C2), propionate (C3), butyrate (C4), valerate (C5), caproate (C6), caprate (C10), laurate (C12), myristate (C14), palmitate (C16) and stearate (C18). The concentrations of substrate dispersions were 5 mM. A lower concentration of p-nitrophenyl stearate (2.5 mM) was used due to its lower solubility. Activity assays were performed as described for p-nitrophenyl butyrate (p-NPB) at pH 7, 4O ⁰ C (Example 1). The specific activities obtained are shown in Figure 4. CcCUT and TrCUT had higher activity on shorter (C2 - C10) than on longer (C16 and C18) fatty acids. Surprisingly, the activities on p- NP acetate (C2) and propionate (C3) were observed to be clearly higher than on p-NPB (C4). The C4/C16 ratio of CcCUT and TrCUT was 1.8 and 3.1 , respectively. Typically, cutinases have high activity on C2 - C8 fatty acids and the ratio C4/C16 ratio is between 1-4. A C4/C16 ratio of about 1 , or <1 indicates no cutinolytic activity (Kolattukudy, 1984).

Lipase and cholesteryl esterase activity

Lipase activity was assayed using olive oil emulsion as substrate according to Kontkanen et al. (2004). The lipase activity of CcCUT and TrCUT is shown in Table 5 . The assay used for determination of cholesteryl esterase (CE) activity was based on the spectrophotometric determination of liberated cholesterol after hydrolysis of 4.3 mM cholesteryl oleate according to Tenkanen et al. (2002). CcCUT preparation showed no cholesteryl esterase activity. Activity in TrCUT preparation was not determined. Protein assay

Protein concentration was determined by the Bio-Rad DC protein assay kit (Bio-Rad, Richmond, Calif.) with bovine serum albumin as standard.

Temperature stability

Temperature stability of CcCUT and TrCUT were investigated by in- cubating the enzymes at 30-80 ⁰ C for 1 , 3 and 20 h at protein concentration of 5 mg/ml and pH 5 (20 mM sodium acetate buffer). After the incubations, the residual activity was measured using p-NPB as substrate (at pH 7 and 40 ⁰ C). CcCUT was rather stable at temperatures up to 50 ⁰ C but residual activity decreased sharply at 60 ⁰ C. TrCUT was somewhat stable retaining 80% of its ac- tivity when incubated at 50 ⁰ C for 20 h or at 60 ⁰ C for 1 h (Table 5). pH stability

The pH stability of CcCUT and TrCUT was determined by incubating the purified enzyme solutions at different pH values at room temperature and at 50 ⁰ C for 20 h. The pH of the solution was adjusted with Mclivaine buffer (0.2 M Na ₂ HPO ₄ and 0.1 M citric acid) at pH 2.2-8.0, 0.2 M Tris-HCI buffer at pH 7.2-9.1 or 0.2 M glycine-NaOH buffer at pH 8.6-10.6 to obtain protein concentration of 5 mg ml ^"1 . The residual activity was measured with p-NPB at pH 7 and 40 ⁰ C. The results are shown in Table 5. It can be seen that both enzymes were active over a wide range of pH including the acidic range. The residual activity of CcCUT was about 80% at a pH of 3 at room temperature, whereas the residual activity at 50°C was about 40% at pH 5, and about 100% at pH 6. TrCUT was shown to retain over 90% of it activity within the pH range of 4-7.

pH optimum

Esterase activities of purified cυtinase preparations were measured at different pH values using Mcllvaine buffer (0.2 M Na2HPC> ₄ and 0.1 M citric acid) at pH 2.3-8, 0.2 M Tris-HCI buffer at pH 7.2-9.1 and 0.2 M glycine-NaOH buffer at pH 8.6-10.6 using p-NPB as substrate. The reaction time was 10 minutes at 40 ⁰ C. The results are shown in Table 5. The pH optimum of CcCUT was around 7-8, whereas TrCLJT was shown to have two clearly different pH optima (around 4 and 8). Thus TrCUT is suitable for treatments in a more acidic range.

Table 5. Biochemical properties of Coprinus cinereus cutiπase 09668 (CcCUT) and Trichoderma reesei cutinase (TrCUT).

a) theoretical Mw n.d. not determined

Example 11, Hydrolysis of isolated apple cutin

Isolated apple cutin was treated with CcCUT and TrCUT. The substrate was treated enzymatically and chemically in order to remove of carbohydrates and pectin as well as noncovalent lipids, respectively. Cutin was suspended in 0.2 M sodium-phosphate buffer, pH 8 in a concentration of 20 mg ml ^"1 and treated with CcCUT and TrCUT at 45 ⁰ C for 20 h. The enzyme dosages were 1000 and 10 000 nkat g ^"1 substrate (p-NPB -activity) and the treatments were performed with and without Triton X-100 addition. The hydrolys- ates were extracted twice with 2 volumes of MTBE in order to recover all fatty acids, both mono- and oligomers, from the solid matrix. Free fatty acids in the

MTBE extract were analysed directly and after alkali hydrolysis of released oligomers using the enzymatic colorimetric method (Free fatty acids, Roche Diagnostics Ltd). Amount of released fatty acids are shown in Table 6. Both cuti- nases were able to hydrolyse apple cutin.

Table 6. Treatment of apple cutin with CcCUT and TrCUT.

* % of substrate, calculated as stearic acid (284.5 g/mol)

Example 12. Hydrolysis of birch bark suberin

Steam exploded birch outer bark suberin was treated with CcCUT and TrCUT similarly as cutin treatments described above. The results are shown in Table 7.

Table 7. Treatment of birch bark suberin with CcCUT and TrCUT.

* o %> of substrate, calculated as stearic acid (284.5 g/mol)

Example 13. Treatment of peeled wheat grain Peeled wheat grains were treated with cutinase (CcCUT) in order to enhance removal of testa which is mainiy composed of unsubstituted linear xy- lan and cutin layers. Grains (2g) were treated in water suspensions with a dry matter content of 20% at 30 ⁰ C for 2 h shaking (100 rpm). Enzyme dosages of

500 and 5000 nkat g ^"1 substrate (as p-NPB -activity) were tested for CcCUT. The effect of two different xylanases and a lipase were also studied. After enzyme treatments, centrifugation (9700g/10 min) was applied to separate the liquid and solid phases. The grains were washed with water (10 ml), and centrifugation was repeated. The grains were freeze-dried and weighed in order to analyze weight loss. The reference treatments were performed under identical conditions but without enzyme additions. The amount of released fatty acids was analysed after MTBE extraction followed by dissolution of fatty acids into EtOH/Triton/water solution. Reducing sugars were analyzed from liquid samples using DNS method (Bernfield, 1955).

The amounts of released fatty acids and solubilised carbohydrates after enzyme treatments are shown in Table 8. It can be seen that CcCUT increased clearly the amount of released fatty acids in used conditions. The treatments performed had no effect on the amount of carbohydrates. No changes on the visual appearance of the grains could be observed after the treatments indicating selective action on cutin.

Table 8. Enzymatic treatment of peeled wheat grains.

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