FIELD

The present invention relates to enzymes. In particular, the present invention relates to enzymes produced by viruses.

BACKGROUND

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Enzymes are large, complex molecules and are currently produced in bioreactors using either the original host organism or appropriate expression vectors for the nucleic acid molecule encoding the enzyme. For example, alpha-amylase is produced by batch fermentation, of Bacillus sp., where the production of the enzyme starts after 10 to 20 hours growth of the bacterial cells and continues for a further 100 to 150 hours. The enzyme is then removed from the cell culture by filtration and concentration using either ultra-filtration or acetone precipitation. The batch nature of the fermentation and the lengthy production and purification times greatly increase the cost of the final enzyme product. In particular a cell lysis step is most often required to isolate an enzyme from within the cell . This is of particular concern in industrial processes, where large amounts of enzyme are required. Once isolated, the enzymes need to be added to a reaction in order for that reaction to

occur.

By way of a commercial example, alpha-amylase from B. licheniformis is used in high temperature processes such as the liquefaction of starch in the initial stages of ethanol, maltose and glucose syrup production, as well as in the paper and textile industries. There are three main steps in the processing of starch to glucose or maltose syrups; gelatinisation, liquefaction, and saccharification. During gelatinisation, the starch slurry and thermostable alpha- amylase are steam injected through pipes at a temperature of 105°C for 5 min. During liquefaction, the starch-amylase mixture is held at 95°C for around two hours in order to reduce the viscosity of the starch. During saccharification, glucose or maltose units are produced by the addition of enzymes such as glucoamylase, pullulanase or fungal alpha- amylase. Accordingly, several enzymes can be required in an enzymatic reaction. Each additional enzyme used increases the cost of the process .

Phage display enables the expression of proteins and peptides on the surface of phage particles, with a direct link between the genotype and the phenotype of the peptide or protein of interest. This method enables vast libraries of peptides or proteins to be screened simultaneously for their ability to interact with other molecules, such as ligands, enzyme substrates and the like. Phage display has been used to screen a phage library for enzymes having a particular activity. In this scenario, phage producing an enzyme may be used in an analytical screening assay to test for activity of the enzyme. However, when a phage expressing a suitable enzyme is identified, the nucleic acids encoding the enzyme of interest are amplified and cloned into an expression system, such as an expression vector in E.coll. In such an expression vector, if the protein is not exported, a cell lysis step is required for isolation of the protein, which

may include the addition of other enzymes for processing; solubilisation may be required if inclusion bodies are formed. This is a time-consuming procedure involving expensive downstream processing technology which contributes to the high cost of the enzyme. Moreover, each phage in a phage library contains nucleic acid encoding only one enzyme. Therefore this procedure must be repeated for each suitable enzyme identified.

Thus there is a need in the art for more cost effective and less time-consuming methods for producing a desired product from an enzymatic reaction.

SUMMARY

In a first aspect, the present invention provides a method of producing a product of an enzymatic reaction with a reactant, the method comprising the steps of: a) providing a recombinant virus or fragment thereof comprising a non-native enzyme or functional fragment, variant, or derivative thereof; and b) contacting the recombinant virus or fragment thereof and the reactant under conditions and for a time suitable to enable the enzyme to catalyze the enzymatic reaction to produce the product.

The first aspect of the invention may optionally include the additional step of recovering the product of the enzymatic reaction, although this will not be necessary in all circumstances, for example if the enzyme is used for bioremediation or if a plurality of enzymes are used to catalyse a pathway and the product is an intermediate in the pathway.

The method according to the first aspect of the invention is useful for carrying out enzymatic reactions on a commercial scale. The use of a recombinant virus comprising a non-native enzyme or functional fragment, variant, or

derivative thereof in a reaction provides an alternative to the use of a purified enzyme, and may reduce the cost and time involved in performing the enzymatic reaction as purified enzymes are not required. The use of a recombinant virus comprising a non- native enzyme or functional fragment, variant, or derivative thereof instead of a purified enzyme allows the non-native enzyme to be produced and the enzymatic reaction to occur in a single reaction vessel. This lends itself to large scale reaction vessels, in excess of 1 litre. It is envisaged that reaction scales of 1-5 or more litres will be achievable using the method of the first aspect of the invention.

It is recognised that either the intact enzyme or a functional fragment, variant, or derivative may be used, as any of these would enable the enzymatic reaction to occur.

It is also recognised that a fragment of the recombinant virus which comprises the non-native enzyme, or functional fragment, variant, or derivative thereof, can be used in place of the virus itself . This virus fragment may be a portion of the viral coat which comprises the recombinant enzyme or functional fragment, variant or derivative thereof.

The non-native enzyme may be encoded by a nucleic acid molecule which has been inserted into the virus, for example into the viral genome, or may be incorporated into the viral particle as a protein.

The virus may comprise the non-native enzyme in the same translation product as a viral protein, for example a coat protein.

In one embodiment, the recombinant virus or fragment thereof comprises a plurality of non-native enzymes, or functional fragments, variants or derivatives of such enzymes. . The non-native enzymes may be incorporated into the viral particle as separate proteins or as a fusion protein. Alternatively, the non-native enzymes may be expressed by

nucleic acid molecules that have been inserted into the virus. The insertion of the nucleic acid molecules into the virus may be such that the enzymes are expressed as the same or separate translation products. The nucleic acid molecules may be inserted into the virus in a cassette incorporating each of the plurality of enzymes, as separate nucleic acid molecules, or a combination of separate nucleic acid molecules and cassettes comprising more than one nucleic acid molecule. Preferably the nucleic acid molecules encoding each enzyme are recombinantly inserted into the viral genome.

The non-native enzyme may be alpha-amylase or xylanase and may produce liquefied starch. Preferably the enzymatic reaction is part of a metabolic pathway which produces maltose or glucose. The plurality of enzymes may act to de-inked mixed office waste.

Due to the large size and complexity of enzymes, and therefore the potential problem of steric hinderance which can effect the activity of an enzyme, and the high specificity which must be retained in a functional enzyme it was unexpected that a plurality of non-native functional enzymes could be provided on a single virus. Furthermore, in traditional phage display, there has not been a need for such plurality to be evaluated.

Moreover, where the enzymatic reaction is one in a series of reactions, each recombinant virus or fragment thereof may express a plurality of non-native enzymes suitable for use in the series of reactions. Thus a series of reactions can be completed with fewer physical operations.

The non-native enzymes or functional fragments, variants or derivatives thereof may be the same or preferably different, and may be encoded by nucleic acid molecules which are the same or preferably different. The enzymes may be located at different locations on or in the virus, and are preferably located on the surface of the virus.

In a second aspect, the invention provides a method of producing a recombinant virus which comprises a plurality of non-native enzymes or functional fragments, variants or derivatives thereof, the method comprising the step of manipulating the virus to comprise a non-native enzyme.

The virus may be manipulated to comprise the non- native enzymes by incorporating the non-native enzymes into the viral particle each as separate proteins or as a fusion protein. Alternatively, the non-native enzymes may be expressed by nucleic acid molecules that have been inserted into the virus . The insertion of the nucleic acid molecules into the virus may be such that each of the enzymes are expressed as the same or separate translation products. The nucleic acid molecules may be inserted into the virus in a cassette incorporating each of the plurality of enzymes, as separate nucleic acid molecules, or a combination of separate nucleic acid molecules and cassettes comprising more than one nucleic acid molecule. Preferably the nucleic acid molecules encoding each enzyme are recombinantIy inserted into the viral genome.

Preferably the plurality of non-native enzymes or functional fragments, variants, or derivatives thereof are different. One or each non-native enzyme, or functional fragment, variant or derivative thereof may act in a metabolic pathway. Regardless of whether the plurality of enzymes are provided as the same or separate translation products, one or each non-native enzyme may comprise part of the same translation product as a coat protein of the virus.

In a third aspect, the invention provides a recombinant virus or fragment thereof comprising a plurality of non-native enzymes or functional fragments, variants or derivatives thereof which enzymes are produced in the same translation product.

In a fourth aspect, the invention provides a

recombinant virus which comprises a plurality of non-native enzymes or functional fragments, variants or derivatives thereof which enzymes are produced as separate translation products . Preferably the plurality of non-native enzymes or functional fragments, variants, or derivatives thereof referred to in the third and fourth aspects of the invention are different.

One or each of the plurality of non-native enzymes, or functional fragment, variant, or derivatives thereof referred to in the third or fourth aspects of the invention may act in a metabolic pathway. Regardless of whether the plurality of enzymes are provided as the same or separate translation products, one or each non-native enzyme may comprise part of the same translation product as a viral protein, particularly a viral coat protein.

According to a preferred embodiment a recombinant virus according to the third or fourth aspects of the invention is produced according to the method of the second aspect of the invention.

The invention further provides a host cell comprising a virus according to the third or fourth aspects of the invention. Preferably the host cell is a bacterial cell, such as Escherichia coli. In a fifth aspect, the present invention provides a method of producing a plurality of enzymes or functional fragments, variants or derivatives thereof, the method comprising the step of producing a recombinant virus of the third or fourth aspect of the invention. This method may allow large scale production of a plurality of enzymes in a single reaction vessel. It is envisaged that scales of reaction in excess of 1 litre, and probably around 5 or more litres would be achievable using this method.

This method may also allow for high concentrations of enzymes to be produced. It is anticipated that concentrations in excess of 10 ¹¹ molecules of enzyme per litre, and probably 10 ¹⁴ molecules of enzyme per litre could be produced in accordance with this method.

In a sixth aspect, the present invention provides enzymes or functional fragments, variants or derivatives thereof, when produced by a recombinant virus of the third or fourth aspect of the invention. In any one of the aspects of the invention preferably the virus is a phage.

A preferred non-native enzyme in any one of the aspects of the invention is alpha-amylase or xylanase. The non-native enzyme may catalyse an enzymatic reaction which produces liquefied starch. The non-native enzyme may catalyse an enzymatic reaction which is part of a metabolic pathway, for example which produces maltose, or glucose.

Particularly the plurality of enzymes referred to in the second to fourth aspects of the invention may act to de-ink mixed office waste.

The invention also encompasses products of the enzymatic reaction produced by a method of the first aspect and a recombinant virus when prepared by a method of the second aspect of the invention. It will be appreciated that a wide range of such products may be produced, and the nature and molecular size of the product will depend inter alia on the enzymes and starting materials used, and the duration of the enzymatic treatment .

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows different ways to produce the recombinant virus. v viral genomic DNA/RNA (eg T7) ; m transcript (mRNA) ; p plasmid DNA; g bacterial genomic DNA (eg E.coli); Φ phage particle with recombinant enzymes.

ι→ transcriptional promoter region; ■ ribosome binding site(rbs); open arrow/box viral protein gene/protein (eg coat-protein 10) ; striped arrow/box enzyme 1 gene/protein (eg alpha-amylase) ; dotted arrow/box enzyme 2 gene/protein (eg xylanase) ; Hashed arrow/box alternate coat-protein gene/protein.

In Figure 1, Ai and Aii show tandem production of enzyme 1 (alpha-amylase) and enzyme 2 (xylanase) in either orientation. B to I show possible ways of producing recombinant virus with enzymes as separate translational products. I shows non-covalent association of enzymes.

Figure 2 shows the expression of alpha-amylase expressed on T7 phage plaques grown in top agarose containing Red Starch (Megazyme) . White zones surrounding plaques (white arrows) indicate hydrolysis of the Red Starch due to the activity of alpha-amylase, and the absence of a clearing zone indicates the absence of alpha-amylase activity (black arrow) .

Figure 3 shows a graph comparing reducing sugar production by phage-displayed enzyme in crude lysate (A) , alpha-amylase (Sigma, A3404) in TB (B) , phage-displayed alpha-amylase in 0.05M glycine-NaOH, pH9.0 (C), and alpha- amylase (Sigma, A3403) in 0.05M glycine-NaOH, pH9.0 (D) at different temperatures using 1% starch (potato, hydrolysed for electrophoresis, Aldrich)

Figure 4 shows a graph of dextrose equivalents obtained using crude alpha-amylase-displaying phage lysate and 15% wheat starch (Sigma), incubated at 93°C.

Figure 5 shows plaque production in 1% Red Starch (Megazyme) (A,C,E and G) and 1% Azo-xylan (Megazyme) (B, D and F) . A: T7-xylanase, no clearing zones. B: T7-xylanase, clearing zones. C: T7-alpha-amylase, clearing zones. D: T7- alpha-amylase, no clearing zones. E: T7-xylanase-α-amylase, clearing zones. F T7-xylanase- α-amylase, clearing zones

present surrounding very small plaques (arrow) . G: T7- ce- amylase-xylanase, clearing zones.

Figure 6 shows the relative % brightness of handsheets made from pulped MOW after treatment with the phage constructs - T7NE T7 with no enzyme (100%) ; T7Amy T7 with amylase; T7Xyn T7 with xylanase; T7AX T7 with both amylase and xylanase; T7NE+com Amy commercially-supplied amylase mixed with T7-NE lysate.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Abbreviations used herein to describe preferred embodiments of the invention are as follows: DE = dextrose equivalent DNS = dinitrosalicylic acid IPTG = isopropl-beta-D-thiogalactopyranoside MOI = multiplicity of infection PCR = polymerase chain reaction PEG = polyethylene glycol PPM = parts per million SEC = size exclusion chromatography TA = Terrific agar TB = Terrific broth

Unless otherwise indicated, the present invention employs conventional chemistry, protein chemistry, molecular biological and enzymological techniques within the capacity of those skilled in the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. It is to be clearly understood that this invention is not limited to the particular materials and methods described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

In the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

As used herein, the singular forms "a" , "an" , and "the" include the corresponding plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an enzyme" includes a plurality of such enzymes, and a reference to "an amino acid" is a reference to one or more amino acids .

As used herein, "plurality" means "plural" or "more than one" .

Where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all values in between these limits . The present invention provides in a first aspect a method of producing a product of an enzymatic reaction. The method intended for commercial scale use of enzyme (s) .

As used herein, "enzymatic reaction" means a reaction catalyzed by an enzyme in which the reactant per se is expended in the reaction to form the product.

This is different to a transcriptional reaction, which is a template dependent process. For example, a polymerase enzyme synthesizes RNA or DNA which is complementary to the template. To read the template, the polymerase must form a complex with the template before it can catalyse the DNA or RNA synthesis. This template is not a reactant of the reaction, even though it is present throughout the reaction, because it is not broken down or used up in the production of one or more products.

As used herein, the term "catalyze" means to accelerate the rate of an enzymatic reaction by a substance which remains chemically unchanged by the reaction.

The enzymatic reaction may be one of a series of enzymatic reactions, such as a metabolic pathway. As used herein a "metabolic pathway" is a series of enzymatic reactions and includes synthesis (anabolism) of a product from a reactant and breakdown (catabolism) of a reactant to produce one or more products . Therefore the enzymatic reactions may build large macromolecules from smaller molecules, or breakdown larger molecules to produce smaller molecules .

An "enzyme" is a molecule or collection of molecules which catalyze an enzymatic reaction. Enzymes are usually specific for the reactions they catalyze and for the reactants that are involved in these reactions. They are generally large complex molecules which contain an active site and a binding site. The active site of an enzyme is the binding site where catalysis occurs. The structure and chemical properties of the active site allow the recognition and binding of the reactant. A binding site is a region on a protein to which specific ligands bind.

This invention suitably relates to protein enzymes and to functional fragments, variants and derivatives thereof. A functional fragment of an enzyme is a portion of the enzyme which retains the desired catalytic activity of the enzyme. For example, where the enzyme is alpha-amylase or xylanase, a functional fragment is any fragment which respectively retains amylase or xylanase activity. A functional variant of an enzyme has one or more substitutions such that the secondary conformation thereof remains unchanged but an activity of the enzyme is retained. Examples of such conservative substitutions include amino acids having substantially the same hydrophobicity, size and

charge as the original amino acid residue. Such substitutions are generally well known to those skilled in the art of protein or peptide chemistry. For example, conservative substitutions include proline for glycine and vice versa; alanine or valine for glycine and vice versa; isoleucine for leucine and vice versa; histidine for lysine and vice versa; threonine for cysteine and vice versa; glutamine for asparagine and vice versa; and arginine for glutamate and vice versa. A functional derivative includes an enzyme with one or several amino acid residues substituted by naturally occurring or synthetic amino acid homologues of the 20 standard amino acids but which retains an activity of the enzyme. Examples of such homologues are 4-hydroxyproline, 5- hydroxyIysine, 3-methylhistidine, homoserine, ornithine, [beta] -alanine and 4-aminobutanoic acid, beta-alanine, norleucine, norvaline, hydroxyproline, thyroxine, gamma-amino butyric acid, homoserine, citrulline, and the like.

The term "enzyme" extends to a homologue of the enzyme. A homologue of an enzyme is a protein sequence that shares a significant degree of sequence similarity to the enzyme. Homologues with greater than 80% sequence similarity are within the scope of the term "enzyme", provided that the homologue retains the catalytic function of the enzyme. Enzymes suitable for use in the invention include lipase, phytase, amylase, xylanase, cellulase, dehalogenase, lactinase, pectinase, formate dehydrogenase, aspartate transaminase, transketolase, lactase, alpha-galactosidase, alkaline phosphatase, pullulanase, isoamylase, alpha-1,6- glucosidase, beta-glucanase, glucoamylase, hydrolytic dehydrogenase, subtilinin, fructose-bisphosphate aldolase and glucose isomerase. Table 1 provides examples of the reactions catalysed by these enzymes and the processes in which they are useful .

TABLE 1 Enzymes and examples of the reactions they catalyse

Examples of specific enzymes suitable for use in this invention are alpha-amylase and xylanase. "Alpha- amylase" is an endohydrolase that cleaves α-1,4- oligosaccharide links to produce α-dextrins, maltose, G ₃ , G ₄ and G ₅ oligosaccharides. A preferred alpha-amylase of the invention is 1,4-α-glucan glucanohydrolase, (EC 3.2.1.1) from Bacillus licheniformis. Commercially, alpha-amylase from B. licheniformis is widely used in high temperature processes such as the liquefaction of starch in the initial stages of ethanol, maltose and glucose syrup production, as well as in the paper and textile industries.

"Xylanase" is an enzyme which catalyses the endohydrolysis of the main chain of xylan, which is a major component of hemi-cellulose. A preferred xylanase of the invention is xylanase A (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) from Bacillus halodurans C125. Xylanase A belongs to Family 10 of the xylanases, and is commercially desirable because of its ability to catalyse reactions at pHs ranging from 6-10. Xylanases are important in the paper and pulp industry for paper bleaching and pulping, in animal feed production and for flour processing in the baking industry.

An enzymatic reaction produces a product from a reactant. A "reactant" is a starting material for an enzymatic reaction and is expended in the enzymatic reaction. A "product" of an enzymatic reaction may be an end product or an intermediate product. As noted above, an enzymatic reaction may produce more than one product. One, two, several or all products produced may be recovered in the recovery step. Preferably, the product is recovered. A "product" as referred to herein to is generally a commercially desirable product. The intention of the first aspect of the invention is to accumulate product, either directly or through the production of intermediates . Accordingly, it will be understood that the method of the

first aspect of the invention is carried out in a non- analytical manner and is entirely different from screening for enzyme activity using phage display.

In addition to requiring an enzyme and one or more reactants in order to produce a product, an enzymatic reaction may require suitable conditions and time to enable the enzyme to catalyse the reaction. Suitable conditions may include specific temperature, pH, salt concentration, or the like, and the requirement for one or more co-factors. The conditions for each reaction must be satisfied, and at least the enzyme and the reactant must be in contact, in order for the enzymatic reaction to occur.

An enzyme of the invention is produced by a recombinant virus. As used herein, a "virus" is an infectious agent that replicates itself only within cells of living hosts, and consists of nucleic acid coated in a thin coat of protein. Examples of viruses suitable for use in this invention are disclosed in Table 2.

The term "recombinant" refers to a particle molecule which is made by the artificial combination of two otherwise separated segments of sequence, i.e. is made by chemical synthesis, genetic engineering, and the like.

Therefore a "recombinant virus" is a virus in which one or more protein(s) not native to the virus is/are present.

Methods for producing a recombinant virus which produces a single enzyme are known in the art. For example, the appropriate nucleic acid molecule may be inserted into the virus by a variety of procedures known in the art, such as recombinantIy inserting the nucleic acid molecule which encodes the enzyme into an appropriate restriction endonuclease site(s) adjacent to a viral gene for the production of a coat protein.

A "non-native enzyme" is an enzyme that is not

expressed by the wild type, naturally occurring virus. For example it may be expressed by a nucleic acid molecule which has been made by the artificial combination of two or more nucleic acid molecules which are not found in combination in nature.

As used herein, "contact" or "contacting" means to bring the reactant and recombinant virus producing an enzyme into contact with each other. The reactant and recombinant virus may be combined in any order. Thus the recombinant virus producing the enzyme may be added to the reactant, or vice versa.

As used herein, "recovered" means to isolate or purify a product from, for example, the enzyme. The product of the enzymatic reaction may be recovered by any known means and at any purity suitable for the intended use of the product. Methods of recovering a product of an enzymatic reaction are known in the art and will depend upon the product being recovered. Suitable methods include affinity chromatography, using one or more purification tags, extraction, precipitation and/or filtration. The recovered product may be subjected to other reactions. The recovered product, optionally following another treatment or reaction step, is suitably ready for its intended use.

The terms "encoded" or "encodes" refer generally to the nucleic acid sequence information being present in a translatable form. An antisense strand is also considered to encode the sequence, since the same informational content is present in a readily accessible form, especially when linked to a sequence which promotes expression of the sense strand. As used herein, a "nucleic acid molecule" may be

DNA or RNA, or an antisense nucleic acid molecule, and includes functional fragments of the nucleic acid molecule. A functional fragment of a nucleic acid molecule is one where, when expressed as a protein, the protein thus

expressed retains an activity of the intact protein.

The nucleic acid molecule may be recombinantly inserted into the genome of the virus. As used herein, "recombinantly inserted" means to artificially insert or add the nucleic acid molecule into another nucleic acid molecule, such as a viral genome.

As used herein, "genome" means the entire genetic complement of an organism, including all of the genes of the organism. Therefore a viral genome includes all of the genes of the virus.

A "gene" is a nucleic acid molecule that contains the information for a specific function. Therefore, viral genes are nucleic acid molecules that encode a particular viral protein, such as a coat protein. Viral genes may also comprise one or more of a signal sequence, an origin of replication, an enhancer element, a promoter, and/or a transcription termination sequence.

The signal sequence may be a component of the viral gene, or it may be a part of the enzyme-encoding nucleic acid that is inserted into the virus. The signal sequence may be a prokaryotic signal sequence, mammalian signal sequence, or insect signal sequence. For example, the signal sequence may be alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leader sequences. A recombinant virus may further comprise a promoter operably linked to an enzyme-encoding nucleic acid sequence. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with bacterial hosts ^' include the p-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter, all of which will be known to the person skilled in the art

A sequence is "operably linked" to a promoter when the functional promoter enhances transcription or expression

of that sequence.

The recombinant virus may also contain a cloned selection gene, also termed a selectable marker, encoding a protein which assists in the isolation of an organism harbouring the virus. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic agents, e.g. ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, for example the gene encoding D-alanine racemase for Bacilli.

The enzyme-encoding nucleic acid molecule or fragment thereof may be expressed as part of the same translation product as a nucleic acid molecule encoding a viral coat protein. A coat protein is a protein which forms part of the coat which encloses the nucleic acid core of a virus . Examples of coat proteins suitable for use in this invention are provided in Table 2. Preferably, this invention uses coat protein 10 of phage T7.

A recombinant virus for use in the method of the first aspect of the invention preferably produces one or more enzyme(s) as part of the same translational product as a protein of the virus. As used herein "same translational product as a protein of the virus" means that the enzyme is produced as part of the same protein as, for example, the coat protein of the virus. A "separate translational product as a protein of the virus" means that the enzyme is produced as a separate protein to, for example, the viral coat protein.

In one embodiment the nucleic acid molecule encoding the enzyme is recombinantly inserted into a nucleic acid molecule encoding a coat protein of the virus, for example coat protein 10 of T7.

TABLE 2. Viruses and coat proteins suitable for use in the invention

Mammalian viruses

Insect viruses

Plant viruses

Fungal Viruses

Bacteriophages

Regardless of whether the virus comprises the enzyme in the same or different translation product to a viral protein, if the recombinant virus comprises a plurality of enzymes these enzymes may be produced as the same or separate translation products.

As shown in Figure IG, the nucleic acid molecule encoding one enzyme may be recombinantly inserted onto a nucleic acid encoding one viral protein, while the nucleic acid molecule encoding another enzyme may be recombinantly inserted onto a nucleic acid molecule encoding another viral protein.

Alternatively, the nucleic acid molecules encoding both enzymes may be recombinantly inserted onto a nucleic acid encoding a viral protein so that they are expressed as part of the same translation product (Figure IAi, ii) . The viral proteins may be different copies of the same protein, or may be different proteins.

Other techniques for introducing a plurality of non-native enzymes into a virus will be appreciated by those skilled in the art (see Figure 1 and the examples section for suggestions) .

The enzyme and viral protein or plurality of enzymes when produced as the same translation product may be separated by a linker, cleavage site, and/or a purification tag or may be directly linked. As used herein, a "linker" is a sequence that adopts a conformation which allows for functional activity of a protein to which it is attached. Preferably the linker is a peptide and may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or 50 or more amino acids. The length of any linker used will depend on the nature of the enzyme and whether it is preferred to have a rigid conformation, in which case a shorter linker or no linker would be preferable, or whether it is preferred for the enzyme to have a flexible confirmation.

A "cleavage site" is an amino acid sequence upon which a protease can act to cleave the enzyme from the coat protein. (TEV, Factor Xa) . A "purification tag" is an amino acid sequence or a non-amino acid sequence such as biotin, which can be used to purify the enzyme and/or recombinant virus from a solution or culture, such as hexHis, Flag™, or I.SPY™.

As indicated in Table 2, viruses are able to infect a range of host cells, including mammalian, insect, plant, fungal, or bacterial cells. Viruses have specific host types. Therefore it will be appreciated by a person skilled in the art that any suitable host cells may be used provided that a suitable virus is also used. Examples of mammalian host cells include BSC-I, HeLa S3, CV-I, RK ₁₃ , Cos-7, HUh-T ₇ , BHK and EB9.

Phage, also known as bacteriophage, are viruses which infect bacterial cells. The phage may be a virulent or temperate phage, filamentous phage, lytic, non-lytic, enveloped, non-enveloped, DNA or RNA. For example, the phage may be from the family Corticoviridae, Cystoviridae,

Inoviridae, Leviviridae, Lipothrixviridae, Microviridae, Myoviridae, Plasmaviridae, Podoviridae, Siphoviridae, Sulpholobus shibatae virus, or Tectiviridae. Examples of specific phage include PM2, Coliphage fd, Acholeplasma phage, Coliphage MS2, Coliphage Qbeta, Coliphage T4, Coliphage T7, Coliphage lambda, bacteriophage SPBc2, phage PRDl, and Bacillus phage φ29. Preferably the invention uses T7 phage. T7 is a lytic phage of E.coli that has an isometric head with 6 short tail fibres, and contains linear double-stranded DNA.

EXAMPLES

The invention will now be described in detail by way of reference to the following non-limiting examples and drawings.

Example 1 Design and construction of recombinant virus

Bacillus licheniformis (strain B2659) was isolated from ropey bread (available from Food Science Australia, North Ryde, NSW, Australia) . T7 phage and Escherichia coli, strain BLT5403, were supplied with the T7Select ^Θ 10-3 Cloning Kit (Novagen, 2000) .

B. licheniformis was grown in TY medium (16 g L ^"1 tryptone (BD) , 10 g L ^"1 yeast extract (Difco) , 5 g L ^'1 NaCl, and 2O g L ^"1 agar (Oxoid) for plates (Martirani et al, 2002)) . Shake flask quantities of BLT5403 were grown in

Terrific Broth (TB) or Terrific Agar (TA) containing 12 g IT ¹ tryptone (Oxoid), 24 g L ^"1 yeast extract (Merck), 0.4% (v/v) glycerol, (plus 20 g L ^"1 agar for plates) . 10% (v/v) sterile phosphate solution, consisting of 23.1 g L ^"1 KH ₂ PO ₄ and 125.4 g L ^"1 K ₂ HPO ₄ ), was added after sterilization of the medium. Carbenicillin (Sigma) was added at a concentration of 50 mg L ^"1 after sterilisation through.a filter (0.2 μm) . Top agarose for plaque production consisted of 10 g L ^"1 tryptone (Oxoid), 5 g L ^"1 yeast extract (Merck), 5 g L ^"1 NaCl, and 6 g L ^"1 agarose LE (Promega) .

Gene amplification

The Bacillus licheniformis alpha-amylase gene

(Accession number X03236; X01386) was amplified using primers 1 and 2 listed in Table 3 in an iCycler (Biorad) . DNA was extracted from 20 mL of overnight culture of B. licheniformis grown in TY medium at 30 ⁰ C with shaking at 200 RMP in an orbital incubator-shaker (INFORS AG CH-4103 Bottmingen) . The cells were pelleted and resuspended in 200 μL lysozyme buffer

(50 mM glucose, 10 ttiM EDTA and 25 mM Tris-HCL (pH 8.0), and 5 g/L lysozyme (Chicken eggwhite [muramidase] , ICM biomedicals)

(Sambrook and Russel, 2001. Molecular Cloning: a laboratory manual, 3 ^rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)), and incubated for 20 min at 37 ⁰ C. After treatment with lysozyme, 500 μL of Trizol reagent

(Invitrogen) was added and the DNA was extracted according to the manufacturer's instructions.

TABLE 3. Primers used for cloning and sequencing of alpha- amylase fused to T7 coat protein 10.

Amplification was carried out in 50 μL reaction volumes containing 240 ng of template DNA, iμM of each primer, 1.25 U of polymerase (Taq DNA Polymerase (Promega), 1 PFU DNA polymerase (Promega) , 0.2 mM of each of dATP, dCTP, dTTP, and dGTP (Promega) , Ix buffer without Mg ²⁺ (Promega) and 2 mM MgCl ₂ (Promega) . The following cycling parameters were used: 1 cycle of denaturation for 2 mins at 95°C

followed by 30 cycles of denaturation at 95 ⁰ C for 1 inins, annealing at 47°C for 30 sees, and extension at 75°C for 3 mins, followed by one cycle of extension at 72°C for 5 rnins. Amplification products were analysed using 1% agarose gels . Following amplification the 1448 bp band was purified using the Wizard ^® PCR Preps DNA Purification System (Promega) .

Cloning of an alpha-amylase-encoding nucleic acid into the. T7Select vector The alpha-amylase amplification product was cloned into the T7Select vector using the Trinucleotide Sticky End Cloning method (Dietmaier and Fabry, 1995. Protocol: Di/Trinucleotide Sticky End Cloning (DI/TRISEC) . In: Boehringer Mannheim PCR Applications Manual. Boehringer Mannheim GmbH, Biochemica. pl36-140) . Purified amplification products were phosphorylated using T4 polynucleotide kinase according to Doyle 1996 (Protocols and Applications Guide, 3 ^rd Edition. USA: Promega Corporation, p 187) , followed by precipitation using 0.5 x volume of 7.5 M ammonium acetate and 3 x volume 100% ethanol. After mixing, the DNA was precipitated by centrifugation in a bench-top microcentrifuge (SIGMA 1-13, B. Braun Biotech International GmbH, Melsungen, Germany) at 4°C for 15 min at maximum speed. After washing with 70% (v/v) ethanol the DNA was resuspended in 10 μL of nuclease-free water (Promega) . The phosphorylated amplification product was then treated with T4 DNA Polymerase (Roche) in the following reaction mixture: 64 ng/35 μL reaction mixture, 1 g L ^""1 Bovine Serum Albumin (Sigma, Fraction V) , 1 x restriction buffer C (Promega) , 1 mM dTTP (Promega) and 3 U/35 μL reaction mixture, at 12°C for 30 min, followed by heat inactivation at 80°C for 15 min and ammonium acetate precipitation as described above. The purified DNA was resuspended in 1 μL sterile dH ₂ O. T7Select 10-3 vector arms (1 μg/ 20 μL reaction volume) were treated with 0.1 mM

dATP (Promega) , 1 x restriction buffer A (Promega) , 4 U/20 μL reaction volume Klenow enzyme (Roche) , for 15 min at room temperature, followed by enzyme inactivation at 75°C for 15 min. The DNA was precipitated using ammonium acetate precipitation as described above, and resuspended in 2 μL sterile dH ₂ O. Ligation was performed as described in the T7Select ^<5 System Manual (Novagen, 2000) . The ligated DNA was packaged and plaque assays were performed according to the T7Select ^β System Manual (Novagen) . Plaques produced from phage displaying alpha- amylase were identified by growth in top agarose containing Red Starch (Megazyme) . Alpha-amylase depolymerises Red Starch into low molecular weight dyed fragments . These low molecular weight fragments were either metabolised by the BLT5403 host cells or diffused away, resulting in clear zones surrounding plaques displaying alpha-amylase, as shown in Figure 2.

Genetic confirmation of positive cloning

PCR amplification and sequencing were used to confirm the presence of the alpha-amylase gene in amylase- positive plaques. Amplification of the alpha-amylase gene resulted in the production of a 1448 nucleotide amplification product using gene-specific primers (primers 1 and 2 listed in Table 3) . For DNA sequence analysis, phage lysate was produced in 60 mL volumes of BLT5403 grown in TB containing 50 mg L ^"1 carbenicillin (Sigma) as described (Novagen, 2000) . The cloned alpha-amylase gene was amplified from phage lysate boiled for 10 min. T7Select ^β (Novagen) forward and reverse primers were used for amplification. The sequences of these primers are provided as primer numbers 5 and 6 in Table 3.

The reaction conditions were as above for the amplification of the gene from B. licheniformis. The amplified product using the T7Select ^" (Novagen) forward and

reverse primers was purified using ammonium acetate precipitation as described above. The T7Select (Novagen) forward and reverse primers, as well as additional primers designed from internal regions of the gene (primers 3 and 4 listed Table 3) , were used for sequencing. The nucleotide sequence of the alpha-amylase gene displayed on the phage was identical to that from the B. licheniformis amyS gene (Accession number M13256) . The T7Select construct containing the amylase gene was designated T7-amy.

Cloning of xylanase-encoding nucleic acid into T7select vector

DNA from an overnight plate culture of Bacillus halodurans C-125 (JCM-9153 - Japanese collection of Microorganisms) grown on Tryptone Soy Agar (Oxoid) at 37 °C, was extracted using TRIZOL (Invitrogen) according to the instructions provided by the supplier. Primers 7 and 8 (Table 4) were used to amplify the xynA gene in the following 50 μL reaction mix using an iCycler (Biorad) : 0.1 μg DNA, iμM of each primer, 1.25 U of polymerase (Taq DNA Polymerase

(Promega) :1 PFU DNA Polymerase (Promega) , 0.2 mM of each of dATP, dCTP, dTTP, and dGTP (Promega) , Ix buffer without Mg ²⁺ (Promega) and 2 mM MgCl ₂ (Promega) . The following cycling parameters were used: 1 cycle of denaturation for 2 min. at 95 ⁰ C followed by 30 cycles of denaturation at 95°C for 1 min., annealing at 53 ⁰ C for 30 sec, and extension at 72 ⁰ C for 3 min., followed by one cycle of extension at 72°C for 5 min. Amplification products were analysed using 1% agarose gels. The amplification product was purified by ammonium acetate precipitation using 0.5 x volume of 7.5 M ammonium acetate and 3 x volume 100% ethanol. After mixing, the DNA was precipitated by centrifugation in a bench-top micro¬ centrifuge (SIGMA 1-13, B. Braun Biotech International GmbH,

Melsungen, Germany) at 4°C for 15 min at maximum speed. After washing with 70% (v/v) ethanol the DNA was resuspended in 10 μh of nuclease-free water (Promega) .

EcoRI (Promega) and HindiII (Promega) were then used to digest the ends of the fragment according to the manufacturer's instructions. The digested DNA was purified by- ammonium acetate precipitation as described above. The resulting fragments were ligated into T7Select vector arms (Novagen) using T4 DNA ligase (Roche) . Ligation was carried out overnight at 4°C. After ligation into the T7 vector arms, the cloned DNA was packaged into virions using the procedure and reagents supplied in the T7Select System (Novagen) . The integrity of the construct (T7-xyn) was confirmed by DNA sequencing.

Table 4 Primers used for cloning nucleic acid encoding xylanase into T7select vector

Example 2 Kinetic analysis of alpha-amylase

K _n , and k _cat determinations

The enzyme activity of the phage-expressed enzyme in its unpurified form and that of the PEG 6000-precipitated phage-expressed enzyme (Novagen, 2000) resuspended in 0.05M glycine-NaOH (pH 9.0) was compared with that of a commercially-supplied alpha-amylase (Sigma, catalogue number A3403) in both TB and 0.05M glycine-NaOH (pH 9.0) . A concentration range of starch (potato starch, hydrolysed for electrophoresis; Aldrich) of 0 % [w/v] - 2.5% [w/v] was used for K _n , and k _cat determinations. All assays were performed in triplicate. Starch was boiled for 15 min in TB or 0.05M glycine-NaOH (pH 9.0) . Equal quantities of starch and lysate/enzyme preparation were equilibrated to 70 ⁰ C in separate tubes for 10 min. Test samples of enzyme and starch were combined and incubated at 70°C for 10 min. Samples of starch and enzyme, to be used as blanks were left at 70°C for 10 min in separate tubes. The starch and enzyme of the blanks were only combined at the start of the colour reaction, in order to minimize maltose production. The dinitrosalicylic acid (DNS) colour reaction was performed according to the method of Bernfeld 1955 (Amylases, alpha and beta. Methods Enzymol 1:149-158.) . The starch-enzyme solution (ImL) was added to ImL of colour reagent and boiled in a boiling water bath for 10 min. After rapid cooling on ice, 10 mL of dH ₂ O was added, and OD ₅₄₀ values were read using a UV-1601 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) . Reducing sugar concentrations were determined using maltose as a standard. K _m and V _max values were determined using Lineweaver-Burk plots. K _cat values were calculated using the following equation:

kcat — JMnax

[El i

where V _max is the maximum velocity calculated from the Lineweaver-Burk plot and [E] i is the initial enzyme concentration) .

Enzyme concentration of the phage was calculated by assuming an average of 10 molecules of enzyme per phage (T7Select ^Θ System Manual, Novagen) , determining the phage concentration in the lysate using plaque assays, and determining the concentration using the following equation:

Mass = number of molecules x molecular weight

Avogadro's constant

The kinetic properties of the phage-expressed alpha-amylase were compared with those of alpha-amylase supplied by Sigma (A3403 from B. licheniformis) and the results are shown in Table 5.

Table 5 Kinetic data obtained for alpha-amylase produced by T7-amy phage and a commercially-supplied alpha-amylase

The crude enzyme produced by the T7-amy phage showed a high affinity for the starch, with a K _1n of 0.05%. The affinity of the crude enzyme for starch was comparable to the commercially-supplied alpha-amylase (Sigma) in TB. Both the crude enzyme produced by T7-amy phage and the commercially-supplied enzyme in TB showed Km values lower than for the same enzymes in 0.05M glycine-NaOH, pH 9.0 buffer. The crude enzyme produced by T7-amy phage had a calculated k _cat of 581 min ^"1 , compared with the slightly lower 315 min ^"1 k _cat value of the commercially-supplied enzyme in TB. Both the enzyme produced by T7-amy phage and the commercially-supplied enzyme had lower k _cat values in 0.05M glycine-NaOH pH 9.0 than in TB. The high k _cat value for the T7-amy phage-expressed enzyme in glycine buffer when compared with the commercially-supplied enzyme may have resulted from

residual medium components present after precipitation of the phage, which enhanced the enzyme activity. The crude enzyme was extremely efficient, with a k _cat /K _π , value of 11614 % ^{" 1} .min ^"1 . This efficiency was almost identical to that of the commercially-supplied enzyme in TB and far higher than both the commercially-supplied enzyme and precipitated enzyme produced by T7-amy phage in 0.05M glycine-NaOH, pH 9.0.

As shown in Fig. 3, both the enzyme produced by T7- amy phage and the commercially-supplied enzyme displayed optimal activity at 70°C, with a sharp decline in activity at 80 ⁰ C and 9O ⁰ C.

Activation energy determinations

For activation energy determinations, starch (potato, hydrolysed for electrophoresis, Aldrich) was used at a final concentration of 10 g L ^'1 . The enzyme assay was as described above, except that the assays were performed at temperatures ranging from 30 ⁰ C (303.IK) to 90 ⁰ C (363.IK) . Activation energies were calculated using the Java Arrhenius calculator (http://members.nuvox.net/~on.jwclymer/arr.html), which uses the equation

E _a = -R*slope

where R is the gas constant [8.314 J/mol K] , and slope is obtained from plotting l/T [K] vs Ln rate [mol/L/sec] , using temperature (K) and rate values . At constant substrate concentrations, rate is proportional to the rate constant, k, since rate = k [substrate]

Only values in the linear portion of the plot l/T (K) vs Ln rate (mol/L/sec) were used in the activation energy calculations.

As shown in Table 5, activation energies for both the commercially-supplied enzyme and the enzyme produced by T7-amy phage were low, indicating that low amounts of energy are required for the hydrolysis of starch to occur in the presence of alpha-amylase. The enzyme produced by T7-amy phage had a slightly higher activation energy, both in the crude lysate and when precipitated and resuspended in 0.05 M glycine-NaOH, pH 9.0, compared with the commercially-supplied amylase enzyme in TB and 0.05 M glycine-NaOH, pH 9.0. These results show that alpha-amylase expressed by

T7 compares very favourably with commercially-supplied alpha- amylase in terms of efficiency (k _cat /K _n ,) , temperature optimum and energy of activation. The results also demonstrate that the activity of alpha-amylase produced by T7 in crude lysate is superior to that in 0.05M glycine-NaOH (pH 9.0), a buffer that has been shown to give optimal activity for purified alpha-amylase. This demonstrates that it is entirely feasible to incorporate the production of enzyme into the starch liquefaction process without the costly and time-consuming process of purifying the enzyme from the phage prior to use.

Example 3 Large-scale phage production

T7-amy phage were produced in a 10 L Biostat ^® C (B. Braun Biotech International GmbH, Melsungen, Germany) fermentor. The fermentation medium consisted of 36 g L ^"1 tryptone (Oxoid) , 72 g L ^"1 yeast extract (Merck), 0.4% (v/v) glycerol, and 10% (v/v) phosphate solution, consisting of 23.1 g L ^"1 KH ₂ PO ₄ and 125.4 g L ^"1 K ₂ HPO _4, added after sterilization. The sterile phosphate solution was pumped into the fermentor after medium sterilization, using a FE411

(B. Braun Biotech International GmbH, Melsungen, Germany) peristaltic pump. Carbenicillin (Sigma) (50 mg L ^"1 ) was added just prior to inoculation by injecting the solution into the fermentor through a sterile 0.2 μτa filter (Sartorius) .

Antifoam (5 mL; Sigma) was added to the initial medium prior to sterilization; additional antifoam was found to be unnecessary. A seed culture of E. coli strain BLT5403 was grown overnight with shaking at 200 RPM in TB containing 50 mg L ^"1 σarbenicillin at 37°C, and was used to inoculate 6 L of fermentation medium to an OD _60O of between 0.3 and 0.4. The culture was maintained at 37 ⁰ C and pH 7.4. pH was measured using a Mettler Toledo 405-DPAS-SC-K8S/120 combination pH probe, and adjusted automatically using a 10% (v/v) ammonium hydroxide solution and a 10% (v/v) ortho-phosphoric acid solution. Dissolved oxygen was set at 20% and measured using a Mettler Toledo InPro 6310/lOO/T/N 02 Sensor. Aeration was maintained using alternating stirrer and airflow. The gas mix was initially set at 40% oxygen, 60% air, and oxygen concentration was increased according to the oxygen demands of the culture. A 0.2 μm Sartofluor Capsule (Sartorius) was used to filter air pumped into the fermentor, and a 0.1 μm Sartofluor Mini Cartridge (Sartorius) was used as the air exhaust filter. Glycerol (Sigma, 99%+) , diluted to 72% (v/v) using deionized water, was fed into the fermentor using a

FE411 (B. Braun Biotech International GmbH, Melsungen, Germany) peristaltic pump after 2.5 h of growth. The feed profile used was as follows:

y = 1.88e ^o - ^llt:

where y is the percentage of the maximum flow rate (100%) of the pump, 1.88 is the starting flow rate (as a percentage of the maximum flow rate), 0.11 is μ which is the slope of the log of the curve and therefore determines the flow rate of the feed, and t is time (h) . The flow rate of 72% (v/v) glycerol through 1.6 mm (inner diameter) x 4.8 mm (outer diameter) silicone rubber tubing (Jehbsil ^R ) follows the equation:

y = 0.2773X + 0.4726

where y is the flow rate (mL/min) and x is the percentage of the maximum flow rate. The software used for the fermentor controls was MFCS/win IFB RS-422 (B. Braun Biotech International GmbH, Melsungen, Germany) .

E. coli strain BLT5403 host cells growing rapidly in exponential phase in TB containing 50 mg L ^"1 carbenicillin were inoculated at an OD ₆ oo of 29.5 (4 h post inoculation) with T7-amy phage at a MOI of between 0.007 and 0.01. Lysis of the culture occurred approximately three hours following inoculation, as evidenced by the rapid decrease in the oxygen demand of the culture. The concentration of the phage in the fermentor following lysis was 6.2xlO ¹⁰ pfu/mL, which equates to approximately 4xlθ ¹⁴ pfu/ total fermentor volume. Assuming 10 enzyme molecules per phage (Novagen, 2000), 0.4mg of enzyme was produced during the fermentation (0.06 mg L ^"1 ) .

The use of phage titre as an indicator of enzyme concentration in the fermentor may have resulted in an underestimation of the enzyme concentration, since the increased shearing in the fermentor created as a result of stirring and oxygenation would have lowered the phage infectivity rates following lysis of the bacterial culture. Phage production may be optimised by the addition of suspended substances such as colloidal clay particles of montmorillonite and attapulgite, which have been shown to reduce viral inactivation due to the adsorption of the phage to the particles .

Small-scale assay of the alpha-amylase activity in the lysate, using 2% starch (Potato, hydrolysed) , revealed the production of 0.111 g reducing sugar/L lysate/min at 70°C.

Example 4 Dextrose equivalent (DE) calculations for fermentor lysate

Wheat starch (Sigma) was slurried in deionized H ₂ O containing 80PPM calcium acetate at a concentration of 30% (w/v) , and made up to a volume of 500 mL in a round-bottomed flask. Fermentor lysate was then added to a final volume of 1 L, and the mixture was heated on an Electromantle MV (Electrothermal, UK) heating mantle, with continual homogenisation using a hand-held homogeniser (Ultraturrax T 25 Basic, IKA. Works, [Asia] , Selangor, Malaysia) in order to shear the starch. The starch mixture was brought to the boil, and allowed to boil for a further 5 min. The solution was then incubated in a water bath at 93°C. Aliquots (1 g) were periodically removed, diluted using 9 mL of distilled water, and tested for reducing sugar concentration (Bernfeld, 1955 supra) against a glucose standard curve. Solid starch was pelleted from the solution after boiling in DNS colour reagent, prior to OD ₅₄₀ readings, by centrifugation for 30 seconds in a bench-top micro-centrifuge (SIGMA 1-13, B. Braun Biotech International GmbH, Melsungen, Germany) . DE was calculated as the percent reducing sugar, expressed as glucose, liberated from the total carbohydrate.

The DE values obtained using 15% wheat starch with 4OPPM calcium ions and fermentor lysate are shown in Fig. 4. A DE of between 8-14 is expected by starch industries following liquefaction using optimised conditions. These conditions were not available at the laboratory scale; however, DE values in the region of those expected commercially were obtained using considerably less enzyme than is currently used industrially. We emphasize that these

DE values were obtained using crude lysate. It was not necessary to purify the enzyme, and neither phage or bacteria were viable after incubation at 93°C for 2 h. Since saccharified syrup obtained from starch is generally filtered

and purified through activated charcoal and ion-exchange resins, any large contaminants resulting from the enzyme production process will be removed prior to use of the final product. When starch is used in the production of ethanol, the liquefied starch is further subjected to batch yeast fermentation; therefore purity of the original sample is not expected to pose problems .

Example 5 Cloning of alpha-amylase encoding nucleic acid and xylanase-encoding nucleic acid into the T7select vector so that they are expressed as the same translation product

Nucleic acid molecules encoding the two enzymes used in this study were cloned into the T7 phage, as shown in Figure IAi and ii, so that they were expressed as the same translation product . Cloning of the two enzymes in the same phage is highly desirable, because many industrial enzymatic reactions require the use of more than one enzyme. These include beer production, bleaching of laundry fabrics, baking, production of laundry detergent, enzymatic bleaching of cotton, separation of the lipid fraction from corn fibre, and the use of fatty acid-oxidizing enzymes for the manufacture of paper materials.

Alpha-amylase (1,4-α-glucan glucanohydrolase, EC 3.2.1.1) from Bacillus licheniformis and xylanase A (1,4- beta-D-xylan xylanohydrolase, EC 3.2.1.8) from Bacillus halodurans C125 (JCM 9153) were cloned into the T7Select vector (Novagen) so that they were produced as a single translational product . The translational product produced also comprised the T7 coat protein 10 (CPlO) . Both orientations of the alpha-amylase and xylanase were made;

CPlO-alpha-amylase-xylanase (T7-AX) and CPlO-xylanase-alpha- amylase (T7-XA) .

The xynA gene was amplified from Bacillus halodurans genomic DNA with either primers 11 and 12 (Table

6) for T7-AX or primers 13 and 14 for T7-XA using the conditions outlined above (with the exception of a reduction in extension time to 2 minutes) . The amyS gene was amplified from 10μl of boiled T7-amyS lysate (described above in Example 1) using almost the same conditions with the exception of an annealing temperature of 49 ⁰ C. Primers 9 and 10 (Table 6) where used for the T7-AX construct and primers 15 and 16 for the T7-XA construct. The PCR products were analysed, purified, digested with appropriate restriction enzymes (BamHl, EcoRl, HindiII) , ligated into T7Select vector arms and packaged into virions using techniques described above. Plaques producing alpha-amylase and/or xylanase were identified by growth in top agarose containing Red Starch (Megazyme) and Birchwood azo-xylan (Megazyme) respectively (Figure 5) . The integrity of the final constructs was confirmed by DNA sequencing using primers 1-17 (Tables 3,5,6) .

Table 6 Primers used for cloning of alpha-amylase and xylanase so that they are produced as the same translation product

Example 6 Kinetic analysis of xylanase and T7 constructs containing alpha-amylase and xylanase as the same translation product

The enzyme activities of the phage-expressed enzymes in their unpurified form were compared together with that of the commercially-supplied alpha-amylase (Sigma, catalogue number A3403) . Alpha-amylase activity was determined using the dinitrosalicylic acid (DNS) colour reaction as described previously.

Xylanase activity was determined using lyophilised oat spelt xylan (Sigma oat spelts, X-0627) . Lyophilised xylan was produced by adding xylan (3 g in 150 πiL) to boiling water followed by the addition of 2 volumes of absolute ethanol and filtration through a 12.5 % Whatman's #4 filter. After filtration, 100 mL of ethanol was passed through the filter followed by 100 mL of 95 % ethanol, then 100 mL of 99.9 % ethanol and then 100 mL of acetone. The xylan was dried overnight in a desiccator. A 1% xylan stock solution was made by adding lyophilized xylan to TB, boiled for 15 min and allowed to cool. The xylanase activity assay was essentially the same as for alpha-amylase (DNS reaction) with the

exception that xylan was the substrate, 4ml of water was added at the end of the reaction, and 1 ml of sample was spun for 1 min at maximum speed in a benchtop centrifuge (SIGMA 1- 13, B. Braun Biotech International GmbH, Melsungen, Germany) prior to OD ₅₄₀ reading. Xylanase assays were carried out at 70 ⁰ C which is the optimum temperature for the Bacillus halodurans cloned enzyme. Reducing sugar concentrations were determined using xylose as a standard. The K _n , and K _cat values for all constructs are shown in Table 7. No equivalent commercially-supplied xylanase enzyme was available to directly compare the phage-based and free xylanase enzyme kinetics.

Table 7 Enzyme Kinetic values for T7Select constructs and commercially-supplied enzymes.

Wo act* - T7-xyn phage showed no activity when starch was used as the substrate

The apparent K _m for the T7-amy, T7-AX and T7-XA phage constructs with starch as the substrate were similar to that obtained under the same conditions using commercially- supplied free alpha-amylase (Sigma), all being between 0.02 and 0.08% starch. T7-xyn showed no activity towards starch. When xylan was used as the substrate, all constructs expressing the xylanase enzyme (T7-xyn, T7-AX and T7-XA) also showed similar Km values of 0.03-0.09%. Alpha-amylase

produced by T7~amy phage and the commercially-supplied enzyme also showed activity towards xylan, but with a slightly higher K _n ,. This is likely to be due to the low purity of the oat spelt xylan used in this study. Although very similar K _n , values were obtained for the enzymes against the corresponding substrate, the highest K _m against starch was seen for T7-AX phage, and the highest against xylan was T7-XA phage (discounting the T7-amy phage and commercially-supplied enzyme) . The enzyme of interest in both the T7-AX and T7-XA phage constructs (i.e. alpha-amylase with starch or xylanase with xylan) was sandwiched between the coat protein of the phage and the second enzyme. This suggests that the affinity of the enzyme for the substrate may be slightly affected by the production of the enzymes as the same translation product. The rate of turnover, or apparent catalytic constant

(K _C at) / varied between constructs. However, it is apparent that constraining the enzymes (i.e. tethering to the coat protein or another enzyme, and/or sandwiching between 2 proteins, effects and possibly improves the turnover rate (K _cat ) .The higher K _cat means a faster reaction with higher turnover. This in turn will affect the specificity constant (Kc _at /Km) ■ Ultimately a high K _cat will result in cost savings.

Example 7 Cloning of alpha-amylase encoding nucleic acid and xylanase-encoding nucleic acid as different translational products in the same T7Select vector

Using standard molecular biology cloning techniques, the following types of constructs could be made:

Single transcriptional unit encoding two different translational products

Nucleic acid molecules (nucleic acid molecules 1 and 2) encoding two different enzymes are inserted separately into phage vector DNA, for example as described above (based

on T7Select kit instructions - Novogen) . Nucleic acid molecule 1, including the stop codon and 3' flanking vector sequences that include enhancer sequence for translation of the adjoining (downstream) gene, is amplified by PCR. Nucleic acid molecule 2, including 5' sequences for the start codon and nucleic acid sequence encoding the CPlO protein, which are in frame with nucleic acid molecule 2, are also amplified by PCR. Appropriate restriction enzyme sites that where incorporated into the 5' and 3' ends of the PCR products are used to ligate nucleic acid molecule 1- and nucleic acid molecule 2- containing PCR fragments into phage vector arms.

As shown in Figure IB, after ligation the vector sequence will include the appropriate promoter and transcriptional start sequences and CPlO coding sequences, all originating from the vector arm, followed in-frame by nucleic acid molecule 1 including a translational stop codon. This is followed by a short intervening sequence, which includes a sequence involved in enhancing translation, and then the start codon and coding sequence of CPlO gene joined in-frame with the coding sequence of nucleic acid molecule 2.

Two transcriptional (tandem) units and two translational products

As shown in Figure 1C, the same procedure as above can be used except that the short intervening region between nucleic acid molecules 1 and 2 is replaced with a long (~100bp) double stranded oligonucleotide, or short PCR product, that encodes a promoter and transcription start site, and a ribosomal binding site for nucleic acid molecule 2. This strategy is similar to that seen in the LIC Duet

Minimal Adapter strategy (Novagen) . A terminator sequence for nucleic acid molecule 1 may also be included in the intervening region.

Two transcriptional (divergent) units and two translational products

Two separate promoters are used to express fusion nucleic acid molecules of interest. The divergent (head-to- head) nature of the promoters reduces expression resulting from readthrough from one transcriptional unit to the next.

This is shown in Figure ID. Philipps et al (2004,

BioTechniques 36:80-83) used this approach to create a novel baculovirus expression system.

One or both enzyme-encoding nucleic acid molecules plasmid born

Nucleic acid molecule 1 and/or 2 which is/are translationally fused to a nucleic acid molecule encoding a coat protein of the virus may be expressed from nucleic acid sequences located within a plasmid vector. After expression within the virus, the expressed products are incorporated with other proteins expressed from the viral genome to form a viral particle. This is shown in Figure IE. Examples of this include the helper phage/ phagmid system (reviewed in

Willats, 2002, Plant Molecular Biology 50:837) or the complementary plasmid which expresses the wildtype CPlOA gene in some of the T7Select systems (Novagen) .

One or both enzyme-encoding nucleic acid molecules genomically expressed

The link between phenotype and genotype of the phage is not important for this methodology as there is no selection process involved. The important outcome of phage production is -to express the enzyme(s) of interest. Therefore the construct encoding the fusion protein can be expressed genomically so long as the nucleic acid molecule encoding the fusion protein is incorporated into the phage particle (Figure IF) . Integration of the fusion construct can be

carried out by methods know in the art.

Two different coat proteins used for fusion products Some phage systems (eg M13) display several different proteins on their surface. This provides the opportunity to express different genes of interest on several different coat proteins (Figure IG) . For example the Ff bacteriophage (eg M13) can display a protein/peptide of interest fused with either the pVIII or pill coat protein (reviewed in Willats, 2002 supra) depending on the size and number of fusion proteins required.

Express each nucleic acid molecule on a separate phage particle and mix Iysate A simple approach to obtaining multiple enzymes is to produce each one as a separate fusion construct. The lysate generated from different fusion constructs can be mixed to create a pool of lysate with multiple enzymic activities in the appropriate ratios (Figure IH) . Castillo et al, (2001, J Immunol Meth 257:117-122) used this approach to express a library of target peptides and a library of target binders (scFv) in two separate phage vectors and successfully isolated candidate binders.

Nucleic acid molecule 1 expressed as fusion product with coat protein, second gene product associates through non-covalent bond

Where the associate of subunits of a multimeric product is relatively strong, only one subunit needs to be anchored to the phage particle via fusion with a coat protein. The assembly of the other subunits can occur within the cell resulting in a multimeric product present on the phage surface (Figure II) . This is a common methodology when various subunits of antibodies are used (eg as reviewed in

Hoogenboom et al, 1998, Iramunotechnology 4:1-20) .

Example 8 De-inking of mixed office waste using phage- expressed enzymes Mixed office waste (MOW) is a large source of recyclable wastepaper, but is considered the most difficult raw material to de-ink, making it desirable to find alternatives to the conventional alkaline deinking processes. A number of studies have shown that enzyme-based deinking is a viable alternative and a range of enzymes have been shown to improve deinking, including lipases and esterases which degrade oil-based inks, and pectinases, hemicellulases/xylanses, cellulases, amylases and other lignolytic enzymes which free the ink particles by altering the fibre surface or bonds that the ink particles are associated with. Xylanase and alpha-amylase have both been shown to improve the deinking process . The T7 constructs containing nucleic acid molecules encoding alpha-amylase and xylanase and attached to a nucleic acid molecule encoding T7 phage coat protein 10 were used in deinking assays and were found to be a viable option for carrying out enzymic deiniking of MOW.

Preparation of pulp MOW with an ink coverage of approximately 22% was shredded (60Og) and pulped according to Technical Association of Paper and Pulp Industries (TAPPI) protocol T525 om-92.

The pulp was partially dried to 75% water:25% fibre and stored at 4 ⁰ C.

Preparation of enzyme

Phage lysate was prepared according to T7Select manufacturers instructions including PEG precipitation

(Novagen) . Approximately equivalent units (eg 20 units) of

activity of either free enzyme or enzyme on phage was resuspended in 170 ml of TB.

Enzyme reaction 170 ml of enzyme preparation (described above) was heated to 70 ⁰ C on a hotplate, followed by the addition of 10 g of oven dried (OD) fibres (40 g pulp) . The mixture was kept at reaction temperature with gentle stirring for 30 min. The temperature was raised to 100 ⁰ C and the mixture was boiled for 10 min. No viable phage remained in the mixture after boiling.

Flotation and Preparation of Handsheets

After boiling for 10 mins, the mixture was diluted to 2 L with water at room temperature then transferred to a flotation device (PaIa et al, 2004. J Biotech 108:79-89) . 3 ml of flotation aid was added ( ¹ Z _5O dilution, Buckman BDR2331) . Flotation occurred with an air flow of 0.5 L min ^"1 for 20 min. At the end of flotation, residual foam was removed and the pulp concentrated using low speed centrifugal filtration and then oven dried.

3 g of OD fibres were wetted in 300 ml water and disintegrated for 1 min (Black and Decker FP15X, setting II) . Handsheets where made (TAPPI protocol T272 om-92) and brightness measured (ColorTouch apparatus (Model ISO) light source D65) , using TAPPI protocol T272 om-92.

Outcome of de-inking process

Figure 6 shows the increase in brightness of handsheets as a result of deinking by the amylase and xylanase enzymes. A T7 phage lysate (T7-NE) that contained no amylase or xylanase activity due to the lack of nucleic acid sequences encoding these enzymes was used as a negative control. Phage containing a nucleic acid molecule encoding a

single enzyme (T7-amy or T7-xyn phage) showed an improvement in brightness over the negative control (both 3.8%) . A combination of both enzymes on the same phage particle (TV- AX) showed a better improvement with an increase of 4.1% in brightness. This trend was observed over several independent reactions. Further optimisation of lysate production should improve the brightness to the levels seen for commercially- supplied amylase in lysate from T7-NE (5.6% improvement) .

Example 9 Production of an enzyme using a mammalian host cell

A plasmid with a synthetic strong promoter is created using techniques known in the art, for example as described by Chakrabarti et al, 1997 (Biotechniques 23 (6) : 1094-1097) . A nucleic acid molecule is generated which encodes the enzyme of interest located between flanking nucleic acids which encode vaccinia sequences, a promoter, and a selection marker, such as TK kinase or an antibiotic selection marker. Suitable promoters and selection markers are known in the art, and are exemplified in Chakrabarti et al. supra.

A recombinant vaccinia virus, such as strain IHDJ is prepared by methods known in the art, for example as described by Earl PL and Moss B 1991 (Generation of Vaccinia viruses pp 16.17.1 - 16.17.16 in FM Ausbal et al, (ed) Current Protocols in Molecular Biology, vol 2. Greene

Publishing Associates and Wiley International Sciences, New York, NY) . This virus comprises the generated nucleic acid inserted into the viral genome translationally fused to the nucleic acids encoding the cytoplasmic and transmembrane domains of the B5R protein of the extracellular envelope of the virus.

Host cells, such as CV-I cells, are infected with the recombinant virus and the recombinant virus will be propagated according to methods known in the art, for example

as described by Earl PL and Moss B 1991 supra. , so that the generated nucleic acid is expressed translationally fused to the nucleic acid encoding part of the B5R protein.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.