WO2003072717A2 - Grain-based enzyme-releasing matrix and method of preparation - Google Patents

Abstract

Thermostable enzymes are incorporated into a grain-based particle-sized matrix for release in aqueous environments, for example, thermostable glycosidase enzymes derived from various Thermococcus, Staphylothermus and Pyrococcus organisms. The grain-based enzyme-releasing matrix is biodegradable and can be used as an adjuvant for animal feeds.

Description

GRAIN-BASED ENZYME-RELEASING MATRIX AND METHOD OF PREPARATION

Related Applications

[0001] This application is a Continuation-in-part application of co-pending U.S. Application Serial No. 09/134,078, filed August 13, 1998, which is a continuation application of U.S. Application Serial No. 08/949,026, filed October 10, 1997, now abandoned, which claims priority from U.S. Provisional application Serial No. 60/056,916, filed December 6, 1996, now abandoned, all of which are hereby incoφorated by reference in their entirety.

Field of the Invention

[0002] This invention relates to new uses for endoglucanase enzymes. More particularly, the invention relates use of endoglucanase enzymes in production of pet litter.

Description of Related Art

[0003] The glycosidic bond of β-galactosides can be cleaved by different classes of enzymes: (i) phospho-β-galactosidases (EC3.2.1.85) are specific for a phosphorylated substrate generated via phosphoenolpyruvate phosphotransferase system (PTS)- dependent uptake; (ii) typical β-galactosidases (EC 3.2.1.23), represented by the Escherichia coli LacZ enzyme, which are relatively specific for β-galactosides; and (iii) β-glucosidases (EC 3.2.1.21) such as the enzymes of Agrobacterium faecalis, Clostridium thermocellum, Pyrococcus furiosus or Sulfolobus solfataricus (Day, A.G. and Withers, S.G., (1986) Purification and characterization of a β-glucosidase from Alcaligenes faecalis. Can. J. Biochem. Cell. Biol. 64, 914-922; Kengen, S.W.M., et al. (1993) Eur. J. Biochem., 213, 305-312; Ait, N., Cruezet, N. and Cattaneo, J. (1982) Properties of β-glucosidase purified from Clostridium thermocellum. J. Gen. Microbiol. 128, 569-577; Grogan, D.W. (1991) Evidence that β-galactosidase of Sulfolobus solfataricus is only one of several activities of a thermostable β-D-glycodiase. Appl. Environ. Microbiol. 57, 1644-1649). Members of the latter group, although highly specific with respect to the β-anomeric configuration of the glycosidic linkage, often display a rather relaxed substrate specificity and hydrolyze β-glucosides as well as β- fucosides and β-galactosides.

[0004] Generally, α-galactosidases are enzymes that catalyze the hydrolysis of galactose groups on a polysaccharide backbone or hydrolyze the cleavage of di- or oligosaccharides comprising galactose.

[0005] Generally, β-mannanases are enzymes that catalyze the hydrolysis of mannose groups internally on a polysaccharide backbone or hydrolyze the cleavage of di- or oligosaccaharides comprising mannose groups, β-mannosidases hydrolyze non-reducing, terminal mannose residues on a mannose-containing polysaccharide and the cleavage of di- or oligosaccaharides comprising mannose groups.

[0006] Guar gum is a branched galactomannan polysaccharide composed of β- 1 ,4 linked mannose backbone with α-1,6 linked galactose side chains. The enzymes required for the degradation of guar are β-mannanase, β-mannosidase and α- galactosidase. β-mannanase hydrolyses the mannose backbone internally and β-mannosidase hydrolyses non-reducing, terminal mannose residues, α-galactosidase hydrolyses α-linked galactose groups.

[0007] Galactomannan polysaccharides and the enzymes that degrade them have a variety of applications. Guar is commonly used as a thickening agent in food and is utilized in hydraulic fracturing in oil and gas recovery. Consequently, galactomannanases are industrially relevant for the degradation and modification of guar. Furthermore, a need exists for thermostable galactomannases that are active in extreme conditions associated with drilling and well stimulation. [0008] There are other applications for these enzymes in various industries, such as in the beet sugar industry. 20-30% of the domestic U.S. sucrose consumption is sucrose from sugar beets. Raw beet sugar can contain a small amount of raffinose when the sugar beets are stored before processing and rotting begins to set in. Raffinose inhibits the crystallization of sucrose and also constitutes a hidden quantity of sucrose. Thus, there is merit to eliminating raffinose from raw beet sugar. α-Galactosidase has also been used as a digestive aid to break down raffinose, stachyose, and verbascose in such foods as beans and other gassy foods.

[0009] β-galactosidases which are active and stable at high temperatures appear to be superior enzymes for the production of lactose-free dietary milk products (Chaplin, M.F. and Bucke, C. (1990) In: Enzyme Technology, pp. 159-160, Cambridge University Press, Cambridge, UK). Also, several studies have demonstrated the applicability of β-galactosidases to the enzymatic synthesis of oligosaccharides via transglycosylation reactions (Nilsson, K.G.I. (1988) Enzymatic synthesis of oligosaccharides. Trends Biotechnol. 6, 156-264; Cote, G.L. and Tao, B.Y. (1990) Oligosaccharide synthesis by enzymatic transglycosylation. Glycoconjugate J. 7, 145-162). Despite the commercial potential, only a few β-galactosidases of thermophiles have been characterized so far. Two genes reported are β-galactoside-cleaving enzymes of the hyperthermophilic bacterium Thermotoga maritima, one of the most thermophilic organotrophic eubacteria described to date (Huber, R., Langworthy, T.A., Kδnig, H., Thomm, M., Woese, C.R., Sleytr, U.B. and Stetter, K.O. (1986) T. martima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90EC, Arch. Microbiol. 144, 324-333) one of the most thermophilic organotrophic eubacteria described to date. The gene products have been identified as a β-galactosidase and a β-glucosidase.

[0010] Pullulanase is well known as a debranching enzyme of pullulan and starch. The enzyme hydrolyzes α-l,6-glucosidic linkages on these polymers. Starch degradation for the production or sweeteners (glucose or maltose) is a very important industrial application of this enzyme. The degradation of starch is developed in two stages. The first stage involves the liquefaction of the substrate with α-amylase, and the second stage, or saccharification stage, is performed by β-amylase with pullalanase added as a debranching enzyme, to obtain better yields.

[0011] Endoglucanases can be used in a variety of industrial applications. For instance, the endoglucanases of the present invention can hydrolyze the internal β-l,4-glycosidic bonds in cellulose, which may be used for the conversion of plant biomass into fuels and chemicals. Endoglucanases also have applications in detergent formulations, the textile industry, in animal feed, in waste treatment, and in the fruit ■ juice and brewing industry for the clarification and extraction of juices.

SUMMARY OF THE INVENTION

[0012] The present invention provides an enzyme delivery matrix in the form of discrete plural particles, each of the particles comprising grain germ that is spent of oil and a thermostable enzyme. The particles readily disperse the glycosidase enzyme contained therein into aqueous media.

[0013] The present invention also provides a process for preparing an enzyme delivery matrix by providing a mixture comprising a grain germ that is spent of oil, a starch and a thermostable glycosidase enzyme. The mixture is compacted into discrete plural particles to form an enzyme delivery matrix.

Brief Description of the Drawings

[0014] The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

[0015] Figure 1 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of Ml 1TL of the present invention. Sequencing was performed using a 378 automated DNA sequencer for all sequences of the present invention (Applied Biosystems, Inc.).

[0016] Figure 2 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of OC1/4V-33B/G.

[0017] Figure 3 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of F1-12G.

[0018] Figure 4 are illustrations of the full-length DNA and corresponding deduced amino acid sequence of 9N2-31B/G.

[0019] Figure 5 are illustrations of the full-length DNA and corresponding deduced amino acid sequence of MSB8-6G.

[0020] Figure 6 are illustrations of the full-length DNA and corresponding deduced amino acid sequence of AEDII12RA-18B/G.

[0021] Figure 7 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of GC74-22G.

[0022] Figure 8 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of VC1-7G1. [0023] Figure 9 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of 37GP 1.

[0024] Figure 10 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of 6GC2.

[0025] Figure 11 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of 6GP2.

[0026] Figure 12 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of 63 GB 1.

[0027] Figure 13 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of OC1/4V (33GP1).

[0028] Figure 14 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of 6GP3.

[0029] Figure 15 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of Thermotoga maritima MSB8-6GP2.

[0030] Figure 16 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of Thermotoga maritima MSB8-6GB4.

[0031] Figure 17 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of Banki gouldi 37GP4.

[0032] Figure 18 is an illustration of the full-length DNA and corresponding deduced amino acid sequence of Pyrococcus furiosus VC1-7EG1.

[0033] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Definitions

[0034] AMonosaccharide≡, as used herein, refers to a single polyhydroxy aldehyde or ketone unit.

[0035] "Oligosaccharide", as used herein, refers to short chains of monosaccharide units joined together by covalent bonds. Of these, the most abundant are the disaccharid es, which have two monosaccharide units.

[0036] "Polysaccharide", as used herein, refers to long chains having many monosaccharide units.

[0037] The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0038] A coding sequence is "operably linked to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences. The coding sequences need not be contiguous to one another so long as the expressed sequences ultimately process to produce the desired protein.

[0039] "Recombinant" enzymes refer to enzymes produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired enzyme. "Synthetic" enzymes are those prepared by chemical synthesis. [0040] A DNA "coding sequence of or a "nucleotide sequence encoding" a particular enzyme, is a DNA sequence which is transcribed and translated into an enzyme when placed under the control of appropriate regulatory sequences.

Detailed Description of the Invention

[0041] The polynucleotides and polypeptides of the present invention have been identified as glucosidases, α-galactosidases, β-galactosidases, β-mannosidases, β-mannanases, endoglucanases, and pullalanases as a result of their enzymatic activity.

[0042] In accordance with one aspect of the present invention, there are provided novel enzymes, as well as active fragments, analogs and derivatives thereof.

[0043] In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the enzymes of the present invention including mRNAs, cDNAs, genomic DNAs as well as active analogs and fragments of such enzymes.

[0044] In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of the enzymes and subsequent recovery of the enzymes.

[0045] In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes, or polynucleotides encoding such enzymes for hydrolyzing lactose to galactose and glucose for use in the food processing industry, the pharmaceutical industry, for example, to treat intolerance to lactose, as a diagnostic reporter molecule, in corn wet milling, in the fruit juice industry, in baking, in the textile industry and in the detergent industry. [0046] In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes for hydrolyzing guar gum (a galactomannan polysaccharide) to remove non-reducing terminal mannose residues. Further polysaccharides such as galactomannan and the enzymes according to the invention that degrade them have a variety of applications. Guar gum is commonly used as a thickening agent in food and also is utilized in hydraulic fracturing in oil and gas recovery. Consequently, mannanases are industrially relevant for the degradation and modification of guar gums. Furthermore, a need exists for thermostable mannases that are active in extreme conditions associated with drilling and well stimulation.

[0047] In accordance with yet a further aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.

[0048] In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes, or polynucleotides encoding such enzymes, for in vitro purposes related to scientific research, for example, to generate probes for identifying similar sequences which might encode similar enzymes from other organisms by using certain regions, i.e., conserved sequence regions, of the nucleotide sequence.

[0049] These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

[0050] The polynucleotides of this invention were originally recovered from genomic gene libraries derived from the following organisms:

[0051] Ml 1TL is a new species of Desulfurococcus isolated from Diamond Pool in Yellowstone National Park. The organism grows optimally at 85-88 °C, pH 7.0 in a low salt medium containing yeast extract, peptone, and gelatin as substrates with a N₂/CO₂ gas phase.

[0052] OC1/4V is from the genus Thermotoga. The organism was isolated from Yellowstone National Park. It grows optimally at 75 °C in a low salt medium with cellulose as a substrate and N₂ in gas phase.

[0053] Pyrococcus furiosus VC1 and (7EG1) is from the genus Pyrococcus. VC1 was isolated from Vulcano, Italy. It grows optimally at 100 °C in a high salt medium (marine) containing elemental sulfur, yeast extract, peptone and starch as substrates and N₂ in gas phase.

[0054] Staphylothermus marinus FI is a from the genus Staphylothermus. FI was isolated from Vulcano, Italy. It grows optimally at 85 °C, pH 6.5 in high salt medium (marine) containing elemental sulfur and yeast extract as substrates and N₂ in gas phase.

[0055] Thermococcus 9N-2 is from the genus Thermococcus 9N-2 was isolated from diffuse vent fluid in the East Pacific Rise. It is a strict anaerobe that grows optimally at

87 °C.

[0056] Thermotoga maritima MSB8 and MSB8 (Clone # 6GP2 and 6GB4) is from the genus Thermotogo, and was isolated from Vulcano, Italy. MSB8 grows optimally at

85 °C, pH 6.5 in a high salt medium (marine) containing starch and yeast extract as substrates and N₂ in gas phase.

[0057] Thermococcus alcaliphilus AEDII12RA is from the genus Thermococcus. AEDII12RA grows optimally at 85 °C, pH 9.5 in a high salt medium (marine) containing polysulfides and yeast extract as substrates and N₂ in gas phase. [0058] Thermococcus chitonophagus GC74 is from the genus Thermococcus. GC74 grows optimally at 85 °C, pH 6.0 in a high salt medium (marine) containing chitin, meat extract, elemental sulfur and yeast extract as substrates and N₂ in gas phase. AEPII la grows optimally at 85 °C at pH 6.5 in marine medium under anaerobic conditions. It has many substrates. Bankia gouldi is from the genus Bankia.

[0059] Accordingly, the polynucleotides and enzymes encoded thereby are identified by the organism from which they were isolated, and are sometimes hereinafter referred to as "Ml 1TL" (Figure 1 and SEQ ID NOS: l and 15), "OC1/4V-33B/G" (Figure 2 and SEQ ID NOS:2 and 16), "F1-12G" (Figure 3 and SEQ ID NOS:3 and 17), "9N2-31B/G" (Figure 4 and SEQ ID NOS:4 and 18), "MSB8" (Figure 5 and SEQ ID NOS:5 and 19), "AEDII12RA-18B/G" (Figure 6 and SEQ ID NOS:6 and 20), "GC74-22G" (Figure 7 and SEQ ID NOS:7 and 21), "VC1-7G1" (Figure 8 and SEQ ID NOS:8 and 22), "37GP1" (Figure 9 and SEQ ID NOS: 9 and 23), "6GC2" (Figure 10 and SEQ ID NOS: 10 and 24), "6GP2" (Figure 11 and SEQ ID NOS:l 1 and 25), "AEPII la" (Figure 12 and SEQ ID NOS:12 and 26), 'OC1/4V" or 33GP1 (Figure 13 and SEQ ID NOS: 13 and 27), and "6GP3" (Figure 14 and SEQ ID NOS:28), "MSB8-6GP2" (Figure 15 and SEQ ID NOS:57 and 61), "MSB8-6GB4"(Figure 16 and SEQ ID NOS:58 and 62),^MVC1- 7EGl"(Figure 17 and SEQ ID NOS:59 and 63), and 37GP4 (Figure 18 and SEQ ID NOS:60 and 64).

[0060] The polynucleotides and polypeptides of the present invention show identity at the nucleotide and protein level to known genes and proteins encoded thereby as shown in Table 1. Table 1

[0061] The polynucleotides and enzymes of the present invention show homology to each other as shown in Table 2.

Table 2

[0062] All the clones identified in Tables 1 and 2 encode polypeptides which have α- glycosidase or β-glycosidase activity.

[0063] This invention, in addition to the isolated nucleic acid molecules encoding the enzymes of the present invention, also provide substantially similar sequences. Isolated nucleic acid sequences are substantially similar if: (i) they are capable of hybridizing under conditions hereinafter described, to the polynucleotides of SEQ ID NOS: 1-14 and 57-60; (ii) or they encode DNA sequences which are degenerate to the polynucleotides of SEQ ID NOS: 1-14 and 57-60. Degenerate DNA sequences encode the amino acid sequences of SEQ ID NOS: 15-28 and 61-64, but have variations in the nucleotide coding sequences. As used herein, substantially similar refers to the sequences having similar identity to the sequences of the instant invention. The nucleotide sequences that are substantially the same can be identified by hybridization or by sequence comparison. Enzyme sequences that are substantially the same can be identified by one or more of the following: proteolytic digestion, gel electrophoresis and/or microsequencing.

[0064] One means for isolating the nucleic acid molecules encoding the enzymes of the present invention is to probe a gene library with a natural or artificially designed probe using art recognized procedures (see, for example: Current Protocols in Molecular Biology, Ausubel F.M. et al. (EDS.) Green Publishing Company Assoc. and John Wiley Interscience, New York, 1989, 1992). It is appreciated to one skilled in the art that the polynucleotides of SEQ ID NOS: 1-14 and 57-60 or fragments thereof (comprising at least 12 contiguous nucleotides), are particularly useful probes. Other particular useful probes for this purpose are hybridizable fragments to the sequences of SEQ ID NOS: 1-14 and 57-60 (i.e., comprising at least 12 contiguous nucleotides).

[0065] With respect to nucleic acid sequences which hybridize to specific nucleic acid sequences disclosed herein, hybridization may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions. As an example of oligonucleotide hybridization, a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45°C in a solution consisting of 0.9 M NaCl, 50 mM NaH2PO4, pH 7.0, 5.0 mM Na2EDTA, 0.5% SDS, 10X Denhardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2 X 107 cpm (specific activity 4-9 X 108 cpm/ug) of 32P end-labeled oligonucleotide probe are then added to the solution. After 12-16 hours of incubation, the membrane is washed for 30 minutes at room temperature in IX SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh IX SET at Tm 1Q°C for the oligonucleotide probe. The membrane is then exposed to auto-radiographic film for detection of hybridization signals.

[0066] Stringent conditions means hybridization will occur only if there is at least 90% identity, preferably at least 95% identity and most preferably at least 97% identity between the sequences. Further, it is understood that a section of a 100 bps sequence that is 95 bps in length has 95% identity with the 1090 bps sequence from which it is obtained. See J. Sambrook et al, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989) which is hereby incoφorated by reference in its entirety. Also, it is understood that a fragment of a 100 bps sequence that is 95 bps in length has 95% identity with the 100 bps sequence from which it is obtained.

[0067] As used herein, a first DNA (RNA) sequence is at least 70% and preferably at least 80% identical to another DNA (RNA) sequence if there is at least 70% and preferably at least a 80% or 90% identity, respectively, between the bases of the first sequence and the bases of the another sequence, when properly aligned with each other, for example when aligned by BLASTN.

[0068] "Identity" as the term is used herein, refers to a polynucleotide sequence which comprises a percentage of the same bases as a reference polynucleotide (SEQ ID NOS: 1-14 and 57-60). For example, a polynucleotide which is at least 90% identical to a reference polynucleotide, has polynucleotide bases which are identical in 90% of the bases which make up the reference polynucleotide and may have different bases in 10% of the bases which comprise that polynucleotide sequence.

[0069] The present invention relates polynucleotides which differ from the reference polynucleotide such that the changes are silent changes, for example the change do not alter the amino acid sequence encoded by the polynucleotide. The present invention also relates to nucleotide changes which result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference polynucleotide. In a preferred aspect of the invention these polypeptides retain the same biological action as the polypeptide encoded by the reference polynucleotide.

[0070] It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe. Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. The probes are thus useful to isolate complementary copies of DNA from other sources or to screen such sources for related sequences. [0071] The polynucleotides of this invention were recovered from genomic gene libraries from the organisms listed in Table 1. For example, gene libraries can be generated in the Lambda ZAP II cloning vector (Stratagene Cloning Systems). Mass excisions can be performed on these libraries to generate libraries in the pBluescript phagemid. Libraries are thus generated and excisions performed according to the protocols/methods hereinafter described.

[0072] The excision libraries are introduced into the E. coli strain BW 14893 F'kanlA. Expression clones are then identified using a high temperature filter assay. Expression clones encoding several glucanases and several other glycosidases are identified and repurified. The polynucleotides, and enzymes encoded thereby, of the present invention, yield the activities as described above.

[0073] The coding sequences for the enzymes of the present invention were identified by screening the genomic DNAs prepared for the clones having glucosidase or galactosidase activity.

[0074] An example of such an assay is a high temperature filter assay wherein expression clones were identified by use of high temperature filter assays using buffer Z (see recipe below) containing 1 mg/ml of the substrate 5-bromo-4-chloro-3-indolyl-β-D- glucopyranoside (XGLU) (Diagnostic Chemicals Limited or Sigma) after introducing an excision library into the E. coli strain BW 14893 F'kanlA. Expression clones encoding XGLUases were identified and repurified from Ml 1TL, OC1/4V, Pyrococcus furiosus VC1, Staphylothemus marinus FI, Thermococcus 9N-2, Thermotoga maritima MSB8, Thermococcus alcaliphilus AEDII12RA, and Thermococcus chitonophagus GC74. [0075] Z-buffer: (referenced in M _ iller, , J _ .H. (1 —99 _ 2_ ) A Short Course in Bacterial

;s, p. 445.)

per liter:

Na₂HPO₄-7H₂O 16.1g

NaH₂PO₄-7H₂O 5.5g

KCl 0.75g

MgSO₄-7H₂O 0.246g β-mercaptoethanol 2.7ml

Adjust pH to 7.0

Hi2h Temperature Filter Assay

(1) The f factor fkan (from E. coli strain CSH118)(1) was introduced into the pho-pnh-lac-strain BW14893(2). BW13893(2). The filamentous phage library was plated on the resulting strain, BW 14893 F'kan. (Miller, J.H. (1992) A Short Course in Bacterial Genetics; Lee, K.S., Metcalf, et al., (1992) Evidence for two phosphonate degradative pathways in Enterobacter Aerogenes, J. Bacteriol., 174:2501-2510.

(2) After growth on 100 mm LB plates containing 100 μg/ml ampicillin,

80 μg ml nethicillin and ImM IPTG, colony lifts were performed using Millipore HATF membrane filters.

(3) The colonies transferred to the filters were lysed with chloroform vapor in 150 mm glass petri dishes.

(4) The filters were transferred to 100 mm glass petri dishes containing a piece of Whatman 3MM filter paper saturated with buffer. a) when testing for galactosidase activity (XGALase), 3MM paper was saturated with Z buffer containing 1 mg/ml XGAL (ChemBridge Corporation). After transferring filter bearing lysed colonies to the glass petri dish, placed dish in oven at 80-85 °C.

b) when testing for glucosidase (XGLUase), 3MM paper was saturated with Z buffer containing 1 mg/ml XGLU. After transferring filter bearing lysed colonies to the glass petri dish, placed dish in oven at 80-85 °C.

(5) 'Positives' were observed as blue spots on the filter membranes. Used the following filter rescue technique to retrieve plasmid from lysed positive colony. Used pasteur pipette (or glass capillary tube) to core blue spots on the filter membrane. Placed the small filter disk in an Eppendorf tube containing 20 μl water. Incubated the Eppendorf tube at 75 C for 5 minutes followed by vortexing to elute plasmid DNA off filter. This DNA was transformed into electrocompetent E. coli cells DH10B for Thermatoga maritima MSB8-6G, Staphylothermus marinus F1-12G, Thermococcus AEDII12RA-18B/G, Thermococcus chitonophagus GC74-22G, Ml 1T1 and OC1/4V. Electrocompetent BW 14893 F'kanlA E. coli were used for Thermococcus 9N2-31B/G, and Pyrococcus furiosus VC1-7G1. Repeated filter-lift assay on transformation plates to identify 'positives'. Return transformation plates to 37 GC incubator after filter lift to regenerate colonies. Inoculate 3 ml LB liquid containing 100 μg/ml ampicillin with repurified positives and incubate at 37°C overnight. Isolate plasmid DNA from these cultures and sequence plasmid insert. In some instances where the plates used for the initial colony lifts contained non-confluent colonies, a specific colony corresponding to a blue spot on the filter could be identified on a regenerated plate and repurified directly, instead of using the filter rescue technique.

[0076] Another example of such an assay is a variation of the high temperature filter assay wherein colony-laden filters are heat-killed at different temperatures (for example, 105EC for 20 minutes) to monitor thermostability. The 3MM paper is saturated with different buffers (i.e., 100 mM NaCl, 5 mM MgCl₂, 100 mM Tris-Cl (pH 9.5)) to determine enzyme activity under different buffer conditions.

[0077] A β-glucosidase assay may also be employed, wherein GlcpβNp is used as an artificial substrate (aryl-β-glucosidase). The increase in absorbance at 405 nm as a result of p-nitrophenol (pNp) liberation was followed on a Hitachi U-l 100 spectrophotometer, equipped with a thermostatted cuvette holder. The assays may be performed at 80°C or 90°C in closed 1-ml quartz cuvette. A standard reaction mixture contains 150 mM trisodium substrate, pH 5.0 (at 80°C), and 0.95 mM pNp derivative pNp = 0.561 mM-1 cm-1). The reaction mixture is allowed to reach the desired temperature, after which the reaction is started by injecting an appropriate amount of enzyme (1.06 ml final volume).

[0078] 1 U β-glucosidase activity is defined as that amount required to catalyze the formation of 1.0 Φmol pNp/min. D-cellobiose may also be used as a substrate.

[0079] An ONPG assay for β-galactosidase activity is described by Miller, J.H. (1992) A Short Course in Bacterial Genetics and Mill, J.H. (1992) Experiments in Molecular Genetics, the contents of which are hereby incoφorated by reference in their entirety.

[0080] A quantitative fluorometric assay for β-galactosidase specific activity is described by : Youngman P., (1987) Plasmid Vectors for Recovering and Exploiting Tn917 Transpositions in Bacillus and other Gram-Positive Bacteria. In Plasmids: A Practical approach (ed. K. Hardy) pp 79-103. IRL Press, Oxford. A description of the procedure can be found in Miller (1992) p. 75-77, the contents of which are incoφorated by reference herein in their entirety.

[0081] The polynucleotides of the present invention may be in the form of DNA which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequences which encodes the mature enzymes may be identical to the coding sequences shown in Figures 1-8 (SEQ ID NOS: 1-14 and 57-60) or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature enzymes as the DNA of Figures 1-18 (SEQ ID NOS: 1-14 and 57-60).

[0082] The polynucleotide which encodes for the mature enzyme of Figures 1-18 (SEQ ID NOS: 15-28 and 61-64) may include, but is not limited to: only the coding sequence for the mature enzyme; the coding sequence for the mature enzyme and additional coding sequence such as a leader sequence or a proprotein sequence; the coding sequence for the mature enzyme (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature enzyme.

[0083] Thus, the term "polynucleotide encoding an enzyme (protein)" encompasses a polynucleotide which includes only coding sequence for the enzyme as well as a polynucleotide which includes additional coding and/or non-coding sequence.

[0084] The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the enzymes having the deduced amino acid sequences of Figures 1-18 (SEQ ID NOS: 15-28 and 61-64). The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-narurally occurring variant of the polynucleotide.

[0085] Thus, the present invention includes polynucleotides encoding the same mature enzymes as shown in Figures 1-18 (SEQ ID NOS: 15-28 and 61-64) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the enzymes of Figures 1-18 (SEQ ID NOS: 15-28 and 61-64). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

[0086] As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in Figures 1-18 (SEQ ID NOS: 1-14 and 57-60). As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded enzyme.

[0087] Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA or a genomic library to isolate the full length DNA and to isolate other DNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 10, preferably at least 15, and even more preferably at least 30 bases and may contain, for example, at least 50 or more bases. The probe may also be used to identify a DNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of genomic DNA to determine which members of the library the probe hybridizes to.

[0088] The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode enzymes which either retain substantially the same biological function or activity as the mature enzyme encoded by the DNA of Figures 1-18 (SEQ ID NOS: 1-14 and 57-60).

[0089] Alternatively, the polynucleotide may have at least 15 bases, preferably at least 30 bases, and more preferably at least 50 bases which hybridize to any part of a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotides of SEQ ID NOS: 1-14 and 57-60, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

[0090] Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% identity and more preferably at least a 95% identity to a polynucleotide which encodes the enzymes of SEQ ID NOS: 15-28 and 61-64 as well as fragments thereof, which fragments have at least 15 bases, preferably at least 30 bases and most preferably at least 50 bases, which fragments are at least 90% identical, preferably at least 95% identical and most preferably at least 97% identical under stringent conditions to any portion of a polynucleotide of the present invention.

[0091] The present invention further relates to enzymes which have the deduced amino acid sequences of Figures 1-18 (SEQ ID NOS: 15-28 and 61-64) as well as fragments, analogs and derivatives of such enzyme.

[0092] The terms "fragment," "derivative" and "analog" when referring to the enzymes of Figures 1-18 (SEQ ID NOS: 15-28 and 61-64) means enzymes which retain essentially the same biological function or activity as such enzymes. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature enzyme. [0093] The enzymes of the present invention may be a recombinant enzyme, a natural enzyme or a synthetic enzyme, preferably a recombinant enzyme.

[0094] The fragment, derivative or analog of the enzymes of Figures 1-18 (SEQ ID NOS: 15-28 and 61-64) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature enzyme is fused with another compound, such as a compound to increase the half-life of the enzyme (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature enzyme, such as a leader or secretory sequence or a sequence which is employed for purification of the mature enzyme or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

[0100] The enzymes and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

[0101] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or enzyme present in a living animal is not isolated, but the same polynucleotide or enzyme, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or enzymes could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

[0102] The enzymes of the present invention include the enzymes of SEQ ID NOS: 15-28 and 61-64 (in particular the mature enzyme) as well as enzymes which have at least 70% similarity (preferably at least 70% identity) to the enzymes of SEQ ID NOS: 15-28 and 61-64 and more preferably at least 90% similarity (more preferably at least 90% identity) to the enzymes of SEQ ID NOS: 15-28 and 61-64 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the enzymes of SEQ ID NOS: 15-28 and 61-64 and also include portions of such enzymes with such portion of the enzyme generally containing at least 30 amino acids and more preferably at least 50 amino acids.

[0103] As known in the art "similarity" between two enzymes is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one enzyme to the sequence of a second enzyme.

[0104] A variant, i.e. a "fragment", "analog" or "derivative" polypeptide, and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.

[0105] Among preferred variants are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

[0106] Most highly preferred are variants which retain the same biological function and activity as the reference polypeptide from which it varies.

[0107] Fragments or portions of the enzymes of the present invention may be employed for producing the corresponding full-length enzyme by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length enzymes. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

[0108] The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of enzymes of the invention by recombinant techniques.

[0109] Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0110] The polynucleotides of the present invention may be employed for producing enzymes by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing an enzyme. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

[0111] The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art. [0112] The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp_, the phage lambda P_L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

[0113] In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

[0114] The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

[0115] As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomvces. Bacillus subtilis; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

[0116] More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pDIO, psiX174, pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

[0117] Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include IacI, lacZ, T3, T7, gpt, lambda P_R, P_L and tφ. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

[0118] In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, ( 1986)).

[0119] The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the enzymes of the invention can be synthetically produced by conventional peptide synthesizers. [0120] Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incoφorated by reference.

[0121] Transcription of the DNA encoding the enzymes of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0122] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated enzyme. Optionally, the heterologous sequence can encode a fusion enzyme including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. [0123] Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella rvphimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

[0124] As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

[0125] Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

[0126] Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

[0127] Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art. [0128] Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23: 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C 127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

[0129] The enzyme can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

[0130] The enzymes of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the enzymes of the present invention may be glycosylated or may be non-glycosylated. Enzymes of the invention may or may not also include an initial methionine amino acid residue.

[0131] β-galactosidase hydrolyzes lactose to galactose and glucose. Accordingly, the OC1/4V, 9N2-31B/G, AEDII12RA-18B/G and F1-12G enzymes may be employed in the food processing industry for the production of low lactose content milk and for the production of galactose or glucose from lactose contained in whey obtained in a large amount as a by-product in the production of cheese. Generally, it is desired that enzymes used in food processing, such as the aforementioned β-galactosidases, be stable at elevated temperatures to help prevent microbial contamination.

[0132] These enzymes may also be employed in the pharmaceutical industry. The enzymes are used to treat intolerance to lactose. In this case, a thermostable enzyme is desired, as well. Thermostable β-galactosidases also have uses in diagnostic applications, where they are employed as reporter molecules.

[0133] Glucosidases act on soluble cellooligosaccharides from the non-reducing end to give glucose as the sole product. Glucanases (endo- and exo-) act in the depolymerization of cellulose, generating more non-reducing ends (endo-glucanases, for instance, act on internal linkages yielding cellobiose, glucose and cellooligosaccharides as products), β-glucosidases are used in applications where glucose is the desired product. Accordingly, Ml 1TL, F1-12G, GC74-22G, MSB8-6G , OC1/4V, VC1-7G1, 9N2-31B/G and AEDII12RA18B/G may be employed in a wide variety of industrial applications, including in corn wet milling for the separation of starch and gluten, in the fruit industry for clarification and equipment maintenance, in baking for viscosity reduction, in the textile industry for the processing of blue jeans, and in the detergent industry as an additive. Such enzymes, particularly glucanases, are especially useful in animal feeds. For these and other applications, thermostable enzymes are desirable.

[0134] Antibodies generated against the enzymes corresponding to a sequence of the present invention can be obtained by direct injection of the enzymes into an animal or by administering the enzymes to an animal, preferably a nonhuman. The antibody so obtained will then bind the enzymes itself. In this manner, even a sequence encoding only a fragment of the enzymes can be used to generate antibodies binding the whole native enzymes. Such antibodies can then be used to isolate the enzyme from cells expressing that enzyme. [0135] For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0136] Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic enzyme products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic enzyme products of this invention.

[0137] Antibodies generated against the enzyme of the present invention may be used in screening for similar enzymes from other organisms and samples. Such screening techniques are known in the art, for example, one such screening assay is described in "Methods for Measuring Cellulase Activities", Methods in enzymology, Vol 160, pp. 87- 1 16, which is hereby incoφorated by reference in its entirety.

[0138] The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

[0139] In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

[0140] "Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

[0141] "Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical puφoses, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the puφose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

[0142] Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).

[0143] "Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

[0144] "Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated. [0145] The invention also provides a biodegradable enzyme delivery matrix which readily releases the enzyme in aqueous media, such as, for example, the digestive fluid of an animal. In accordance with the invention, the matrix is provided in the form of discrete plural particles of matrix.

[0146] In yet another embodiment, the invention provides an enzyme delivery matrix which releases active enzyme in aqueous media. The invention enzyme delivery matrix is prepared from a grain-based substrate, which substrate may comprise or be derived from any suitable grain. While grains such as wheat, corn, soy, sorghum, alfalfa, barley, and the like, are suitable for use in conjunction with the invention, the grain-based substrate preferably is a corn-based substrate. The substrate may be derived from any suitable part of the grain. Most preferably, the grain-based substrate is a grain germ approved for animal feed use, such as corn germ that is obtained in a wet or dry milling process.

[0147] The grain germ preferably comprises spent germ, which is grain germ from which oil has been expelled, such as by pressing or hexane or other solvent extraction. Alternatively, the grain germ is expeller extracted, that is, the oil has been removed by pressing.

[0148] The enzyme delivery matrix of the invention is in the form of discrete plural particles. In accordance with a preferred embodiment of the invention, the particles are in the form of compressed or compacted particles, i.e., in the form of granules. By "granules" is meant particles that are compressed or compacted, such as by a pelletizing, extrusion, or similar compacting to remove water from the matrix. Such compression or compacting of the particles also promotes intraparticle cohesion of the particles. For example, the granules can be prepared by pelletizing the grain-based substrate in a pellet mill. The pellets prepared thereby are ground or crumbled to a granule size suitable for use as an adjuvant in animal feed. Preferably, the enzyme delivery matrix is in the form of granules having a granule size ranging from about 4 to about 400 mesh (USS); more preferably, about 8 to about 80 mesh; and most preferably about 14 to about 20 mesh. If the grain germ is spent via solvent extraction, use of a lubricity agent such as corn oil may be necessary in the pelletizer, but such a lubricity agent ordinarily is not necessary if the germ is expeller extracted. In other embodiments of the invention, the matrix is prepared by other compacting or compressing processes such as, for example, by extrusion of the grain-based substrate through a die and grinding of the extrudate to a suitable granule size.

[0149] The enzyme delivery matrix preferably further includes a polysaccharide component as a cohesiveness agent to enhance the cohesiveness of the matrix granules. The cohesiveness agent is believed to provide additional hydroxyl groups, which enhance the bonding between grain proteins within the matrix granule. It is further believed that the additional hydroxyl groups so function by enhancing the hydrogen bonding of proteins to starch and to other proteins. The cohesiveness agent may be present in any amount suitable to enhance the cohesiveness of the granules of the enzyme delivery matrix.

[0150] In accordance with the invention, the cohesiveness agent preferably is a polysaccharide. Any suitable polysaccharide may be used in conjunction with the invention and thus, for example, the cohesiveness agent may comprise one or more polysaccharides such as dextrins, maltodextrins, starches, flours, cellulosics, hemicellulosics, and the like. Preferably, the cohesiveness agent comprises a starch, and most preferably, the cohesiveness agent comprises a com starch. The preferred percentage of grain germ and cohesiveness agent in the matrix (not including the enzyme) is 78% com germ meal and 20% by weight of com starch.

[0151] Non-gelatinized, unmodified starches are especially suitable for use as cohesiveness agents in conjunction with the invention. When unmodified starches are employed, it is believed that such starches may contribute to a small extent to inteφarticle adhesion in the matrix. [0152] Because the enzyme-releasing matrix of the invention is made from biodegradable materials, the matrix may be subject to spoilage, such as by molding. To prevent or inhibit such molding, the matrix may include a mold inhibitor, which may be present in any amount sufficient to inhibit the molding of the enzyme-releasing matrix. Any suitable mold inhibitor may be employed in conjunction with the invention. The mold inhibitor preferably is selected from among the propionate salts, and most preferably is selected from the group consisting of sodium propionate and calcium propionate. Thus, the invention enzyme delivery matrices provide the advantage of a stable formulation which does not require refrigeration.

[0153] Preferably, the grain-based component of the matrix is present in the enzyme- delivery matrix in a dry basis amount of at least about 45% by weight of the matrix. More preferably, the grain-based component is present in an amount ranging from about 50% to about 85% by weight; even more an amount ranging from about 60% to about 75% by weight; and most preferably, about 67.5% by weight. The remaining dry components of the enzyme-releasing matrix preferably include a cohesiveness agent, the enzyme, and, when used, a mold inhibitor. The cohesiveness agent may be present in a dry basis amount ranging up to about 45% by weight of the matrix; preferably, an amount ranging from about 3% to about 35% by weight; more preferably, an amount ranging from about 10% to about 25% by weight; and most preferably, inclusive of the supplemental cohesiveness agent, an amount of about 18% by weight, of which about 16.2% by weight comprises the cohesiveness agent.

[0154] To maintain the cohesiveness and soφtion capacity of the enzyme-releasing matrix, the matrix preferably has a moisture content sufficiently high to prevent the granules of the enzyme delivery matrix from becoming friable and sufficiently low to allow additional moisture to be absorbed. Preferably, the moisture content is less than about 30%. More preferably, the moisture content ranges from about 5% to about 25%; even more preferably, the moisture content ranges from about 10% to about 20%; even more preferably, the moisture content ranges from about 12% to about 16%, and most preferably the moisture content is about 14.5%. It is contemplated that some of the moisture in the enzyme-releasing matrix will be present originally in the enzyme, the grain-based substrate and/or cohesiveness agent.

[0155] The enzyme contained in the invention enzyme delivery matrix is a thermostable enzyme so as to resist inactivation of the enzyme during manufacture where steam may be employed. Preferred thermostable enzymes to be included when the enzyme delivery matrix is intended to be used as an adjuvant for animal feed are thermostable glucanases (glucanases assist in the digestion of cellulose-containing feed ingredients), such as an endoglucanase or an exoglucanase, or a combination thereof. During digestion of feed containing the invention enzyme delivery matrix, aqueous digestive fluids will cause release of the active enzyme. In a few cases the thermostability of endoglucanases have been determined, one of the most studied species is Clostridium thermocellum which have proven stable up to 80° C. Thermoanaerobacter cellulyticus produces at least two endoglucanases with stability up to 80°C. The OC1/V endoglucanase derived from Clostridium thermocellum (33GP1), an endo-l,4-β-endoglucanase, is thermostable up to about 82 ° C or higher and is the glucase preferred for use in the invention enzyme matrix. The amino acid sequence of 33GP1 (SEQ ID NO:27) and the nucleotide sequence encoding 33GP1 (SEQ ID NO: 13) are shown in Figures 13A and B. Another preferred endoglucanase obtained from Dictyoglomus exhibits optimum activity as a temperature above 85°C as disclosed in U.S. Patent No. 6,043,075, which is incoφorated herein by reference in its entirety.

[0156] In alternative embodiments, the invention enzyme delivery matrices can be used to stably incoφorate other types of thermostable enzymes for release under any type of aqueous conditions. For example, Aspergillus ficuum (A.ficuum) produces a phytase which has a particularly high specific activity and thermostability. Phytases, more properly referred to as myo-inositol hexaphosphate phosphohydrolases, are a family of enzymes which catalyze the step-wise removal of inorganic orthophosphate from phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate). The economic interest in phytase is due to its ability to increase the bio-availability of inorganic phosphorous in phytate-containing non-ruminant animal feeds. Currently, feed for non-ruminant animals must be supplemented with inorganic phosphorous because these animal cannot utilize the phosphorous present as phytate. Mixture of the invention enzyme matrix with traditional meal diets, such as the corn-soybean meal diets, typically fed to swine is believed to significantly improve the utilization of phosphorous from such feeds and also to decrease the amount of phosphorous excreted from swine by up to 50% (See U.S. Patent 5,900,525, which is incoφorated hereby by reference in its entirety.)

[0157] In addition, since the enzyme is incoφorated within the body of the matrix, it is possible to coat the particles with any type of coating that is suitable to the intended puφose without risk that the enzyme will be inactivated by the coating.

[0158] Because the enzyme-releasing matrix of the invention is used to release the enzyme in an aqueous environment, the matrix should have a soφtion capacity sufficient to allow the enzyme-releasing matrix to so function. The soφtion capacity of the preferred matrices of the invention typically is at least about 0.04 ml water per gram of matrix, and more typically ranges from about 0.04 to about 2.4 ml/g; more typically, the soφtion capacity is from about 1.0 to about 1.4 ml/g. The soφtion capacity is determined per gram of matrix, and is based on the soφtion of deionized water.

[0159] Other desired properties include biodegradability and digestibility, for example when included as an adjuvant in an animal feed.

[0160] The invention is also directed towards a process for preparing an enzyme- releasing matrix. In accordance with the invention, the process comprises providing discrete plural particles of a grain-based substrate in a particle size suitable for use as an enzyme-releasing matrix, as previously described. Preferably, the process includes compacting or compressing the particles of enzyme-releasing matrix into granules, which most preferably is accomplished by pelletizing. The mold inhibitor and cohesiveness agent, when used, can be added at any suitable time, and preferably are mixed with the grain-based substrate in the desired proportions prior to pelletizing of the grain-based substrate. Moisture content in the pellet mill feed preferably is in the ranges set forth above with respect to the moisture content in the finished product, and preferably is about 14-15%. Preferably, moisture is added to the feedstock in the form of an aqueous preparation of the enzyme to bring the feedstock to this moisture content. The temperature in the pellet mill preferably is brought to about 82° C. with steam. The pellet mill may be operated under any conditions that impart sufficient work to the feedstock to provide pellets. In one preferred embodiment, the pellet mill is operated with a 1/8 in. by 2 in. die at 100 lb./min. pressure at 82°C. to provide pellets, which then are crumbled in a pellet mill crumbier to provide discrete plural particles having a particle size capable of passing through an 8 mesh screen but being retained on a 20 mesh screen.

[0161] The following examples demonstrate various embodiments of the invention, but should not be construed as limiting the invention in scope.

[0162] Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Example 1 Bacterial Expression and Purification of Glycosidase Enzymes

[0163] DNA encoding the enzymes of the present invention, SEQ ID NOS: 1-14 and 57-60 were initially amplified from a pBluescript vector containing the DNA by the PCR technique using the primers noted herein. The amplified sequences were then inserted into the respective PQE vector listed beneath the primer sequences, and the enzyme was expressed according to the protocols set forth herein. The 5' and 3' primer sequences for the respective genes are as follows:

[0164] Thermococcus AEDII 12RA - 18B/G

5' CCGAGAATTCATTAAAGAGGAGAAATTAACTATGGTGAATGCTATGATTGTC 3' (SEQ ID NO:29) 3' CGGAAGATCTTCATAGCTCCGGAAGCCCATA 5' (SEQ ID NO:30)

Vector: pQE12; and contains the following restriction enzyme sites 5' EcoRI and 3' Big II.

[0165] OC1/4V-33B/G

5' CCGAGAATTCATTAAAGAGGAGAAATTAACTATGATAAGAAGGTCCGATTTTCC 3'

(SEQ lD NO:31)

3' CGGAAGATCTTTAAGATTTTAGAAATTCCTT 5' (SEQ ID NO:32)

Vector: pQE12; and contains the following restriction enzyme sites 5' EcoRI and 3' Bgl II.

[01 6] Thermococcus 9N2 - 31 B/G

5' CCGAGAATTCATTAAAGAGGAGAAATTAACTATGCTACCAGAAGGCTTTCTC 3"

(SEQ ID NO:33)

3' CGGAGGTACCTCACCCAAGTCCGAACTTCTC 5' (SEQ ID NO:34)

Vector: pQE30; and contains the following restriction enzyme sites 5' EcoRI and 3' KpnI.

[0167] Staphylothermus marinus FI - 12G

5' CCGAGAATTCATTAAAGAGGAGAAATTAACTATGATAAGGTTTCCTGATTAT 3'

(SEQ ID NO:35)

3' CGGAAGATCTTTATTCGAGGTTCTTTAATCC 5' (SEQ ID NO:36)

Vector: pQE12; and contains the following restriction enzyme sites 5' EcoRI and 3' Bgl II.

[0168] Thermococcus chitonophagus GC74 - 22G

5' CCGAGAATTCATTCATTAAAGAGGAGAAATTAACTATGCTTCCAGGAGAACTTTCTC 3'

(SEQ ID NO:37)

3' CGGAGGATCCCTACCCCTCCTCTAAGATCTC 5' (SEQ ID NO:38) Vector: pQE12; and contains the following restriction enzyme sites 5' EcoRI and 3' BamHI.

[0169] M11TL

5' AATAATCTAGAGCATGCAATTCCCCAAAGACTTCATGATAG 3' (SEQ ID NO:39) 3" AATAAAAGCTTACTGGATCAGTGTAAGATGCT 5' (SEQ ID NO:40)

Vector: pQE70; and contains the following restriction enzyme sites 5' SphI and 3' Hind III.

[0170] Thermotoga maritima MSB8-6G

5' CCGACAATTGATTAAAGAGGAGAAATTAACTATGGAAAGGATCGATGAAATT 3' (SEQ ID NO:41) 3' CGGAGGTACCTCATGGTTTGAATCTCTTCTC 5' (SEQ ID NO:42)

Vector: pQE12; and contains the following restriction enzyme sites 5' EcoRI and 3' KpnI.

[0171] Pyrococcus furiosus VC1 - 7G1

5' CCGACAATTGATTAAAGAGGAGAAATTAACTATGTTCCCTGAAAAGTTCCTT 3' (SEQ ID NO:43) 3' CGGAGGTACCTCATCCCCTCAGCAATTCCTC 5' (SEQ ID NO:44)

Vector: pQE12; and contains the following restriction enzyme sites 5' EcoRI and 3' Kpn I.

[0172] Bankia gouldi endoglucanase (37GP 1 )

5' AATAAGGATCCGTTTAGCGACGCTCGC 3' (SEQ ID NO:45)

3' AATAAAAGCTTCCGGGTTGTACAGCGGTAATAGGC 5' (SEQ IDNO:46)

Vector: pQE52; and contains the following restriction enzyme sites 5' Bam HI and 3' Hind III.

[0173] Thermotoga maritima α-galactosidase (6GC2)

5' TTTATTGAATTCATTAAAGAGGAGAAATTAACTATGATCTGTGTGGAAATATTCGGAAAG 3'

(SEQ ID NO:47)

3' TCTATAAAGCTTTCATTCTCTCTCACCCTCTTCGTAGAAG 5' (SEQ ID NO:48)

Vector: pQET; and contains the following restriction enzyme sites 5' EcoRI and 3' Hind III. [0174] Thermotoga maritima β-mannanase (6GP2)

5' TTTATTCAATTGATTAAAGAGGAGAAATTAACTATGGGGATTGGTGGCGACGAC 3' (SEQ ID NO:49)

3' TTTATTAAGCTTATCTTTTCATATTCACATACCTCC 5' (SEQ ID NO:50)

Vector: pQEt; and contains the following restriction enzyme sites 5' Hind III and 3' EcoRI.

[0175] AEPII la β-mannanase (63GB 1)

5' TTTATTGAATTCATTAAAGAGGAGAAATTAACTATGCTACCAGAAGAGTTCCTATGGGGC 3'

(SEQ ID NO:51 )

3' TTTATTAAGCTTCTCATCAACGGCTATGGTCTTCATTTC 5' (SEQ ID NO:52)

Vector: pQEt; and contains the following restriction enzyme sites 5' Hind III and 3' EcoRI.

[0176] OC1/4V endoglucanase (33GP1)

5*

AAAAAACAATTGAATTCATTAAAGAGGAGAAATTAACTATGGTAGAAAGACACTTCAGATATGTTCTT

3" (SEQ ID NO:53)

3" TTTTTCGGATCCAATrCTTCATTTACTCTTTGCCTG 5' (SEQ ID NO:54)

Vector: pQEt; and contains the following restriction enzyme sites 5' BamHI and 3' EcoRI.

[0177] Thermotoga maritima pullalanase (6GP3)

5' TTTTGGAATTCATTAAAGAGGAGAAATTAACTATGGAACTGATCATAGAAGGTTAC 3'

(SEQ ID NO:55)

3' ATAAGAAGCTTTTCACTCTCTGTACAGAACGTACGC 5' (SEQ ID NO:56)

Vector: pQEt; and contains the following restriction enzyme sites 5' EcoRI and 3' Hind III.

[0178] The restriction enzyme sites indicated correspond to the restriction enzyme sites on the bacterial expression vector indicated for the respective gene (Qiagen, Inc. Chatsworth, CA). The pQE vector encodes antibiotic resistance (Amp¹), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites.

[0179] The pQE vector was digested with the restriction enzymes indicated. The amplified sequences were ligated into the respective pQE vector and inserted in frame with the sequence encoding for the RBS. The ligation mixture was then used to transform the E. coli strain M15/pREP4 (Qiagen, Inc.) by electroporation. M15/pREP4 contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kan^r). Transformants were identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown overnight (O N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture was used to inoculate a large culture at a ratio of 1 : 100 to 1 :250. The cells were grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final concentration of 1 mM. IPTG induces by inactivating the lad repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation.

[0180] The primer sequences set out above may also be employed to isolate the target gene from the deposited material by hybridization techniques described above.

Example 2 Isolation of A Selected Clone From the Deposited genomic clones

[0181] A clone is isolated directly by screening the deposited material using the oligonucleotide primers set forth in Example 1 for the particular gene desired to be isolated. The specific oligonucleotides are synthesized using an Applied Biosystems DNA synthesizer. The oligonucleotides are labeled with ³²P-ATP using T4 polynucleotide kinase and purified according to a standard protocol (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY, 1982). The deposited clones in the pBluescript vectors may be employed to transform bacterial hosts which are then plated on 1.5% agar plates to the density of 20,000- 50,000 pfu/150 mm plate. These plates are screened using Nylon membranes according to the standard screening protocol (Stratagene, 1993). Specifically, the Nylon membrane with denatured and fixed DNA is prehybridized in 6 x SSC, 20 mM NaH₂PO₄, 0.4%SDS, 5 x Denhardt's 500 μg/ml denatured, sonicated salmon sperm DNA; and 6 x SSC, 0.1% SDS. After one hour of prehybridization, the membrane is hybridized with hybridization buffer 6xSSC, 20 mM NaH₂PO₄, 0.4%SDS, 500 ug/ml denatured, sonicated salmon sperm DNA with lxlO⁶ cpm/ml ³²P-probe overnight at 42EC. The membrane is washed at 45-50EC with washing buffer 6 x SSC, 0.1% SDS for 20-30 minutes dried and exposed to Kodak X-ray film overnight. Positive clones are isolated and purified by secondary and tertiary screening. The purified clone is sequenced to verify its identity to the primer sequence.

[0182] Once the clone is isolated, the two oligonucleotide primers corresponding to the gene of interest are used to amplify the gene from the deposited material. A polymerase chain reaction is carried out in 25 μl of reaction mixture with 0.5 ug of the DNA of the gene of interest. The reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaruration at 94 °C for 1 min; annealing at 55EC for 1 min; elongation at 72EC for 1 min) are performed with the Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the gene of interest by subcloning and sequencing the DNA product. The ends of the newly purified genes are nucleotide sequenced to identify full length sequences. Complete sequencing of full length genes is then performed by Exonuclease III digestion or primer walking.

Example 3 Screening for Galactosidase Activity

[0183] Screening procedures for α-galactosidase protein activity may be assayed for as follows:

[0184] Substrate plates were provided by a standard plating procedure. Dilute XL 1 -Blue MRF E coli host of (Stratagene Cloning Systems, La Jolla, CA) to O.D.βoo = 1.0 with NZY media. In 15 ml tubes, inoculate 200 μl diluted host cells with phage. Mix gently and incubate tubes at 37 °C for 15 min. Add approximately 3.5 ml LB top agarose (0.7%) containing ImM IPTG to each tube and pour onto all NYZ plate surface. Allow to cool and incubate at 37 °C overnight. The assay plates are obtained as substrate p-Nitrophenyl α-galactosidase (Sigma) (200 mg/100 ml) (100 mM NaCl, 100 mM Potassium-Phosphate) 1% (w/v) agarose. The plaques are overlayed with nitrocellulose and incubated at 4 °C for 30 minutes whereupon the nitrocellulose is removed and overlayed onto the substrate plates. The substrate plates are then incubated at 70 °C for 20 minutes.

Example 4 Screening of Clones for Mannanase Activity

[0185] A solid phase screening assay was utilized as a primary screening method to test clones for β-mannanase activity.

[0186] A culture solution of the Y 1090-E. coli host strain (Stratagene Cloning Systems, La Jolla, CA) was diluted to O.D.₆oo=l 0 with NZY media. The amplified library from Thermotoga maritima lambda gtll library was diluted in SM (phage dilution buffer): 5 x 10⁷ pfu μl diluted 1 :1000 then 1 :100 to 5 x 10² pfu/μl. Then 8 μl of phage dilution (5 x 10 pfu μl) was plated in 200 μl host cells. They were then incubated in 15 ml tubes at 37 °C for 15 minutes.

[0187] Approximately 4 ml of molten, LB top agarose (0.7%) at approximately 52 °C was added to each tube and the mixture was poured onto the surface of LB agar plates.

The agar plates were then incubated at 37 °C for five hours. The plates were replicated and induced with 10 mM IPTG-soaked Duralon-UV™ nylon membranes (Stratagene Cloning Systems, La Jolla, CA) overnight. The nylon membranes and plates were marked with a needle to keep their orientation and the nylon membranes were then removed and stored at 4 °C.

[0188] An Azo-galactomannan overlay was applied to the LB plates containing the lambda plaques. The overlay contains 1% agarose, 50 mM potassium-phosphate buffer pH 7, 0.4% Azocarob-galactomannan. (Megazyme, Australia). The plates were incubated at 72 °C. The Azocarob-galactomannan treated plates were observed after 4 hours then returned to incubation overnight. Putative positives were identified by clearing zones on the Azocarob-galactomannan plates. Two positive clones were observed.

[0189] The nylon membranes referred to above, which correspond to the positive clones were retrieved, oriented over the plate and the portions matching the locations of the clearing zones for positive clones wre cut out. Phage was eluted from the membrane cut-out portions by soaking the individual portions in 500 μl SM (phage dilution buffer) and 25 μl CHC1₃.

Example 5 Screening of Clones for Mannosidase Activity

[0190] A solid phase screening assay was utilized as a primary screening method to test clones for β-mannosidase activity.

[0191] A culture solution of the Y 1090-E. coli host strain (Stratagene Cloning Systems, La Jolla, CA) was diluted to O.D.6oo=l-0 with NZY media. The amplified library from AEPII la lambda gtll library was diluted in SM (phage dilution buffer): 5 x 10⁷ pfu/μl diluted 1:1000 then 1 :100 to 5 x 10² pfu μl. Then 8 μl of phage dilution (5 x 10² pfu/μl) was plated in 200 μl host cells. They were then incubated in 15 ml tubes at 37 °C for 15 minutes.

[0192] Approximately 4 ml of molten, LB top agarose (0.7%) at approximately 52 °C was added to each tube and the mixture was poured onto the surface of LB agar plates. The agar plates were then incubated at 37 °C for five hours. The plates were replicated and induced with 10 mM IPTG-soaked Duralon-UV™ nylon membranes (Stratagene Cloning Systems, La Jolla, CA) overnight. The nylon membranes and plates were marked with a needle to keep their orientation and the nylon membranes were then removed and stored at 4 °C.

[0193] A p-nitrophenyl-^β-D-manno-pyranoside overlay was applied to the LB plates containing the lambda plaques. The overlay contains 1% agarose, 50 mM potassium- phosphate buffer pH 7, 0.4% p-nitrophenyl-^β-D-manno-pyranoside. (Megazyme,

Australia). The plates were incubated at 72 °C. The p-nitrophenyl-β-D-manno- pyranoside treated plates were observed after 4 hours then returned to incubation overnight. Putative positives were identified by clearing zones on the p-nitrophenyl-β- D-manno-pyranoside plates. Two positive clones were observed.

[0194] The nylon membranes referred to above, which correspond to the positive clones were retrieved, oriented over the plate and the portions matching the locations of the clearing zones for positive clones wre cut out. Phage was eluted from the membrane cut-out portions by soaking the individual portions in 500 μl SM (phage dilution buffer) and 25 μl CHClj.

Example 6 Screening for Pullulanase Activity

[0195] Screening procedures for pullulanase protein activity may be assayed for as follows:

[0196] Substrate plates were provided by a standard plating procedure. Host cells are diluted to O.D.600 = 1.0 with NZY or appropriate media. In 15 ml tubes, inoculate 200 Φl diluted host cells with phage. Mix gently and incubate tubes at 37 °C for 15 min. Add approximately 3.5 ml LB top agarose (0.7%) is added to each tube and the mixture is plated, allowed to cool, and incubated at 37°C for about 28 hours. Overlays of 4.5 mis of the following substrate are poured:

100 ml total volume

0.5g Red Pullulan Red (Megazyme, Australia) l.Og Agarose

5ml Buffer (Tris-HCL pH 7.2 @ 75 °C)

2ml 5M NaCl

5ml CaCl₂ (100mM)

85ml dH₂O

[0197] Plates are cooled at room temperature, and thenm incubated at 75EC for 2 hours. Positives are observed as showing substrate degradation.

Example 7 Screening for Endoglucanase Activity

[0198] Screening procedures for endoglucanase protein activity may be assayed for as follows:

1. The gene library is plated onto 6 LB/GelRite/0.1 % CMC/NZY agar plates («4,800 plaque forming units/plate) in E.coli host with LB agarose as top agarose. The plates are incubated at 37EC overnight.

2. Plates are chilled at 4EC for one hour.

3. The plates are overlayed with Duralon membranes (Stratagene) at room temperature for one hour and the membranes are oriented and lifted off the plates and stored at 4 °C. 4. The top agarose layer is removed and plates are incubated at 37 °C for about 3 hours.

5. The plate surface is rinsed with NaCl.

6. The plate is stained with 0.1% Congo Red for 15 minutes.

7. The plate is destained with 1M NaCl.

8. The putative positives identified on plate are isolated from the Duralon membrane (positives are identified by clearing zones around clones). The phage is eluted from the membrane by incubating in 500μl SM + 25μl CHC1₃ to elute.

9. Insert DNA is subcloned into any appropriate cloning vector and subclones are reassayed for CMCase activity using the following protocol:

i) Spin 1ml overnight miniprep of clone at maximum speed for 3 minutes.

ii) Decant the supernatant and use it to fill "wells" that have been made in an LB/GelRite/0.1% CMC plate.

iii) Incubate at 37EC for 2 hours.

iv) Stain with 0.1 % Congo Red for 15 minutes.

v) Destain with 1M NaCl for 15 minutes.

vi) Identify positives by clearing zone around clone. Example 8 Preparation of Biodegradable Enzyme Delivery Matrix

[0199] An enzyme-releasing matrix is prepared by pelletizing washed, expeller- extracted spent com germ, a cohesiveness agent, and a supplemental cohesiveness agent in a California Pellet Mill, then crumbling the pellets thus formed to form discrete plural particles of an enzyme-releasing matrix.

[0200] To prepare the matrix, com germ meal, com starch were blended simultaneously .in a ratio of 78:20, moisture-adjusted to 15% by weight with the addition 600u/ml of 33GP1 enzyme (an endoglucase). The resulting blend was introduced into a California Pellet Mill. The mixture was extruded through a 1/8 in. by 2 in. die at 100 lb./min. to form pellets, which were then crumbled with a pellet mill crumbier to form discrete plural particles having a particle size capable of passing through an 8 mesh screen but retained on a 20-mesh screen. There was about a 90% recovery of active enzyme with no steam applied in the conditioning section of the pellet mill. The experiment was repeated except that the temperature in the conditioning section of the pellet machine was adjusted to 82° C by adding steam to the conditioning section of the pellet mill. In this embodiment, the enzyme recovered was about 80% active.

[0201] Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

Claims

What is claimed is:

1. An enzyme delivery matrix in the form of discrete plural particles, each of the particles comprising grain germ that is spent of oil and a thermostable enzyme, the particles readily dispersing the glycosidase enzyme contained therein into aqueous media.

2. The enzyme delivery matrix of claim 1 , wherein the thermostable enzyme is a glycosidase.

3. The enzyme delivery matrix of claim 2, wherein the thermostable glycosidase is an endoglucanase derived from Clostridium thermocellum.

4. The enzyme delivery matrix of claim 2, wherein the thermostable endoglucanase is 33GPl.

5. The enzyme delivery matrix of claim 1 wherein the thermostable enzyme is a phytase.

6. The enzyme delivery matrix of claim 1 , wherein the grain germ is expeller-extracted corn germ.

7. The enzyme delivery matrix of claim 1, wherein the corn germ is present in the delivery matrix in an amount of at least about 45% by weight of the delivery matrix.

8. The enzyme delivery matrix of claim 1 , wherein the com germ is present in the delivery matrix in an amount of from about 50% to about 85% by weight of the delivery matrix.

9. The enzyme delivery matrix of claim 1, wherein the corn germ is present in the delivery matrix in an amount of from about 60% to about 75% by weight of the delivery matrix.

10. The enzyme delivery matrix of claim 1, wherein the delivery matrix has a bulk density of from about 15 to about 45 lbs./cu. ft.

11. The enzyme delivery matrix of claim 1 , wherein the delivery matrix has a bulk density of from about 25 to about 40 lbs./cu. ft.

12. The enzyme delivery matrix of claim 1, wherein the delivery matrix has a moisture content of less than about 30% by weight.

13. The enzyme delivery matrix of claim 12, wherein the delivery matrix has a moisture content of from about 5% to about 25% by weight of the delivery matrix.

14. The enzyme delivery matrix of claim 10, wherein the delivery matrix has a moisture content of from about 10% to about 20% by weight of the delivery matrix.

15. The enzyme delivery matrix of claim 1 , wherein the delivery matrix has a soφtion capacity of at least about 0.04 ml water/g litter.

16. The enzyme delivery matrix of claim 15, wherein the delivery matrix has a soφtion capacity of from about 0.04 to about 2.4 ml water/g litter.

17. The enzyme delivery matrix of claim 1, wherein the particles are in the form of compacted granules, each of the granules further comprising a polysaccharide-containing cohesiveness agent in at least an amount effective to enhance the cohesiveness of the granules.

18. The enzyme delivery matrix of claim 17, wherein the compacted granules are in the form of crumbled pelletized particles.

19. The enzyme delivery matrix of claim 17, wherein the cohesiveness agent is present in an amount ranging from about 3% to about 35% by weight of the delivery matrix.

20. The enzyme delivery matrix of claim 19, wherein the cohesiveness agent is present in an amount ranging from about 10% to about 25% by weight of the delivery matrix.

21. The enzyme delivery matrix of claim 17, wherein the enzyme is embedded within the particles and the particles further comprise a coating.

22. The enzyme delivery matrix of claim 17, wherein the polysaccharide-containing cohesiveness agent includes a starch.

23. The enzyme delivery matrix of claim 26, wherein the starch is co starch.

24. The enzyme delivery matrix of claim 1, wherein the enzyme delivery matrix further comprises a non-toxic mold inhibitor.

25. The enzyme delivery matrix of claim 24, wherein the mold inhibitor is selected from the group consisting of the propionate salts of calcium and sodium.

26. The enzyme delivery matrix of claim 25, wherein the mold inhibitor is present in the delivery matrix in an amount ranging from about 0.02% to about 3.5% by weight of the delivery matrix.

27. The enzyme delivery matrix of claim 1, wherein the grain germ is solvent- extracted corn germ.

28. The enzyme delivery matrix of claim 1, wherein the grain germ is obtained in a wet milling process.

29. The enzyme delivery matrix of claim 1, wherein the grain germ is obtained in a dry milling process.

30. A process preparing an enzyme delivery matrix, said process comprising: providing a mixture comprising a grain germ that is spent of oil, a starch and a thermostable glycosidase enzyme, and compacting the mixture into discrete plural particles to form an enzyme delivery matrix.

31. The process of claim 30, wherein the grain germ is com germ meal.

32. The process of claim 30, wherein the enzyme is derived from Clostridium thermocellum.

33. The process of claim 32, wherein the enzyme is 33GP1.

34. The process of claim 33, wherein the compacting comprises pelletizing the mixture.

35. The process of claim 33, the process further comprising reducing pellets of the matrix formed in the pelletizing to a granule size suitable for use as an enzyme delivery matrix.

36. The process of claim 35, wherein the pelletizing includes application of steam.

37. The process of claim 35, wherein the granule size ranges from about 4 mesh to about 400 mesh.

38. The process of claim 35, wherein the granule size ranges from about 8 mesh to about 80 mesh.