EP0807180A1 - Method for the production of thermostable xylanase and beta-glucosidase from bacteria - Google Patents

Abstract

The invention relates to the production of thermostable enzymes, including xylanase and β-glucosidase. These enzymes are expressed and secreted by bacteria, transformed with the structural genes encoding the enzymes. Expression and secretion of the enzymes occurs in the absence of the signal peptide, typically required for the translocation of secreted proteins across cell membranes.

Description

HT E

METHOD FOR THE PRODUCTION OF

THERMOSTABLE XYLANASE AND

BETA-GLUCOSIDASE FROM BACTERIA

FIELD OF THE INVENTION The invention relates to the production of thermostable enzymes, such as xylanase and β-glucosidase, from recombinant bacteria using expression vectors devoid of a signal sequence.

BACKGROUND Thermostable enzymes (TE) , are important in the manufacture of petrochemicals and in the paper and pulp industry where they are used for the hydrolysis of cellulose and hemicellulose. In nature, thermostable enzymes are produced both intracellularly and extracellularly in small amounts by thermophilic bacteria and fungi. The production of commercial quantities of thermostable enzymes is costly due to the high temperatures needed to culture the microorganisms and the complex purification procedures used for enzyme isolation. Production of thermostable enzymes by easily cultured mesophilic fungi or bacteria would increase the cost effectiveness of production. Additionally, exocellular production of the enzymes would facilitate purification and further contribute to the cost effectiveness of the production process.

One of the most common thermostable enzymes is xylanase which is useful in the conversion of hemicellulose into fermentable carbohydrates. Xylanase production has been reported for many microorganisms including both fungi and bacteria. Typical xylanase producers include the fungi Trichoderma reesei and Trichoder a harzianum as well as bacteria of the genera Bacillus and Cai ocelluin.

Recombinant production of xylanase and other thermostable enzymes by mesophilic fungi and bacteria is known. For example, Schuelein et al. (EP 507723) disclose the expression of an H. insolens xylanase by Asperg±lus oryzae wherein the A. oryzae has been transformed with H. Insole s . Schofield et al. ( Int . J. Biochem. , 25, 609, (1993)) report the expression and purification of a .beta.-l, -xylanase from an E. coli strain carrying a xylanase gene from Caldocellum saccharolytlcum. Additionally, Luthi et al., teach the cloning of genes encoding xylan-degrading enzymes from C. saccharolytlcum (Appl . Environ . Microblol . , 56, 1017, (1990) and the expression and purification of these enzymes form recombinant E. coll . (Appl . Environ . Microblol . 56, 1017, (1990). Okadad, in Microbiol . Appl . . Food Blotechnol . [Proc. Congr. Singapore Soc. Microbiol.] 2nd, Meeting Date 1989, 1-12 Nga et al., Eds. Elsevier:London, UK., review the expression of the xylanase gene of B. pumllus in a variety of organisms including E. coll, B. subtllis and S. cerevisiae . These methods are useful for thermostable enzyme production as they do not require the harsh conditions needed for enzyme production from native sources. However, the desired enzymes are generally produced intracellularly and must be subjected to expensive and time consuming purification processes. A preferred method of thermostable enzyme production would involve the secretion or release of the desired enzyme into the growth media allowing for a simpler and less expensive purification.

In both gram negative and gram positive bacteria most extracellular proteins are synthesized as precursors with a signal peptide covalently fused to the N-Terminus of the mature protein. The signal peptide retards folding of the precursor (Park et al.. Science, 239, 1033 (1988)) and targets the precursor to the export apparatus (Thorn et al., J. Bacteriol . , 170, 5654, (1988)). The signal peptide is cleaved from the mature protein prior to or upon completion of its translocation across the membrane. However, the exact mechanism of precursor translocation, as well as the issues surrounding the cleavage of the signal peptide from the mature protein, are not fully understood. Thus, recombinant production of secreted proteins relies on the inexact science of providing the appropriate signal sequence needed for the secretion of the desired biologically active protein. A limitation of this method is that the signal peptide is sometimes improperly cleaved from the mature protein rendering an incorrectly folded and thus inactive or partially active protein.

In spite of the difficulties associated with the secretion of recombinant proteins, various thermophilic enzymes have been secreted from mesophilic fungi and bacteria. Morosoli et al. (Prog. Biotencnol . , 7, 247, (1992)) disclose the secretion into the growth media of a xylanase from C. alblduy by Saccharomyces sp. and Hamamoto et al. (Agric. Biol . Chem . 51, 3133, (1987)) have demonstrated that recombinantly produced xylanase from Alkalophilic Bacillus is secreted through the outer member of the E. coll host.

Typical examples of the use of Bacillus sp . for the secretion of recombinantly produced thermophilic enzymes are taught by Joergensen et al. (WO 9310248) who disclose the use of genes of thermophilic micro¬ organisms, expressed in Bacillus licheniformis and Bacillus subtllis under the control of a variant B . licheiformis .alpha.-amylase promoter. Another example is seen in Jung et al. (Biotechnol . Lett . 15, 115, (1993) who teach the expression of a Clostridium xylanase gene in B. subtllis under the control of a strong B . subtilis promoter. Additionally, Hirata et al. (U.S. 4861718) teach the secretion of a thermo¬ stable, B . stearothermophilus β-galactosidase in

B. subtilis under the control of a strong B. stearothermophilus promoter.

The above cited methods of recombinant expression and secretion of thermostable enzymes are useful, however all suffer from the need to use a signal peptide for membrane translocation and correct post-secretional processing. As mentioned above, in recombinant systems, the selection of an appropriate signal peptide for the protein to be secreted is unpredictable and further complicates the method of production. A preferred method would allow for the secretion of the desired protein in the absence of the signal protein.

Proteins that can be secreted without the signal peptide while maintaining biological activity are known, but are rare. There are several examples of proteins containing an C-terminal gene extension that appears to function in membrane translocation (Koronakis et al., EMBO, 8, 595, (1989)). Even more rare are proteins which lack any distinguishable export signal (Rubartelli et al., EMBO, 9, 1503, (1990)), and yet are translocated. Genes encoding two proteins, a xylanase and a β-glucosidase of the thermophile C. saccharolytlcum, have been cloned and sequenced and analysis of the predicted amino acid sequence indicate that both of these proteins lack a conventional signal peptide. (Perlman et al., J. Mol . Biol . 167, 391, (1983))

Applicants have made the unexpected discovery that these thermostable enzymes may be produced exocellularly in the absence of the generally required signal peptide. In one embodiment, recombinant mesophilic Bacillus subtilis was used to achieve exocellular enzyme production of both xylanase and β-glucosidase.

Applicants finding is surprising since the lack of recognized signal sequences on these genes would lead to the expectation that the protein products from the expression of theses genes would be intracellular.

However, Applicants have found that proteins are present extracellulary. To date, there is no report in the art of a thermostable enzyme that has been secreted from a recombinant host in the absence of an appropriate signal sequence.

SUMMARY OF THE INVENTION

The present invention provides a method for the exocellular production of thermostable proteins from bacteria. The method for the exocellular production of a thermostable protein from bacteria, preferably Bacillus sp . , comprises the steps of:

(i) creating a DNA fragment comprising (a) a suitable promoter, further comprising transcription and translation initiation sites; and (b) a gene encoding a biologically active thermostable enzyme, wherein said promoter transcription and translation initiation sites are operably linked to the 5' end of the gene; (ii) cloning said DNA fragment into an appropriate transformation vector;

(iii) transforming said bacteria with said vector; and (iv) growing said transformed bacteria under conditions whereby the thermostable protein is expressed.

The invention also concerns a method for making a transformation vector for transforming bacteria, so as to exocellularly produce a thermostable protein, comprising the steps of: (i) creating a DNA fragment comprising

(a) a suitable promoter and

(b) a gene encoding a biologically active thermostable enzyme, wherein said DNA fragment does not contain a signal sequence; and

(ii) cloning said DNA fragment into an appropriate transformation vector. It is further within the scope of the invention to provide transformed SaciiJus subtllis bacteria for the enhanced expression, synthesis and exocellular production of thermostable enzymes, particularly xylanase and β-glucosidase. BRIEF DESCRIPTION OF THE DRAWINGS,

SEQUENCE LISTINGS AND BIOLOGICAL DEPOSITS

Figure 1 illustrates the construction of plasmid pBE119 containing the xylanase gene (xynA) C. saccharo- lyticu-τι under the control of the Bacillus apr promoter (aprp) .

Figure 2 illustrates the construction of plasmid pBE145 containing the xylanase gene of C. saccharo¬ lytlcum under the control of the Bacillus npr promoter (nprp) .

Figure 3 illustrates the construction of plasmid pBE164 containing the β-glucosidase (β-glu) gene under the control of the bacillus apr promoter.

Figure 4 illustrates the construction of plasmid pBE158 containing the xynA gene downstream of the bacillus apr promoter fused to the apr signal sequence

(aprss) .

Figure 5 is an SDS-PAGE gel, stained with commassie blue comparing the accumulation of xylanase in the supernatant of pBE119 (apr-xynA) transformed Bacillus grown in three different media.

Figure 6 is a Western blot using anti-xylanase antiserum as the primary antibody. This figure demonstrates exocellular production of xylanase in B. subtilis without a signal peptide and reduced production of xylanase associated with the apr_Ss .

Figure 7 is a coomassie-stained PAGE gel of the supernatant fraction and cell associated fraction of cells transformed with pBE164 illustrating exocellular β-glucosidase production is possible without a signal sequence.

As used herein, the designation "ATCC" refers to the American Tissue Culture Collection depository located at 12301 Parklawn Drive, Rockville, MD 20852 U.S.A. The "ATCC No." is the accession number to the following cultures on deposit under terms of the

Budapest Treaty at the ATCC: Identifi cation Reference ATCC Desiςmatinn Deposit Date

Transformed Bacillus 69572 22 February 1994 subtilis, BE3000/pBE145

Transformed Bacillus 69573 22 February 1994 subtilis, BE3000/pBE164

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the exocellular production of commercially useful thermostable enzymes in high yields from recombinant bacteria. The present invention also provides vectors for the transformation of host bacteria wherein these vectors are devoid of the signal sequence typically required for translocation of proteins across the cell membrane.

The following definitions are used herein and should be referred to for interpretation of the claims and the specification.

The term "thermostable enzyme" refers to an enzyme capable of withstanding temperatures in the range of 45°C-115°C without significant loss of biological activity (Bergquist et al., Biotech Genet . Engin . Rev. , 5, 199, (1987)). Typical thermostable enzymes may include but are not limited to xylanases, amylases, transferases, glucosidases, galactosidases, dehydrogenases, polymerases and Upases.

The term "thermophilic microorganism" or "thermo¬ philic bacteria" or "thermophile" will refer to microorganisms which produce enzymes and are capable of living at elevated temperatures of between 45°C and

115°C. It should be noted that many of the thermostable enzymes isolated to date are similar in function to the more typical mesophilic enzymes with the exception of being able to function at unusually high temperatures. The term "xylanase" will refer to a thermostable enzyme capable of the hydrolysis of cellulose and hemicellulose and typically produced by a variety of fungi and bacteria. Sources of xylanase and xylanase genes may include but are not limited to members of the genera Caldocellum, Bacillus, Trichoderma and Clostrldium.

The term "β-glucosidase" will refer to a thermostable enzyme capable of the hydrolysis of glucose and glucosides as well as cellulose-based substrates. The term "exocellular protein" or "extracellular protein" will refer to any protein produced by a microorganism which is secreted, transported or released in either an active or passive fashion through the cellular membrane to an exocellular location such as the growth media.

The term "precursor protein" will refer to a protein which includes the signal peptide and mature protein. The term "mature protein" will refer to the final protein product resulting from cleavage of the signal peptide from the precursor.

The term "signal peptide" will refer to an amino terminal polypeptide preceding the mature protein. The signal peptide is cleaved from and is therefore not present in the mature protein. Signal peptides direct secreted proteins across cell membranes. Signal peptide may also be referred to as "signal protein".

The term "signal sequence" will refer to the DNA fragment encoding the signal peptide.

The terms "peptide", "polypeptide" and "protein" are used interchangeably.

The terms "promoter" and "promoter region" refer to a sequence of DNA, usually 5' to the protein coding sequence of a structural gene, which promotes proper transcription.

The term "suitable promoter" will refer to any promoter capable of driving the expression of a gene encoding a thermostable enzyme. A "fragment" or "DNA fragment" will constitute a fraction of the DNA sequence of the particular region.

The term "construction" or "construct" refers to a plasmid, virus, autonomously replicating sequence, phage or nucleotide sequence, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of DNA fragments have been joined or recombined into a unique entity which is capable of introducing an operably linked promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

As used herein, "transformation" is the acquisition of new genes in a cell by the incorporation of nucleic acid.

The term, "operably linked" refers to the fusion of two fragments of DNA in a proper orientation and reading frame to lead to the transcription of functional RNA.

The term "expression" as used herein is intended to mean the transcription and translation to gene product from a gene encoding the gene product.

The term "plasmid" or "vector" as used herein refers to an extra-chromosomal element which is usually in the form of circular double-stranded DNA molecules. The term "restriction endonuclease" refers to an enzyme which catalyzes hydrolytic cleavage within a specific nucleotide sequence in double-stranded DNA.

The term "compatible restriction sites" refers to different restriction sites that when cleaved yield nucleotide ends that can be ligated without any additional modification.

The term "Ap-" refers to ampicillin and the term "Kan-" or "Km-" refers to Kanamycin.

The terms "apr", aprp and "Apr" refer to alkaline protease gene, promoter and protein respectively and the terms "npr", nprp and "Npr" refer to neutral protease gene, promoter and protein respectively.

Thermostable enzymes are currently of great commercial usefulness and methods for their reliable production are in high demand. Thermostable xylanases are of increasing importance since they are superior to their thermolabile counterparts for the enzymatic bleach-boosting of wood pulps, because of the high temperature of the incoming pulp. Thermostable enzymes are also used in the food industry where high temperature hydrolysis of carbohydrates is a key process. Thermostable enzymes suitable for expression and exocellular production in the present invention are known in nature and are produced by a variety of thermophilic microorganisms. Suitable thermostable enzymes may include but are not limited to xylanases, amylases, transferases, β-glucosidases, galactosidases dehydrogenases and Upases.

Detection of thermostable enzyme activity in the supernatant and cell fractions of cell cultures may be accomplished by any means well know in the art. In particular, a variety of methods are available for the determination and quantitation of xylanase. For example. Khan et al. (Enzyme Microb . Technol . , 8(6), 373-7, (1986)) present an analysis of assay methods for xylanase and xylosidase activities in bacterial and fungal cultures. Additionally Tang, et al. ( Wood Agric . Residues : Res. Use Feed, Fuels, Chem., Proc. Conf. Feed, Fuels, Chem., Wood Agric. Residues, Meeting Date 1982,287-301; Edited by: Soltes, Ed J. Academic: New York, NY) discuss an assay for xylanase using trinitrophenyl-xylan as substrate where reducing sugars produced were detected by measurements at 345 nm. A preferred method, used in the present invention, for analyzing exocellular xylanase activity involves measuring the release of reducing sugars. Briefly, cells are removed from a culture and a sample of the resulting supernatant is added to a substrate solution containing 0.5% xylan in 50mM sodium citrate buffer. The enzyme is assayed by heating the substrate-supernatant mixture at 70°C and the reaction terminated by boiling for 5 minutes. The absorbence at 420nm was read and the amount of reducing sugar released was determined from a standard curve generated with xylose. Similarly, assays for β-glucosidase are well known in the art. For example. Woodward et al. (Enzyme Microb . Technol . , 7(9), 449-53, (1985)) teach a method for assaying β-glucosidase by the coupling of glucose oxidase and a Fenton's reaction. Alternatively, Sarathchandra et al. (Soil Sci . , 138, 15, (1984)) reports a method for the assay of .beta.-glucosidase involving the incubation of soils with p-nitrophenyl. beta.-D-glucopyranoside (PNPG) extraction of the p-nitrophenol (PNP) released with EtOH, and colorimetric detection of the PNP in this solution in the presence of soil. Preferred in the instant application is a method essentially as described by Love et al. (Biotechnol . 5, 384, (1987)). The method of Love et al. relies on the release of p-nitrophenol from the substrate p-nitro- phenol-β-D-glyucopyranoside.

In one embodiment of this invention, suitable host bacteria for the vectors comprise gram positive bacteria such as Bacillus sp. and particularly Bacillus subtilis . Specifically BE3000 was used as a transformation host.

BE3000 can be obtained from its parent strain 1A40 which may be obtained from the Bacillus Genetics Stock Center (BGSC) , the Ohio State University, Columbus, Ohio 43210, U.S.A. Genes encoding thermostable enzymes useful in the present invention may be derived from a variety of sources. Preferred sources are thermophilic microorganisms such as bacteria and fungi. Typical thermophiles will include but are not limited to members of the Caldocellum genus (C. saccharolytlcum) , thermophilic sulfate-reducing bacteria

(ArchaeogloJ us sp . , Sulfolobus sp. , Desulfuroloccus sp . , and rherjnodesulfo-acteriujn sp . , ) thermophilic Bacillus sp . (B. Stearothermophilus, and B. lichenlformls) , Thermococcus sp. , thermophilic Clostridium sp . , ( C. thermocellum) as well as thermophilic fungi such as thermophilic Apsergillus sp. , (A. foetidus) and Trichoderma sp. , (Trichoderma reesei, and Trichoderma harzlanum) .

The present invention provides a variety of plasmids or vectors suitable for the cloning of portions the DNA required for the expression and exocellular production of the thermostable enzymes. Suitable vectors will be those which are compatible with the bacterium employed. Suitable vectors can be derived, for example, from a bacteria, a plasmid and/or a virus (such as bacteriophage T7 or a M-13 derived phage) .

Protocols for obtaining and using such vectors are known to those in the art. (Sambrook et al. Molecular Cloning: A Laboratory Manual - volumes 1,2,3 (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989)).

Vectors suitable for B. subtllis will have compatible regulatory sequences and origins of replication. They will be multicopy and have a selective marker gene, for example, a gene coding for antibiotic resistance. Vectors of the present invention will be either autonomously replicated or capable of integration into the host genome. In embodiments of the invention vectors compatible with the Bacillus sp. are preferred. Suitable expression vectors will contain a DNA fragment comprising a regulatable promoter sequence which controls transcription, a sequence for a ribosome binding site which controls translation and a heterologous DNA fragment encoding a thermostable enzyme. Notably absent in these vectors are the signal sequences typically necessary for signal peptide expression. Generally, the vectors are constructed so that the promoter region is 5' of the heterologous DNA. Optionally, the vector may also include a region 3' of the heterologous DNA which controls transcriptional termination. It is most preferred when both the promoter and the transcriptional termination regions are derived from genes homologous to the host bacterium employed, however, it is to be understood that such control regions may be derived from sources other than the host bacterium. Further it will be appreciated by one of skill in the art that a termination control region may be unnecessary for expression of the desired protein.

Promoters which are useful for driving the expression of heterologous DNA fragments in Bacillus are numerous and familiar to those skilled in the art. Virtually any promoter capable of transcribing the gene encoding the desired thermostable enzyme is suitable for the present invention, where promoters native to Bacillus sp . are preferred. The promoters in the DNA sequences may be either constitutive or inducible. Suitable promoters may include but are not limited to the alkaline protease promoter (aprp) , the neutral protease promoter (nprp) , and the barnase promoter (iarp) . Methods of isolating DNA sequences encoding the promoter, ribosome binding site and termination control regions are well known to those skilled in the art and illustrative examples are documented in the literature. (See Biotechnology Handbook 2 Bacillus, C. R. Harwood, Ed., Plenum Press, New York, New York (1989)).

The addition of restriction endonuclease cleavage sites to the 3' or 5' ends of DNA for the purposes of vector construction or modification is also easily accomplished by means well known to those skilled in the art and is described by Sambrook et al., supra . Any restriction endonuclease site may be used but the use of a restriction site unique to that vector is desirable. Suitable compatible restriction sites are well known in the art. (See, for example the Restriction Fragment Compatibility Table of the New England Biolabs 1988-1989 Catalog, New England Biolabs Inc., Beverly, MA 01915 (1988).) Preferred for use herein are Xbal , Ndel, Nhel , Sail and Kpnl . The combined DNA sequences encoding a promoter, ribosome binding site and termination control regions with a restriction site at its 3' end and the DNA sequences encoding heterologous polypeptides or proteins with a compatible restriction site at its 5' end can be operably integrated by conventional techniques (Sambrook et al., supra; Harwood, supra) . Optionally, it may be useful to obtain desired genes or promoters from readily available plasmids or vectors by DNA amplification of the appropriate regions of the plasmid. Such amplifications may be accomplished by any of several schemes known in this art, including but not limited to the polymerase chain reaction (PCR) U.S. Patent 4,683,202 (1987, Mullis et al.); or the ligase chain reaction (LCR) (Tabor et al. (Proc. Acad. Sci . USA 82, 1074-1078) (1985)).

Once suitable vectors are constructed they are used to transform suitable bacterial hosts. Introduction of desired DNA fragments into B . subtilis may be accomplished by known procedures such as by transformation, electroporation, or by transfection using a recombinant phage virus. (Sambrook et al., supra) .

Construction of Bacillus vectors pertinent to the present invention are illustrated in Figures 1-4. The C. saccharolytlcum xylanase gene (xynA) encoding xylanase was isolated from the plasmid pNZl448 using a PCR protocol and engineered to include a Ndel site and an Xbal site. The Bacillus expression vectors were either pBE20 or pBE60 based (Nagarajan et al.. Gene, 114, 121, (1992) ) and contained either the Bacillus aprp or nprp which are bounded by a 5* Kpnl site and a 3' Ndel site. All expression vectors were created by inserting the xynA PCR fragment at the Ndel site 3' of the promoter. Hence, pBE119 consists of the xynA gene downstream of the aprp (Figure 1), pBE145 contains the xynA gene downstream of the nprp (Figure 2) and pBE158 contains the xynA gene downstream of the aprp-apr_as, also containing the signal sequence (Figure 4) . For the expression of β-glucosidase, the β-glucosidase (β-glu) gene was amplified by PCR from an M13 clone and engineered to incorporate a 5' Ndel site and a 3^• Xbal site. The β-glu gene was inserted 3' of the aprp to form pBE164 (Figure 3) .

The following non-limiting examples are meant to illustrate key embodiments of the invention but should not be construed to be limiting in any way.

EXAMPLES GENERAL METHODS

Restriction enzyme digestions, phosphorylations, ligations, transformations and other suitable methods of genetic engineering employed herein are described in Sambrook et al.,supra, and in the instructions accompanying commercially available kits for genetic engineering. Polyclonal rabbit anti-xylanase was prepared from purified xylanase by Hazleton Labs,

Denver, PA. Isopropyl-b-D-thiogalactoside (IPTG) and all other standard reagents and solutions were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. Enzyme assays:

Xylanase: Xylanase activity was measured according to the method of Luthi et al., Appl . Environ . Microbiol . , 56, 2677, (1990) which measures the release of reducing sugars. Cells are removed from an aliquot of culture by centrifugation and a sample of the resulting supernatant is added to a substrate solution containing 0.5% xylan in 50mM sodium citrate buffer. The enzyme is assayed by heating the substrate- supernatant mixture at 70°C for 15 minutes. An aliquot of 50mM hydroxy CaC12, 20mM sodium hydroxide was added and the reaction terminated by boiling for 5 minutes. The absorbance at 420nm was read on a Milton Roy spectrophotometer, model 1001 and the amount of reducing sugar released was determined from a standard curve generated with xylose. β-glucosidase: β-glucosidase was assayed according to a modification of the method described by Love et al. (Biotechnol . 5, 384, (1987)). The method of Love et al. relies on the release of p-nitrophenol from the su_bstrate p-nitro-phenol-β-D-glyucopyranoside (PNPG,

Sigma Chemical Co.). Cells were pelleted by centrifugation as above. A 1:1000 dilution of the supernatant was made in buffer and 20 ul of the dilution was added to 300 ul of a 0.5 mg/ml PNPG solution in 50 mM sodium citrate buffer pH 6.0 and heated at 70°C for 10 min. An equal volume of 1 M Na2C03 was added to stop the reaction. The sample absorbance at 400 nm was read on a Milton Roy spectrophotometer (model 1001) . Purified β-glucosidase was used as a reference for activity.

Protein Extraction:

Cultures were separated into cell pellet and supernatant fractions by centrifugation. The cell pellet was re-suspended in 5% TCA while 50% TCA was added to the supernatant fraction for a final of 5% TCA. After a one hour incubation at 4°C the samples were centrifuged and the pellet was washed with acetone and allowed to dry. To the supernatant and cell fractions TEP (50 mM Tris, 1 mM PMSF, 1 mM EDTA) or TEP with 1 mg/ml lysozyme, respectively, was added and the samples were incubated at 37°C for 15 min. An equal volume of 2% SDS in TEP was added and the sample boiled for 5 min. An aliquot was then mixed with sample buffer, heated and analyzed on an SDS-PAGE gel. Bacil lus host strains:

Bacillus host strains were of the species subtilis and included BE3000 ( trpC2, lys3, ΔaprE66, Δnpr82, xynA sacB : : ermC) . BE3000 may be derived from its parent strain 1A40 obtainable from the Bacillus Genetics Stock Center (BGSC) , The Ohio State University, Columbus, OH 43210 U.S.A. Bacterial growth conditions: Bacillus strains were grown in S7 minimal medium containing 50 ug/ml kanamycin, with 25 mM sodium citrate and yeast extract at concentrations of 0.05-1.00%. The components of S7 media are as follows: 50 mM KPO₄, pH 7.0 10 mM NH₄SO₄

20 mM glutamic acid, pH 7.0 2 mM MgCl2 0.7 mM CaCl₂

0.05 mM MnCl₂ 1 uM FeCl₃

1 uM ZnCl₃

2 nM thiamine HC1 50 ug/ml lysine

50 ug/ml tryptophan Cultures were inoculated at 0.15 O.D.βoo ^and grown overnight at 37°C. Final O.D.₆₀₀ or the cultures was between 4 and 8. The cells were harvested by centrifugation and the culture supernatant was analyzed for the presence of xylanase or β-glucosidase according to the protocol described in the general methods.

EXAMPLE 1 Construction of expression Vectors All figures of plasmid constructions are meant to illustrate the events of the construction; however DNA constructs are not drawn to scale. Plasmid pBETIQ:

The outline of the scheme used to construct pBE119 used for the transformation of B. subtilis is given in Figure 1.

DNA containing the xylanase gene from C. saccharolytlcum was obtained via PCR amplification of the appropriate regions of plasmid pNZ1448 (a kind gift of Peter Bergquist of Cent. Gene Technol., Univ.

Auckland, Auckland, N.Z.), fully described in Luthi et al., Appl . Environ . Microbiol . , 56, 2677, (1990)) . PCR amplification was accomplished according to the protocol of the manufacturer (GeneAmp PCR Reagent Kit, Perkin-Elmer Cetus, Norwalk, CT) using the following primers: Primer 1, Upper:

5•-CCTAACCATATGTGCGAAAATTTAGAGATGCTA-3' SEQ ID NO. :1

Primer 2, Lower:

5'-ACTGATTCTAGATTTCTTCGTTAAAAAATCTT-3' SEQ ID NO.:2

Reagent concentrations were: IX Buffer 200uM dATP 200uM dCTP 200uM dGTP

200uM dTTP 2.5units Amplitaq® Polymerase (Perkin-Elmer

Cetus) lOOpM Upper Primer lOOpM Lower Primer lng pNZ1448 dH₂θ to lOOul The reaction was performed using a Perkin-Elmer Cetus GeneAmp PCR System 9600 thermal cycler programmed as follows:

Melting: 94 °C for 1 min Annealing: 55 °C for 1 min Extension: 72 °C for 1 min Cycles: 25 The amplified product contains the entire xynA gene encoding the xylanase gene, bounded on the 5' end by a Ndel site and on the 3' end by a Xbal site. Briefly, plasmid pBE1020 (Collier, D., J. Bact., 176, 3013, (1994)) and the xynA PCR fragment were digested with Ndel and Xbal and the appropriate fragments ligated to yield the plasmid pBE105 (Figure 1) . Plasmid pBE240 and pBE60 (Nagarajan et al.. Gene, supra) are digested with Kpnl and Xbal and the appropriate fragments ligated to form the high copy number plasmid pBE113 (Figure 1) . pBE105 and pBE113 were digested with Ndel and Xbal and the appropriate fragments were ligated yielding plasmid pBE119 which contains the xylanase gene downstream of the aprp (Figure 1) . Plasmid pBEl45:

Construction of pBE145, containing the xynA gene under the control of the Bacillus nprp is illustrated in Figure 2. The plasmids pBE105 (containing the xynA gene) and pBE146 (containing the nprp) were digested with the restriction enzymes Ndel and Kpnl. The full nucleic acid sequence of pBE146 is given in SEQ ID NO.3) . The large fragment of pBE105 and the 250 bp fragment of pBE146 were ligated, yielding plasmid, pBE147 (Figure 2) . The nprp-xynA fusion was then transferred from pBE147 to a high copy plasmid by digestion of pBE147 with Kpnl and Xbal and ligation of the appropriate fragment with pBE113 cut with the same enzymes. The resultant plasmid is pBE145 (Figure 2). Plasmid pBE164:

Construction of plasmid pBE164 containing the β-glucosidase gene under the control of the aprp is illustrated in Figure 3. The C. saccharolytlcum β-glucosidase gene was obtained by PCR amplification of the gene from an M13 clone, kindly provided by Peter Bergquist of Cent. Gene Technol., Univ. Auckland, Auckland, N.Z. PCR amplification was done essentially according to the protocols described above. The complete DNA sequence of the gene is known and is disclosed in Love et al. (Biotechnol . 5, 384, (1987)). The primers used for the PCR amplification of the gene were as follows:

5' region of β-glucosidase, primer 3: 5'-GTT TAT GCA TAT GAG TTT CCC AAA AGG-3' SEQ ID NO.:4

3' region of β-glucosidase, primer 4: 5'-GCC CGC TCT AGA TTA TAT TTA CGA ATT TTC C-3' SEQ ID NO.:5

PCR of the β-glucosidase gene using the above primers resulted in a fragment carrying a Ndel site at the 5' end and an Xbal site at the 3' end. The high copy number plasmid pBE60 contains three Ndel sites (Figure 3) . pBE60 was first digested with Ndel and the two large fragments of plasmid DNA were isolated and treated with the Klenow fragment of DNA polymer as klenow to create blunt ends. The two large fragments were ligated and resulted in pBE928. The loss of the Ndel sites were verified (Figure 3) . pBE928 and pBE119 were digested with Kpnl and Xbal. Ligation of the small Kpnl-Xbal fragment from pBE119 with the large Kpnl-Xbal fragment from pBE928 resulted in pBE163. The β-glu PCR product was digested with Ndel and Xbal and ligated with the large Ndel-Xbal fragment of Pbel63 resulting in pBE164 (Figure 3) . Plasmid pBElSB:

The construction of pBE158 containing xynA fused to a bacillus signal sequence is illustrated in Figure 4. Briefly the xynA gene was amplified from the plasmid pBE105 using the PCR protocol listed above and primers 5 and 6 given below:

5' region of xynA gene, Primer 5: 5'-GCG GCA GCT AGC GCG ATG TGC GAA AAT TTA GAG ATG-3' SEQ ID NO. :6 3' region of xynA gene Primer 6: 5'-CAT GCC TGC AGG TCG AC-3' SEQ ID NO.:

PCR amplification using the above primers resulted in a fragment containing a Nhel restriction site at the 5' end and a Sail site at the 3' end. After amplification the fragment was digested with Nhel and Sail and ligated to the large fragment of pBE92 which contains the aprp and apr_ss (Figure 4) . The full sequence of pBE92 is given in SEQ ID NO.:8. The resulting plasmid is pBE158 containing the aprp-apr_ss-xynA fusion.

All plasmid manipulations were accomplished in E. coli XLl-Blue (Bullock et al., Biotechniques, 5, 376, (1987)) and selected on the basis of ampicillin resistance. Colonies were screened for xylanase activity by overlaying plates with a 0.8% (wt/vol) Difco Bactor^® Agar and 0.8% (wt/vol) xylan coupled to REMAZOL® Brilliant blue. A positive result is indicated by clearing of the blue color from the plate. (Luthi et al., Appl . Environ, Microblol . 56, 1017, (1990) .) Presence of the desired plasmid construction was confirmed by restriction enzyme analysis of plasmid DNA removed from transformed bacteria.

EXAMPLE 2 Transfnrma ion of B. subtllis Transformation with pBE119. DBE145 and PBE158 Plasmids pBE119 (aprp-xynA) , pBE145 (nprp-xynA) and pBE158 (aprp-apr_ss-xynA) were introduced into the B. subtilis strain BE3000 by standard transformation protocols, and bacteria were selected on kanamycin plates and screened by the coupled xylan method described above.

Transformation with pBE164:

Plasmid pBE164 (aprp-β-glu) was introduced into the B. su_tilis strain BE3000 by standard transformation protocols, and bacteria were selected on kanamycin plates and screened by the release of p-nitrophenol as described above.

EXAMPLE 3 Expression of xylanase from pBE119. pBE145 and PBE158 Transfor pd Bacillus hosts Coomassie stained SDS-PAGE analysis:

Transformed Bacillus host cells containing plasmid pBE119 (aprp-xynA) were grown overnight in various media, the cultures fractionated and the culture supernatants analyzed by SDS-PAGE according to the protocols described in the GENERAL METHODS. Figure 5 shows an SDS-PAGE gel, stained with commassie blue, comparing the accumulation of protein in the supernatant of pBE119 (aprp-xynA) transformed Bacillus grown in S7 media containing 1% glucose (lane 1) ; S7 media containing 25 mM sodium citrate (lane 2) ; or Am3 rich media [equivalent to Penassay Broth, Difco Laboratories, Detroit, MI (lane 3) ] . Lane 4 contains culture supernatant from a Bacillus host cell transformed with an aprp-aprss-phoA (alkaline protease) gene. Figure 5 demonstrates that the apparent molecular weight of the predominant protein band in the culture supernatants of a Bacillus host harboring pBE119, but not the control plasmid, was the same as is expected for the xynA gene product. Hence xylanase is the major extracellular protein despite the fact it was synthesized without a signal peptide. Transformants grown in either the S7+ sodium citrate or Am3 rich media produced more extracellular xylanase than those grown in the S7+glucose media. Anti-xvlanase immuno-hlo :

Immuno-blotting (Western blot analysis) was used to demonstrate that the major extracellular protein produced in Bacillus hosts transformed with pBE119 (aprp-xynA) was indeed xylanase. Cultures of BE3000 harboring a vector control [pBE60 (panel A) ] , pBE158 [aprp-apr_ss-xynA (panel B) ] , pBE119 [aprp-xynA (panel C) ] or pBE145 [nprp-xynA (panel D) ] were grown for 14 hours at 37°C in S7 medium supplemented with yeast extract (0.5%), sodium citrate (25 mm) and tryptophan and lysine (50 ug/ml each), then separated into cell (c) and supernatant (s) fractions as described above. The proteins contained in each fraction were resolved by SDS-PAGE, transferred to a nitrocellulose filter and xylanase was identified by probing with rabbit anti- xylanase anti-serum as the primary antibody. The vector control [pBE60 (panel A) ] demonstrated a lack of endogenous xylanase antigen in B . subtilis strain BE3000. Cells harboring the arp-apr_Ss~xynA gene [pBE158 (panel B) ] produced modest levels of xylanase. Approximately 50% of the total xylanase was found in the culture supernatant (s) . Both aprp-xynA [pBE119 (panel C) ] and nprp-xynA [pBE145 (panel d) ] sponsored production of 10-20 fold more xylanase than did apr-apr_ss-xynA (panel B) . Between 20-40% of the xylanase produced in cells harboring aprp-xynA or nprp-xynA was recovered in the supernatant fraction. Xvlanase activity:

Cells transformed with either pBE145, pBE119, pBE158 or the control plasmid pBE60 were grown in S7 media supplemented with sodium citrate as described in the GENERAL METHODS, the culture supernatants were collected and placed on ice and the xylanase assay described in the GENERAL METHODS was used to determine the xylanase activity contained in each supernatant.

The values are given in Table 1.

TABLE 1

Xylanase Activity

S rain fper ml supernatant) nprp -xynA (pBE145) 1110 U aprp -xynA (pBE119) 272 U apr-apr_ss-xynA (pBE158) 37 U vector control (pBE60) 0 U

These data indicate that enzymatically active xylanase accumulates in the growth media (i.e. extracellular) of Bacillus host strains carrying xynA fused to a Bacillus promoter, even in the absence of a signal peptide. In fact extracellular xylanase activity is at least 7 fold greater in the absence of a signal peptide.

EXAMPLE 4 Expression of β-glucosidase from pBE!64

Transformed Bacillus hosts Coomassie stained SDS-PAGE analysis:

Cells transformed with pBE164 (aprp-β-glu) or the control plasmid pBE60 were grown and fractionated as described in the GENERAL METHODS. Proteins in the cell and supernatant fractions were resolved by SDS-PAGE and visualized by coomassie staining. Figure 7 demonstrates that a major new protein band with the expected molecular weight of β-glucosidase, which is not present in cells harboring pBE60 (panel A) , is readily identified in cells harboring pBE164 (panel B) . Between 20-40% of this protein was found in the culture supernatant fraction (S) . β-glucpsit-flRft activity:

The supernatant fractions were tested for β-glucosidase activity employing the assay described in the GENERAL METHODS and activity is shown in Table 2.

TABLE 2 β-glucosidase Activity Strain (per ml supernatant) aprp-β-glu (pBE164) 15.5 U

Control plasmid (pBE60) 0 U

The values reported in Table 2 indicate that, as was the case for xylanase, enzymatically active β-glucosidase is recovered in culture supernatants of

Bacillus, despite the absence of a signal peptide.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: JACKSON, ETHEL N.

NAGARAJAN, VASANTHA COLLIER, DAVID N. LIU, GSEPING

(ii) TITLE OF INVENTION: METHOD FOR THE

PRODUCTION OF THERMO¬ STABLE ENZYMES FROM BACTERIA

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDING ADDRESS:

(A) NAME: E. I. DU PONT DE NEMOURS AND

COMPANY

(B) STREET: 1007 MARKET STREET

(C) CITY: WILMINGTON

(D) STATE: DELAWARE

(E) COUNTRY : UNITED STATES OF AMERICA

(F) POSTAL CODE (ZIP) : 19898

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: DISKETTE, 3.50 INCH

(B) COMPUTER: MACINTOSH

(C) OPERATING SYSTEM: MACINTOSH, 6.0

(D) SOFTWARE: MICROSOFT WORD, 4.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: SIEGELL, BARBARA C.

(B) REGISTRATION NUMBER: 30,684

(C) REFERENCE/DOCKET NUMBER: CR-9501

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 302-992-4931

(B) TELEFAX: 302-773-0164

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CCTAACCATA TGTGCGAAAA TTTAGAGATG CTA 33

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ACTGATTCTA GATTTCTTCG TTAAAAAATC TT 32

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8808 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GAATTCGAGC TCGGTACCGA TCTTAACATT TTTCCCCTAT CATTTTTCCG TCTTCATTTG 60

TCATTTTTTC CAGAAAAAAT CGCGTCATTC GACTCATGTC TAATCCAACA CGTGTCTCTC 120

GGCTTATCCC CTGACACCGC CCGCCGACAG CCCGCATGGG ACGATTCTAT CAATTCAGCC 180

GCGGAGTCTA GTTTTATATT GCAGAATGCG AGATTGCTGG TTTATTATAA CAATATAAGT 240

TTTCATTATT TTCAAAAAGG GGGATTTCAT ATGAGAGGCA AAAAAGTATG GATCAGTTTG 300

CTGTTTGCTT TAGCGTTAAT CTTTACGATG GCGTTCGGCA GCACATCCTC TGCCCAGGCG 360

GCAGGGGATA TCGTGTGCGA AAATTTAGAG ATGCTAAACT TATCATTAGC AAAAACATAC 420

AAAGATTACT TTAAAATAGG TGCTGCAGTA ACTGCGAAAG ATTTAGAAGG AGTTCATAGG 480 GATATTCTTT TGAAGCATTT TAATAGCCTC ACACCAGAAA ATGCCATGAA GTTTGAAAAT 540

ATTCATCCAG AAGAGCAGAG ATATAATTTT GAAGAGGTTG CCAGGATAAA AGAGTTTGCA 600

ATTAAAAATG ACATGAAGTT AAGAGGACAT ACATTTGTTT GGCATAATCA AACTCCGGGG 660

TGGGTGTTTT TAGATAAGAA TGGGGAAGAA GCCTCAAAAG AGTTAGTTAT TGAAAGGTTA 720

AGAGAGCATA TAAAAACTTT GTGTGAGAGA TACAAGGATG TAGTATATGC GTGGGATGTG 780

GTGAACGAAG CAGTAGAAGA TAAAACAGAλ AAGCTTTTGC GAGAATCAAA CTGGAGAAAA 840

ATTATTGGAG ATGATTATAT TAAAATTGCT TTTGAGATAG CAAGAGAATA TGCAGGAGAT 900

GCAAAGTTAT TTTATAACGA TTATAACAAT GAAATGCCTT ATAAATTAGA AAAAACCTAC 960

AAAGTTCTAA AAGAGCTTTT AGAAAGAGGT ACTCCAATAG ATGGAATTGG TATACAAGCA 1020

CACTGGAATA TATGGGATAA AAATCTTGTT AGTAATTTAA AAAAGGCTAT AGAAGTATAT 1080

GCTTCCTTAG GTTTAGAAAT TCATATTACA GAACTTGACA TTTCAGTATT TGAGTTTGAA 11 0

GATAAGAGGA CTGACTTGTT TGAACCAACC CCGGAAATGC TTGAACTACA AGCAAAAGTA 1200

TATGAAGATG TATTTGCAGT TTTTCGAGAA TATAAAGATG TAATAACTTC TGTTACATTA 1260

TGGGGTATTA GCGACAGACA CACATGGAAA GATAACTTCC CTGTAAAGGG TCGAAAAGAT 1320

TGGCCTCTCT TATTCGACGT AAATGGAAAA CCAAAAGAAG CCTTGTACAG GATATTAAGA 1380

TTTTAAAGAT TTTTTAACGA AGAAATCTAG AGTCGACCTG CAGGCATGCA AGCTTACTCC 1440

CCATCCCCTC CAGTAATGAC CTCAGAACTC CATCTGGATT TGTTCAGAAC GCTCGGTTGC 1500

CGCCGGGCGT TTTTTATTGG TGAGAATCGC AGCAACTTGT CGCGCCAATC GAGCCATGTC 1560

GTCGTCAACG ACCCCCCATT CAAGAACAGC^"AAGCAGCATT GAGAACTTTG GAATCCAGTC 1620

CCTCTTCCAC CTGCTGAGGG CAATAAGGGC TGCACGCGCA CTTTTATCCG CCTCTGCTGC 1680

GCTCCGCCAC CGTAGTTAAA TTTATGGTTG GTTATGAAAT GCTGGCAGAG ACCCAGCGAG 1740

ACCTGACCGC AGAACAGGCA GCAGAGCGTT TGCGCGCAGT CAGCGATACC CCGGTTGATA 1800

ATCAGAAAAG CCCCAAAAAC AGGAAGATTG TATAAGCAAA TATTTAAATT GTAAACGTTA 1860

ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG 1920

CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGCC CGAGATAGGG TTGAGTGTTG 1980

TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA 2040

AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCAAATCA AGTTTTTTGG 2100

GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT 2160

GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG 2220

CTAGGGCGCG AGCAAGTGTA GCGGTCACGC GCGCGTAACC ACCACACCCG CCGCGCTTAA 2280

TGCGCCGCTA CAGGGCGCGT ATCCATTTTC GCGAATCCGG AGTGTAAGAA ATGAGTCTGA 2340 AAGAAAAAAC ACAATCTCTG TTTGCCAACG CATTTGGCTA CCCTGCCACT CACACCATTC 2400

AGGTGCGTCA TATACTGACT GAAAACGCCC GCACCGTTGA AGCTGCCAGC GCGCTGGAGC 2460

AAGGCGACCT GAAACGTATG GGCGAGTTGA TGGCGGAGTC TCATGCCTCT ATGCGCGATG 2520

ATTTCGAAAT CACCGTGCCG CAAATTGACA CTCTGGTAGA AATCGTCAAA GCTGTGATTG 2580

GCGACAAAGG TGGCGTACGC ATGACCGGCG GGGGATTTGG CGGCTGTATC GTCGCGCTGA 2640

TCCCGGAAGA GCTGGTGCCT GCCGCACAGC AAGCTGTCGC TGAACAATAT GAAGCAAAAA 2700

CAGGTATTAA AGAGACTTTT TACGTTTGTA AACCATCACA AGGAGCAGGA CAGTGCTGAA 2 60

CGAAACTCCC GCACTGGCAC CCGATGGCAG CCGTACCGAC TGTTCTGCCT CGCGCGTTTC 2820

GGTGATGACG GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG 2880

TAAGCGGATG CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT 2940

CGGGGCGCAG CCATGACCCA GTCACGTAGC GATAGCGGAG TGTATACTGG CTTAACTATG 3000

CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA CCGCACAGAT 3060

GCGTAAGGAG AAAATACCGC ATCAGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC 3120

GCTCGGTCGT TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT 3180

CCACAGAATC AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA 3240

GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC 3300

ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC 3360

AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG 3420

GATACCTGTC CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCAATGC TCACGCTGTA 3480

GGTATCTCAG TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG 3540

TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC 3600

ACGACTTATC GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG 3660

GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA AGGACAGTAT 3720

TTGGTATCTG CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT 3780

CCGGCAAACA AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC 3840

GCAGAAAAAA AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT 3900

GGAACGAAAA CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT 3960

AGATCCTTTT AAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT 4020

GGTCTGACAG TTACCAATGC TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC 4080

GTTCATCCAT AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC 4140 CATCTGGCCC CAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT 4200

CAGCAATAAA CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA ACTTTATCCG 4260

CCTCCATCCA CTCTATTAAT TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA 4320

GTTTGCGCAA CGTTGTTGCC ATTGCTACAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA 4380

TGGCTTCATT CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT 4440

GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG 4500

TGTTATCACT CATGGTTATG GCAGCACTGC ATAATTCTCT TACTGTCATG CCATCCGTAA 4560

GATGCTTTTC TGTGACTGGT GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC 4620

GACCGAGTTG CTCTTGCCCG GCGTCAACAC GGGATAATAC CGCGCCACAT AGCAGAACTT 4680

TAAAAGTGCT CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC 4740

TGTTGAGATC CAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA 4800

CTTTCACCAG CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AAAAAGGGAA 4860

TAAGGGCGAC ACGGAAATGT TGAATACTCA TACTCTTCCT TTTTCAATAT TATTGAAGCA 4920

TTTATCAGGG TTATTGTCTC ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC 4980

AAATAGGGGT TCCGCGCACA TTTCCCCGAA AAGTGCCACC TGACGTCTAA GAAACCATTA 5040

TTATCATGAC ATTAACCTAT AAAAATAGGC GTATCACGAG GCCCTTTCGT CTTCAAGCCC 5100

GAGGTAACAA AAAAACAACA GCATAAATAA CCCCGCTCTT ACACATTCCA GCCCTGAAAA 5160

AGGGCATCAA ATTAAACCAC ACCTATGGTG TATGCATTTA TTTGCATACA TTCAATCAAT 5220

TGTTATCTAA GGAAATACTT ACATATGGTT^"CGTGCAAACA AACGCAACGA GGCTCTACGA 5280

ATCGATGCAT GCAGCTGATT TCACTTTTTG CATTCTACAA ACTGCATAAC TCATATGTAA 5340

ATCGCTCCTT TTTAGGTGGC ACAAATGTGA GGCATTTTCG CTCTTTCCGG CAACCACTTC 5400

CAAGTAAAGT ATAACACACT ATACTTTATA TTCATAAAGT GTGTGCTCTG CGAGGCTGTC 5460

GGCAGTGCCG ACCAAAACCA TAAAACCTTT AAGACCTTTC TTTTTTTTAC GAGAAAAAAG 5520

AAACAAAAAA ACCTGCCCTC TGCCACCTCA GCAAAGGGGG GTTTTGCTCT CGTGCTCGTT 5580

TAAAAATCAG CAAGGGACAG GTAGTATTTT TTGAGAAGAT CACTCAAAAA ATCTCCACCT 5640

TTAAACCCTT GCCAATTTTT ATTTTGTCCG TTTTGTCTAG CTTACCGAAA GCCAGACTCA 5700

GCAAGAATAA AATTTTTATT GTCTTTCGGT TTTCTAGTGT AACGGACAAA ACCACTCAAA 5760

ATAAAAAAGA TACAAGAGAG GTCTCTCGTA TCTTTTATTC AGCAATCGCG CCCGATTGCT 5820

GAACAGATTA ATAATAGATT TTAGCTTTTT ATTTGTTGAA AAAAGCTAAT CAAATTGTTG 5880

TCGGGATCAA TTACTGCAAA GTCTCGTTCA TCCCACCACT GATCTTTTAA TGATGTATTG 5940

GGGTGCAAAA TGCCCAAAGG CTTAATATGT TGATATAATT CATCAATTCC CTCTACTTCA 6000 ATGCGGCAAC TAGCAGTACC AGCAATAAAC GACTCCGCAC CTGTACAAAC CGGTGAATCA 6060

TTACTACGAG AGCGCCAGCC TTCATCACTT GCCTCCCATA GATGAATCCG AACCTCATTA 6120

CACATTAGAA CTGCGAATCC ATCTTCATGG TGAACCAAAG TGAAACCTAG TTTATCGCAA 6180

TAAAAACCTA TACTCTTTTT AATATCCCCG ACTGGCAATG CCGGGATAGA CTGTAACATT 62 0

CTCACGCATA AAATCCCCTT TCATTTTCTA ATGTAAATCT ATTACCTTAT TATTAATTCA 6300

ATTCGCTCAT AATTAATCCT TTTTCTTATT ACGCAAAATG GCCCGATTTA AGCACACCCT 6360

TTATTCCGTT AATGCGCCAT GACAGCCATG ATAATTACTA ATACTAGGAG AAGTTAATAA 6420

ATACGTAACC AACATGATTA ACAATTATTA GAGGTCATCG TTCAAAATGG TATGCGTTTT 6480

GACACATCCA CTATATATCC GTGTCGTTCT GTCCACTCCT GAATCCCATT CCAGAAATTC 65 0

TCTAGCGATT CCAGAAGTTT CTCAGAGTCG GAAAGTTGAC CAGACATTAC GAACTGGCAC 6600

AGATGGTCAT AACCTGAAGG AAGATCTGAT TGCTTAACTG CTTCAGTTAA GACCGAAGCG 6660

CTCGTCGTAT AACAGATGCG ATGATGCAGA CCAATCAACA TGGCACCTGC CATTGCTACC 6720

TGTACAGTCA AGGATGGTAG AAATGTTGTC GGTCCTTGCA CACGAATATT ACGCCATTTG 6780

CCTGCATATT CAAACAGCTC TTCTACGATA AGGGCACAAA TCGCATCGTG GAACGTTTGG 6840

GCTTCTACCG ATTTAGCAGT TTGATACACT TTCTCTAAGT ATCCACCTGA ATCATAAATC 6900

GGCAAAATAG AGAAAAATTG ACCATGTGTA AGCGGCCAAT CTGATTCCAC CTGAGATGCA 6960

TAATCTAGTA GAATCTCTTC GCTATCAAAA TTCACTTCCA CCTTCCACTC ACCGGTTGTC 7020

CATTCATGGC TGAACTCTGC TTCCTCTGTT GACATGACAC ACATCATCTC AATATCCGAA 7080

TAGGGCCCAT CAGTCTGACG ACCAAGAGAG CCATAAACAC CAATAGCCTT AACATCATCC 7140

CCATATTTAT CCAATATTCG TTCCTTAATT TCATGAACAA TCTTCATTCT TTCTTCTCTA 7200

GTCATTATTA TTGGTCCATT CACTATTCTC ATTCCCTTTT CAGATAATTT TAGATTTGCT 7260

TTTCTAAATA AGAATATTTG GAGAGCACCG TTCTTATTCA GCTATTAATA ACTCGTCTTC 7320

CTAAGCATCC TTCAATCCTT TTAATAACAA TTATAGCATC TAATCTTCAA CAAACTGGCC 7380

CGTTTGTTGA ACTACTCTTT AATAAAATAA TTTTTCCGTT CCCAATTCCA CATTGCAATA 74 0

ATAGAAAATC CATCTTCATC GGCTTTTTCG TCATCATCTG TATGAATCAA ATCGCCTTCT 7500

TCTGTGTCAT CAAGGTTTAA TTTTTTATGT ATTTCTTTTA ACAAACCACC ATAGGAGATT 7560

AACCTTTTAC GGTGTAAACC TTCCTCCAAA TCAGACAAAC GTTTCAAATT CTTTTCTTCA 7620

TCATCGGTCA TAAAATCCGT ATCCTTTACA GGATATTTTG CAGTTTCGTC AATTGCCGAT 7680

TGTATATCCG ATTTATATTT ATTTTTCGGT CGAATCATTT GAACTTTTAC ATTTGGATCA 7740

TAGTCTAATT TCATTGCCTT TTTCCAAAAT TGAATCCATT GTTTTTGATT CACGTAGTTT 7800 TCTGTATTCT TAAAATAAGT TGGTTCCACA CATACCAATA CATGCATGTG CTGATTATAA 7860 GAATTATCTT TATTATTTAT TGTCACTTCC GTTGCACGCA TAAAACCAAC AAGATTTTTA 7920 TTAATTTTTT TATATTGCAT CATTCGGCGA AATCCTTGAG CCATATCTGA CAAACTCTTA 7980 TTTAATTCTT CGCCATCATA AACATTTTTA ACTGTTAATG TGAGAAACAA CCAACGAACT 8040 GTTGGCTTTT GTTTAATAAC TTCAGCAACA ACCTTTTGTG ACTGAATGCC ATGTTTCATT 8100 GCTCTCCTCC AGTTGCACAT TGGACAAAGC CTGGATTTAC AAAACCACAC TCGATACAAC 8160 TTTCTTTCGC CTGTTTCACG ATTTTGTTTA TACTCTAATA TTTCAGCACA ATCTTTTACT 8220 CTTTCAGCCT TTTTAAATTC AAGAATATGC AGAAGTTCAA AGTAATCAAC ATTAGCGATT 8280 TTCTTTTCTC TCCATGGTCT CACTTTTCCA CTTTTTGTCT TGTCCACTAA AACCCTTGAT 8340 TTTTCATCTG AATAAATGCT ACTATTAGGA CACATAATAT TAAAAGAAAC CCCCATCTAT 8400 TTAGTTATTT GTTTAGTCAC TTATAACTTT AACAGATGGG GTTTTTCTGT GCAACCAATT 8460 TTAAGGGTTT TCAATACTTT AAAACACATA CATACCAACA CTTCAACGCA CCTTTCAGCA 8520 ACTAAAATAA AAATGACGTT ATTTCTATAT GTATCAAGAT AAGAAAGAAC AAGTTCAAAA 8580 CCATCAAAAA AAGACACCTT TTCAGGTGCT TTTTTTATTT TATAAACTCA TTCCCTGATC 8640 TCGACTTCGT TCTTTTTTTA CCTCTCGGTT ATGAGTTAGT TCAAATTCGT TCTTTTTAGG 8700 TTCTAAATCG TGTTTTTCTT GGAATTGTGC TGTTTTATCC TTTACCTTGT CTACAAACCC 8760 CTTAAAAACG TTTTTAAAGG CTTTTAAGCC GTCTGTACGT TCCTTAAG 8808 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GTTTATGCAT ATGAGTTTCC CAAAAGG 27 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GCCCGCTCTA GATTATATTT ACGAATTTTC C 31

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GCGGCAGCTA GCGCGATGTG CGAAAATTTA GAGATG 36

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

CATGCCTGCA GGTCGAC 17

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10140 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GAATTCGAGC TCGGTACCCG GGCGTCATTT AATATGATCG TTTTCTTCCC GGAAGCTGTC 60

TGGATTGCAT ATGTAAACCC CGTGAGCGCC AAAAGCCGGG ACGTGAAAAA CTGATCATTC 120

TTTACGGAAA TGCCGCCATT TGCAGCGTCG ATTAATAAAA TCGAGCCGCC TTCTGCATCC 180

AGCTTTCTTT TGATTTCATT CAGGTTTTCT TCCTGAGCCG ATAATTGTTT CTCGATGATG 240

TCCTTTTTAC CGACCGCTTT TTCAATGGTC AAGGCACGTC AACCGCATCC TGATAATCCG 300

CTACTTCATT TTCAAAGCGA TTGTCGGAGC GATCTTCGTC AGCTTATTGT ATATCCCTCT 360

GTGCAGGCTA GAATCTGCAA TGCTGAGATC GGGTTTTAAA GAAGCGATCT TTTTCAGGTC 420 GGGATGTGAT CGGGTGCCGA CAGACTTTGA CGGGTCATCG GCCGGCTTTA TGCCGAGATC 480

ATGGAGGGTA TCAATAAAGC CCGGATTCAA TACGGCGAGC CGCTGCGGGT GCGCTGCCAC 540

ATGCGCGGTC CCGAGATCAT GTCTGATGGT GGTCTCATGT TCCGGTTCTG AACATGCCGG 600

TATGAAGAGG ACGGATAAGA TCAGCAGCAT ACTGAAAATC GGTTTCATCA GTTCCCCCTC 660

TTTCGTTTTT CCGCAATTAT ATCATTGACA ATATCAACAT CAATGATATT CATTATCATT 720

ATTTTTATAA AATGGTTTCA CAGCTTTTCT CGGTCAAGAA AGCCAAAGAC TGATTTCGCT 780

TACGTTTCCA TCAGTCTTCT GTATTCAACA AAAGATGACA TTTATCCTGT TTTTGGAACA 840

ACCCCCAAAA ATGGAAACAA ACCGTTCGAC CCAGGAAACA AGCGAGTGAT TGCTCCTGTG 900

TACATTTACT CATGTCCATC CATCGGTTTT TTCCATTAAA ATTTAAATAT TTCGAGTTCC 960

TACGAAACGA AAGAGAGATG ΛTATACCTAA ATAGAAATAA AACAATCTGA AAAAAATTGG 1020

GTCTACTAAA ATATTATTCC ATACTATACA ATTAATACAC AGAATAATCT GTCTATTGGT 1080

TATTCTGCAA ATGAAAAAAA GGAGAGGATA AAGAGTGAGA GGCAAAAAAG TATGGATCAG 1140

TTTGCTGTTT GCTTTAGCGT TAATCTTTAC GATGGCGTTC GGCAGCACAT CCTCTGCTAG 1200

CGCGGATATC CGGACACCAG AAATGCCTGT TCTGGAAAAC CGGGCTGCTC AGGGCGATAT 1260

TACTGCACCC GGCGGTGCTC GCCGTTTAAC GGGTGATCAG ACTGCCGCTC TGCGTGATTC 1320

TCTTAGCGAT AAACCTGCAA AAAATATTAT TTTGCTGATT GGCGATGGGA TGGGGGACTC 1380

GGAAATTACT GCCGCACGTA ATTATGCCGA AGGTGCGGGC GGCTTTTTTA AAGGTATAGA 1440

TGCCTTACCG CTTACCGGGC AATACACTCA CTATGCGCTG AATAAAAAAA CCGGCAAACC 1500

GGACTACGTC ACCGACTCGG CTGCATCAGC AACCGCCTGG TCAACCGGTG TCAAAACCTA 1560

TAACGGCGCG CTGGGCGTCG ATATTCACGA AAAAGATCAC CCAACGATTC TGGAAATGGC 1620

AAAAGCCGCA GGTCTGGCGA CCGGTAACGT TTCTACCGCA GAGTTGCAGG ATGCCACGCC 1680

CGCTGCGCTG GTGGCACATG TGACCTCGCG CAAATGCTAC GGTCCGAGCG CGACCAGTGA 1740

AAAATGTCCG GGTAACGCTC TGGAAAAAGG CGGAAAAGGA TCGATTACCG AACAGCTGCT 1800

TAACGCTCGT GCCGACGTTA CGCTTGGCGG CGGCGCAAAA ACCTTTGCTG AAACGGCAAC 1860

CGCTGGTGAA TGGCAGGGAA AAACGCTGCG TGAACAGGCA CAGGCGCGTG GTTATCAGTT 1920

GGTGAGCGAT GCTGCCTCAC TGAATTCGGT GACGGAAGCG AATCAGCAAA AACCCCTGCT 1980

TGGCCTGTTT GCTGACGGCA ATATGCCAGT GCGCTGGCTA GGACCGAAAG CAACGTACCA 2040

TGGCAATATC GATAAGCCCG CAGTCACCTG TACGCCAAAT CCGCAACGTA ATGACAGTGT 2100

ACCAACCCTG GCGCAGATGA CCGACAAAGC CATTGAATTG TTGAGTAAAA ATGAGAAAGG 2160

CTTTTTCCTG CAAGTTGAAG GTGCGTCAAT CGATAAACAG GATCATGCTG CGAATCCTTG 2220 TGGGCAAATT GGCGAGACGG TCGATCTCGA TGAAGCCGTλ CAACGGGCGC TGGAATTCGC 2280

TAAAAAGGAG GGTAACACGC TGGTCATAGT CACCGCTGAT CACGCCCACG CCAGCCAGAT 2340

TGTTGCGCCG GATACCAAAG CTCCGGGCCT CACCCAGGCG CTAAATACCA AAGATGGCGC 2400

AGTGATGGTG ATGAGTTACG GGAACTCCGA AGAGGATTCA CAAGAACATA CCGGCAGTCA 2460

GTTGCGTATT GCGGCGTATG GCCCGCATGC CGCCAATGTT GTTGGACTGA CCGACCAGAC 2520

CGATCTCTTC TACACCATGA AAGCCGCTCT GGGGCTGAAA TAAAACCGCG CCCGGCAGTG 2580

AATTTTCGCT GCCGGGTGGT TTTTTTGCTG TTAGCAACCA GACTTAATGG CAGATCACGG 2640

GCGCATACGC TCATGGTTAA AACATGAAGA GGGATGGTGC TATGAAAATA ACATTACTGG 2700

TTACATCAAA TCAAACCGGG GGAGACCGGC CAGATCCTCT AGAGTCGACC TGCAGGCATG 2760

CAAGCTTACT CCCCATCCCC TCCAGTAATG ACCTCAGAAC TCCATCTGGA TTTGTTCAGA 2820

ACGCTCGGTT GCCGCCGGGC GTTTTTTATT GGTGAGAATC GCAGCAACTT GTCGCGCCAA 2880

TCGAGCCATG TCGTCGTCAA CGACCCCCCA TTCAAGAACA GCAAGCAGCA TTGAGAACTT 2 40

TGGAATCCAG TCCCTCTTCC ACCTGCTGAG GGCAATAAGG GCTGCACGCG CACTTTTATC 3000

CGCCTCTGCT GCGCTCCGCC ACCGTAGTTA AATTTATGGT TGGTTATGAA ATGCTGGCAG 3060

AGACCCAGCG AGACCTGACC GCAGAACAGG CAGCAGAGCG TTTGCGCGCA GTCAGCGATA 3120

CCCCGGTTGA TAATCAGAAA AGCCCCAAAA ACAGGAAGAT TGTATAAGCA AATATTTAAA 3180

TTGTAAACGT TAATATTTTG TTAAAATTCG CGTTAAATTT TTGTTAAATC AGCTCATTTT 3240

TTAACCAATA GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG CCCGAGATAG 3300

GGTTGAGTGT TGTTCCAGTT TGGAACAAGA^" GTCCACTATT AAAGAACGTG GACTCCAACG 3360

TCAAAGGGCG AAAAACCGTC TATCAGGGCG ATGGCCCACT ACGTGAACCA TCACCCAAAT 3420

CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA GGGAGCCCCC 3480

GATTTAGAGC TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA 3540

AAGGAGCGGG CGCTAGGGCG CGAGCAAGTG TAGCGGTCAC GCGCGCGTAA CCACCACACC 3600

CGCCGCGCTT AATGCGCCGC TACAGGGCGC GTATCCATTT TCGCGAATCC GGAGTGTAAG 3660

AAATGAGTCT GAAAGAAAAA ACACAATCTC TGTTTGCCAA CGCATTTGGC TACCCTGCCA 3720

CTCACACCAT TCAGGTGCGT CATATACTGA CTGAAAACGC CCGCACCGTT GAAGCTGCCA 3780

GCGCGCTGGA GCAAGGCGAC CTGAAACGTA TGGGCGAGTT GATGGCGGAG TCTCATGCCT 3840

CTATGCGCGA TGATTTCGAA ATCACCGTGC CGCAAATTGA CACTCTGGTA GAAATCGTCA 3900

AAGCTGTGAT TGGCGACAAA GGTGGCGTAC GCATGACCGG CGGGGGATTT GGCGGCTGTA 3960

TCGTCGCGCT GATCCCGGAA GAGCTGGTGC CTGCCGCACA GCAAGCTGTC GCTGAACAAT 4020

ATGAAGCAAA AACAGGTATT AAAGAGACTT TTTACGTTTG TAAACCATCA CAAGGAGCAG 4080 GACAGTGCTG AACGAAACTC CCGCACTGGC ACCCGATGGC AGCCGTACCG ACTGTTCTGC 4140

CTCGCGCGTT TCGGTGATGA CGGTGAAAAC CTCTGACACA TGCAGCTCCC GGAGACGGTC 4200

ACAGCTTGTC TGTAAGCGGA TGCCGGGAGC AGACAAGCCC GTCAGGGCGC GTCAGCGGGT 4260

GTTGGCGGGT GTCGGGGCGC AGCCATGACC CAGTCACGTA GCGATAGCGG AGTGTATACT 4320

GGCTTAACTA TGCGGCATCA GAGCAGATTG TACTGAGAGT GCACCΛTATG CGGTGTGAAA 380

TACCGCACAG ATGCGTAAGG AGAAAATACC GCATCAGGCG CTCTTCCGCT TCCTCGCTCA 4440

CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG 4500

TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC 4560

AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC 4620

CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC 4680

TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC 4740

TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCAAT 4800

GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC 4860

ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA 4920

ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG 4980

CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA 5040

GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG 5100

GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC 5160

AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT 5220

CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA 5280

GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT 5340

ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA 5400

TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC 5460

GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG 5520

CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG 5580

CAACTTTATC CGCCTCCATC CACTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT 5640

CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT 5700

CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT 5760

CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA 5820

AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA 5880 TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT 5940

AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAC ACGGGATAAT ACCGCGCCAC 6000

ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA 6060

GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT 6120

CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG 6180

CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT 6240

ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT 6300

AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT 6360

AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC 6420

GTCTTCAAGC CCGAGGTAAC AAAAAAACAA CAGCATAAAT AACCCCGCTC TTACACATTC 6480

CAGCCCTGAA AAAGGGCATC AAATTAAACC ACACCTATGG TGTATGCATT TATTTGCATA 65 0

CATTCAATCA ATTGTTATCT AAGGAAATAC TTACATATGG TTCGTGCAAA CAAACGCAAC 6600

GAGGCTCTAC GAATCGATGC ATGCAGCTGA TTTCACTTTT TGCATTCTAC AAACTGCATA 6660

ACTCATATGT AAATCGCTCC TTTTTAGGTG GCACAAATGT GAGGCATTTT CGCTCTTTCC 6720

GGCAACCACT TCCAAGTAAA GTATAACACA CTATACTTTA TATTCATAAA GTGTGTGCTC 6780

TGCGAGGCTG TCGGCAGTGC CGACCAAAAC CATAAAACCT TTAAGACCTT TCTTTTTTTT 6840

ACGAGAAAAA AGAAACAAAA AAACCTGCCC TCTGCCACCT CAGCAAAGGG GGGTTTTGCT 6900

CTCGTGCTCG TTTAAAAATC AGCAAGGGAC AGGTAGTATT TTTTGAGAAG ATCACTCAAA 6960

AAATCTCCAC CTTTAAACCC TTGCCAATTT TTATTTTGTC CGTTTTGTCT AGCTTACCGA 7020

AAGCCAGACT CAGCAAGAAT AAAATTTTTA TTGTCTTTCG GTTTTCTAGT GTAACGGACA 7080

AAACCACTCA AAATAAAAAA GATACAAGAG AGGTCTCTCG TATCTTTTAT TCAGCAATCG 7140

CGCCCGATTG CTGAACAGAT TAATAATAGA TTTTAGCTTT TTATTTGTTG AAAAAAGCTA 7200

ATCAAATTGT TGTCGGGATC AATTACTGCA AAGTCTCGTT CATCCCACCA CTGATCTTTT 7260

AATGATGTAT TGGGGTGCAA AATGCCCAAA GGCTTAATAT GTTGATATAA TTCATCAATT 7320

CCCTCTACTT CAATGCGGCA ACTAGCAGTA CCAGCAATAA ACGACTCCGC ACCTGTACAA 7380

ACCGGTGAAT CATTACTACG AGAGCGCCAG CCTTCATCAC TTGCCTCCCA TAGATGAATC 7440

CGAACCTCAT TACACATTAG AACTGCGAAT CCATCTTCAT GGTGAACCAA AGTGAAACCT 7500

AGTTTATCGC AATAAAAACC TATACTCTTT TTAATATCCC CGACTGGCAA TGCCGGGATA 7560

GACTGTAACA TTCTCACGCA TAAAATCCCC TTTCATTTTC TAATGTAAAT CTATTACCTT 7620

ATTATTAATT CAATTCGCTC ATAATTAATC CTTTTTCTTA TTACGCAAAA TGGCCCGATT 7680

TAAGCACACC CTTTATTCCG TTAATGCGCC ATGACAGCCA TGATAATTAC TAATACTAGG 7740 AGAAGTTAAT AAATACGTAA CCAACATGAT TAACAATTAT TAGAGGTCAT CGTTCAAAAT 7800

GGTATGCGTT TTGACACATC CACTATATAT CCGTGTCGTT CTGTCCACTC CTGAATCCCA 7860

TTCCAGAAAT TCTCTAGCGA TTCCAGAAGT TTCTCAGAGT CGGAAAGTTG ACCAGACATT 7920

ACGAACTGGC ACAGATGGTC ATAACCTGAA GGAAGATCTG ATTGCTTAAC TGCTTCAGTT 7980

AAGACCGAAG CGCTCGTCGT ATAACAGATG CGATGATGCA GACCAATCAA CATGGCACCT 8040

GCCATTGCTA CCTGTACAGT CAAGGATGGT AGAAATGTTG TCGGTCCTTG CACACGAATA 8100

TTACGCCATT TGCCTGCATA TTCAAACAGC TCTTCTACGA TAAGGGCACA AATCGCATCG 8160

TGGAACGTTT GGGCTTCTAC CGATTTAGCA GTTTGATACA CTTTCTCTAA GTATCCACCT 8220

GAATCATAAA TCGGCAAAAT AGAGAAAAAT TGACCATGTG TAAGCGGCCA ATCTGATTCC 8280

ACCTGAGATG CATAATCTAG TAGAATCTCT TCGCTATCAA AATTCACTTC CACCTTCCAC 8340

TCACCGGTTG TCCATTCATG GCTGAACTCT GCTTCCTCTG TTGACATGAC ACACATCATC 8400

TCAATATCCG AATAGGGCCC ATCAGTCTGA CGACCAAGAG AGCCATAAAC ACCAATAGCC 8460

TTAACATCAT CCCCATATTT ATCCAATATT CGTTCCTTAA TTTCATGAAC AATCTTCATT 8520

CTTTCTTCTC TAGTCATTAT TATTGGTCCA TTCACTATTC TCATTCCCTT TTCAGATAAT 8580

TTTAGATTTG CTTTTCTAAA TAAGAATATT TGGAGAGCAC CGTTCTTATT CAGCTATTAA 8640

TAACTCGTCT TCCTAAGCAT CCTTCAATCC TTTTAATAAC AATTATAGCA TCTAATCTTC 8700

AACAAACTGG CCCGTTTGTT GAACTACTCT TTAATAAAAT AATTTTTCCG TTCCCAATTC 8760

CACATTGCAA TAATAGAAAA TCCATCTTCA TCGGCTTTTT CGTCATCATC TGTATGAATC 8820

AAATCGCCTT CTTCTGTGTC ATCAAGGTTT AATTTTTTAT GTATTTCTTT TAACAAACCA 8880

CCATAGGAGA TTAACCTTTT ACGGTGTAAA CCTTCCTCCA AATCAGACAA ACGTTTCAAA 8940

TTCTTTTCTT CATCATCGGT CATAAAATCC GTATCCTTTA CAGGATATTT TGCAGTTTCG 9000

TCAATTGCCG ATTGTATATC CGATTTATAT TTATTTTTCG GTCGAATCAT TTGAACTTTT 9060

ACATTTGGAT CATAGTCTAA TTTCATTGCC TTTTTCCAAA ATTGAATCCA TTGTTTTTGA 9120

TTCACGTAGT TTTCTGTATT CTTAAAATAA GTTGGTTCCA CACATACCAA TACATGCATG 9180

TGCTGATTAT AAGAATTATC TTTATTATTT ATTGTCACTT CCGTTGCACG CATAAAACCA 9240

ACAAGATTTT TATTAATTTT TTTATATTGC ATCATTCGGC GAAATCCTTG AGCCATATCT 9300

GACAAACTCT TATTTAATTC TTCGCCATCA TAAACATTTT TAACTGTTAA TGTGAGAAAC 9360

AACCAACGAA CTGTTGGCTT TTGTTTAATA ACTTCAGCAA CAACCTTTTG TGACTGAATG 9420

CCATGTTTCA TTGCTCTCCT CCAGTTGCAC ATTGGACAAA GCCTGGATTT ACAAAACCAC 9480

ACTCGATACA ACTTTCTTTC GCCTGTTTCA CGATTTTGTT TATACTCTAA TATTTCAGCA 9540 CAATCTTTTA CTCTTTCAGC CTTTTTAAAT TCAAGAATAT GCAGAAGTTC AAAGTAATCA 9600

ACATTAGCGA TTTTCTTTTC TCTCCATGGT CTCACTTTTC CACTTTTTGT CTTGTCCACT 9660

AAAACCCTTG ATTTTTCATC TGAATAAATG CTACTATTAG GACACATAAT ATTAAAAGAA 9720

ACCCCCATCT ATTTAGTTAT TTGTTTAGTC ACTTATAACT TTAACAGATG GGGTTTTTCT 9780

GTGCAACCAA TTTTAAGGGT TTTCAATACT TTAAAACACA TACATACCAA CACTTCAACG 9840

CACCTTTCAG CAACTAAAAT AAAAATGACG TTATTTCTAT ATGTATCAAG ATAAGAAAGA 9900

ACAAGTTCAA AACCATCAAA AAAAGACACC TTTTCAGGTG CTTTTTTTAT TTTATAAACT 9960

CATTCCCTGA TCTCGACTTC GTTCTTTTTT TACCTCTCGG TTATGAGTTA GTTCAAATTC 10020

GTTCTTTTTA GGTTCTAAAT CGTGTTTTTC TTGGAATTGT GCTGTTTTAT CCTTTACCTT 10080

GTCTACAAAC CCCTTAAAAA CGTTTTTAAA GGCTTTTAAG CCGTCTGTAC GTTCCTTAAG 10140

Claims

We claim:

1. A method for the exocellular production of a thermostable protein from Bacillus sp. comprising the steps of: (i) creating a DNA fragment comprising:

(a) a suitable promoter, further comprising transcription and translation initiation sites; and

(b) a gene encoding a biologically active thermostable enzyme, wherein said promoter transcription and translation initiation sites are operably linked to the 5' end of the gene; (ii) cloning said DNA fragment into an appropriate transformation vector; (iii) transforming said bacteria with said vector; and (iv) growing said transformed bacteria under conditions whereby the thermostable protein is expressed.

2. A method as recited in Claim 1 wherein said thermostable protein is xylanase.

3. A method as recited in Claim 1 wherein said thermostable protein is β-glucosidase.

4. A method as recited in Claim 1 wherein said bacteria is selected from the group consisting of

B. subtilis, B. liceniformis and B. brevis .

5. The method of Claim 1 wherein the gene encoding a thermostable enzyme for use in step 1(b) is derived from thermophilic bacteria or thermophilic f ngi.

6. The method of Claim 5 wherein the gene for encoding a thermostable enzyme is derived from a member of the group consisting of the Cal ocelJuiTi genus, thermophilic sulfate-reducing bacteria, thermophilic Bacillus sp . , Thermococcus sp . and thermophilic Clostridium sp.

7. The method of Claim 5 wherein the gene for encoding a thermostable enzyme is derived from fungi of the group consisting of thermophilic Aspergillus sp . , and Trichoderma sp.

8. The method as recited in Claim 4 wherein said bacteria is B. subtilis and the gene encoding a biologically active thermostable enzyme is selected from genes encoding thermostable xylanase or β-glucosidase.

9. A transformed Bacillus subtilis which secretes biologically active xylanase enzyme corresponding to the ATCC designation ATCC 69572.

10. A transformed Bacillus subtilis which secretes biologically active β-glucosidase enzyme corresponding to the ATCC designation ATCC 69573.

11. A method for making a transformation vector for transforming bacteria, so as to exocellularly produce a thermostable protein, comprising the steps of:

(i) creating a DNA fragment comprising

(a) a suitable promoter and

(b) a gene encoding a biologically active thermostable enzyme, wherein said DNA fragment does not contain a signal sequence; (ii) cloning said DNA fragment into an appropriate transformation vector.

12. A vector produced by the method of Claim 11.

13. The method of Claim 1 wherein the promoter is selected from the group consisting of the alkaline protease promoter (aprp) , the neutral protease promoter (nprp) , and the barnase promoter (iarp) .

14. The method of Claim 11 wherein the promoter is selected from the group consisting of the alkaline protease promoter (aprp) , the neutral protease promoter (nprp) , and the barnase promoter (J arp) .