| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 08.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 29.08.00 |
| PATENT TITLE |
Orpinomyces cellulase CelE protein and coding sequences |
| PATENT ABSTRACT |
A CDNA designated celE cloned from Orpinomyces PC-2 encodes a polypeptide (CelE) of 477 amino acids. CelE is highly homologous to CelB of Orpinomyces (72.3% identity) and Neocallimastix (67.9% identity), and like them, it has a non-catalytic repeated peptide domain (NCRPD) at the C-terminal end. The catalytic domain of CelE is homologous to glycosyl hydrolases of Family 5, found in several anaerobic bacteria. The gene of celE is devoid of introns. The recombinant proteins CelE and CelB of Orpinomyces PC-2 randomly hydrolyze carboxymethylcellulose and cello-oligosaccharides in the pattern of endoglucanases. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 17.07.98 |
| PATENT REFERENCES CITED |
Beguin, P. (1983) "Detection of Cellulase Activity in Polyacrylamide Gels Using Congo Red-Stained Agar Replicas" Analytical Biochem. 131:333-336. Black et al. (1994) "Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide" Biochem. J. 299:381-387. Borneman et al. (1989) "Fermentation Products and Plant Cell Wall-Degrading Enzymes Produced by Monocentric and Polycentric Anaerobic Ruminal Fungi" Applied and Environ. Microbiol. 55:1066-1073. Chen et al. (1998) "Two genes of the anaerobic fungus Orpinomyces sp. strain PC-2 encoding cellulases with endoglucanase activities may have arisen by gene duplication" FEMS Microbiol. Letts. 159:63-68. Chen et al. (1997) "Sequencing of a 1,3-1,4-.beta.-D-Glucanase (Lichenase) from the Anaerobic Fungus Orpinomyces Strain PC-2: Properties of the Enzyme Expressed in Escherichia coli and Evidence that the Gene Has a Bacterial Origin" J. Bacteriol. 179:6028-6034. Choi, S.-K. And Ljungdahl, L.G. (1996) "Structural Role of Calcium for the Organization of the Cellulosome of Clostridium thermocellum" Biochemistry 35:4906-4910. Dalrymple et al. (1997) "Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases" Microbiology 143:2605-2614. Denman et al. (1996) "Characterization of a Neocallimastix patriciarum Cellulase cDNA (celA) Homologous to Trichoderma reesei Cellobiohydrolase II" Appl. Environ. Microbiol. 62:1889-1896. Durand et al. (1996) "Molecular characterization of xyn3, a member of the endoxylanase multigene family of the rumen anaerobic fungus Neocallimastix frontalis" Curr. Genet. 30:531-540. Fanutti et al. (1995) "The Conserved Noncatalytic 40-Residue Sequence in Cellulases and Hemicellulases from Anaerobic Fungi Functions as a Protein Docking Domain" J. Biol. Chem. 270:29314-29322. GenBank Accession Number AF015248, submitted on Jul. 20, 1997. GenBank Accession Number U97153, released in Apr. 1998. Gilbert et al. (1992) "Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin" Mol. Microbiol. 6:2065-2072. Henrissat, B. and Bairoch, A. (1993) "New families in the classification of glycosyl hydrolases based on amino acid sequence similarities" Biochem. J. 293:781-788. Knowles et al. (1987) "Cellulase families and their genes" Trends Biotechnol. 5:255-261. Li et al. (1997) "Monocentric and Polycentric Anaerobic Fungi Produce Structurally Related Cellulases and Xylanases" Appl. Environ. Microbiol. 63:628-635. Li et al. (1997) "Two Cellulases, CelA and CelC, from the Polycentric Anaerobic Fungus Orpinomyces Strain PC-2 Contain N-Terminal Docking Domains for a Cellulase-Hemicellulase Complex" Appl. Environ. Microbiol. 63:4721-4728. Li et al. (1997) "High Molecular Weight Cellulase/Hemicellulase Complexes of Anaerobic Fungi" Abstr. 97.sup.th Gen. Meet. Am. Soc. Microbiol., American Society for Microbiology, Washington, DC. p. 424. Li, X.-L. And Ljungdahl, L.G. (1994) "Cloning, Sequencing, and Regulation of a Xylanase Gene from the Fungus Aureobasidium pulllans Y-2311-1" Appl. Environ. Microbiol. 60:3160-3166. Liu et al. (1997) "An endoglucanase from the anaerobic fungus Orpinomyces joyonii: characterization of the gene and its product" Can. J. Microbiol. 43:477-485. Millward-Sadler et al. (1996) "Evidence that the Piromyces gene family encoding endo-1,4-mannanases arose through gene duplication" FEMS Microbiol. Letts. 141:183-188. Reymond et al. (1991) "Molecular cloning of genes from the rumen anaerobic fungus Neocallismastix frontalis: expression during hydrolase induction" FEMS Microbiol. Letts. 77:107-112. Xue et al. (1992) "Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli" J. Gen. Microbiol. 138:1413-1420. Zhou et al. (1994) "Intronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanase" Biochem. J. 297:359-364. |
| PATENT GOVERNMENT INTERESTS |
ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT This invention was made, at least in part, with funding from the United States Department of Energy (Grant No. DE-FG05 93ER 20127). Accordingly, the United States Government has certain rights in this invention |
| PATENT CLAIMS |
We claim: 1. A non-naturally occurring recombinant DNA molecule comprising a nucleotide sequence encoding a CelE cellulase, said CelE cellulase having characteristics of an endoglucanase having hydrolytic activity for carboxymethylcellulose, barley .beta.-glucan, lichenin and para-nitrophenyl-.beta.-D-cellobioside and said nucleotide sequence hybridizing to a DNA molecule having a nucleotide sequence as given in SEQ ID NO:1, nucleotides under conditions of medium stringency, wherein conditions of medium stringency are hybridization and wash at a temperature from 50.degree. C. to 65.degree. C., 1.times.SSC, 0.1% sodium dodecyl sulfate. 2. The non-naturally occurring recombinant DNA molecule of claim 1 wherein said nucleotide sequence encodes a CelE cellulase having an amino acid sequence as given in SEQ ID NO:2. 3. The non-naturally occurring recombinant DNA molecule of claim 1 wherein said nucleotide sequence encoding said CelE cellulase is as given in SEQ ID NO:1, nucleotides 39-1469, exclusive of a transcription termination codon. 4. A recombinant host cell comprising the non-naturally occurring recombinant DNA molecule of claim 1. 5. The recombinant host cell of claim 4 wherein the nucleotide sequence encodes a mature CelE cellulase having an amino acid sequence as given in SEQ ID NO:2. 6. The recombinant host cell of claim 5 wherein said nucleotide sequence encoding said CelE cellulase is as given in SEQ ID NO:1, nucleotides 39 to 1462, exclusive of a translation termination codon. 7. A method of producing recombinant CelE cellulase in a recombinant host cell, said method comprising the steps of: (a) transforming or transfecting a host cell to contain and express a non-naturally occurring recombinant DNA molecule comprising a nucleotide sequence encoding a CelE cellulase, said cellulase having characteristics of an endoglucanase having hydrolytic activity for carboxymethylcellulose, barley .beta.-glucan, lichenin and para-nitrophenyl-.beta.-D-cellobioside and nucleotide sequence hybridizing to a DNA molecule having a nucleotide sequence as given in SEQ ID NO:1, said nucleotides under conditions of medium stringency wherein conditions of medium stringency are hybridization and wash at a temperature from 50.degree. C. to 65.degree. C. 1.times.SSC, 0.1% sodium dodecyl sulfate; and (b) culturing the recombinant host cell of step (a) under conditions for expression of the CelE cellulase. 8. The method of claim 7 wherein said nucleotide sequence encodes a CelE cellulase having an amino acid sequence as given in SEQ ID NO:2. 9. The method of claim 8 wherein said nucleotide sequence encoding said CelE cellulase is as given in SEQ ID NO:1, nucleotides 39-1469, exclusive of a transcription termination codon. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
CROSS REFERENCE TO RELATED APPLICATIONS not applicable BACKGROUND OF THE INVENTION The field of the present invention is the area of cellulolytic enzymes, nucleotide sequences encoding them and recombinant host cells and methods for producing them. Cellulosic biomass, photosynthesized by solar energy with CO.sub.2 and H.sub.2 O, is one of the most important renewable energy resources on earth. Its effective utilization through biological processes is one approach to overcoming the shortage of foods, feeds and fuels, expected as a consequence of the explosive increase in human population [Ohmiya et al. (1997)]. Several types of enzymes are required for complete hydrolysis of cellulose to glucose, including endoglucanase, exoglucanase or cellobiohydrolase and .beta.-glucosidase [Filho 1996)]. Obligately anaerobic fungi are part of the natural microflora of the alimentary tract of many herbivorous animals. Since the first anaerobic fungus, Neocallimastix frontalis was isolated in 1975 from the rumen of sheep [Orpin, G. C. (1975) J. Gen. Microbiol. 91, 249-262], at least 17 different anaerobic fungi have been isolated from ruminant and nonruminant herbivores. Anaerobic fungi produce highly active hydrolytic enzymes [Borneman et al.(1989) Appl. Environ. Microbiol. 55, 1066-1073], they physically associate with the lignocellulosic tissue of plant fragments, and their hyphae penetrate the plant tissue in vivo [Akin et al.(1983) Appl. Environ. Microbiol. 46, 738-748; Thedorou et al. (1996) Proc. Nutr. Soc. 55, 913-926]. These fungi are involved in degradation of plant biomass and play an important role in the rumen ecosystem. Several genes coding for hydrolytic enzymes have been cloned and sequenced from the monocentric fungi Neocallimastix patriciarum [Gilbert et al. (1992) Mol. Microbiol. 6, 2065-2072; Zhou et al. (1994) Biochem. J.. 297, 359-364; Black et al. (1994) Biochem. J.. 299, 381-387; Denman et al. (1996) Appl. Environ. Microbiol. 62, 1889-1896; Dalrymple et al. (1997) Microbiology 143, 2605-2614], N. frontalis [Reymond et al. (1991) FEMS Microbiol. Letts. 77, 107-112; Durand et al. (1996) Curr. Genet. 30, 531-540], and Piromyces sp. [Fanutti et al. (1995) J. Biol. Chem. 270, 29314-29322] and from the polycentric fungi Orpinomyces PC-2 [Li et al.(1997) Appl. Environ. Microbiol. 63, 628-635; Chen et al. (1997) J. Bacteriol. 179, 6028-6034] and Orpinomycesjoyonii [Liu et al. (1997) Can. J. Microbiol. 43, 477-485]. It has been suggested that genes encoding three mannanases of a Piromyces sp. [Millward-Sadler et al. (1996) FEMS Microbiol. Letts. 141, 183-188] and two cellobiohydrolases (CelA and CeiC) of Orpinomyces PC-2 [Li et al. (1997) Appl. Environ. Microbiol. 63, 4721-4728; see also WO 98/14597] resulted from gene duplications. CelB is described in WO 98/14597, incorporated by reference herein in its entirety. There is a need in the art for a high-specific-activity cellulase in pure form which degrades cellulosic materials, and for DNA encoding this cellulase to enable methods of producing the cellulase in pure form. SUMMARY OF THE INVENTION This invention provides a novel cellulase (CelE) from Orpinomyces sp. PC-2. CelE has endoglucanase activity, producing primarily cellobiose from carboxymethylcellulose, cellotetraose, .beta.-glucan, lichenin and certain other substrates, and some activity with para-nitrophenyl-.beta.-D-cellobioside as substrate. Avicel, para-nitrophenyl-.beta.-D-glucoside, oat spelt xylan and para-nitrophenyl-.beta.-D-xyloside are not hydrolyzed. This invention provides a cellulase protein termed "CelE" of Orpinomyces PC-2, which has an amino acid sequence as given in Table 3, SEQ ID NO:2. This cellulase is useful for degrading cellulosic materials. The CelE protein as from Orpinomyces PC-2 has a calculated molecular weight of 53,635 Da; however the CelE polypeptide of this invention includes proteins or polypeptides having the same or equivalent amino acid sequence and different amounts of glycosylation. The term CelE refers to the protein or polypeptide having the sequence given in SEQ ID NO:2 herein, equivalent sequences as defined below, and such sequences preceded with a methionine residue immediately preceding the listed sequence. "Substantially pure" CelE is substantially free of naturally associated components when separated from the native contaminants which accompany it in its natural state, either when isolated from Orpinomyces or when recombinantly produced in host cells such as Saccharomyces cerevisiae or Escherichia coli. A chemically synthesized CelE polypeptide protein is considered an "isolated" protein as is the protein isolated from Orpinomyces PC-2 or other host cell in which it is recombinantly produced. CelE as used herein refers to a polypeptide product which exhibits similar biological activities, i.e., has similar specific activity to natural CelE isolated from Orpinomyces PC-2 or chemically synthesized in accordance with the sequence provided in SEQ ID NO:2, enzymatic activity as measured in recognized bioassays, and has substantially the same or "equivalent" amino acid sequence as native CelE (SEQ ID NO:2). It will be understood that polypeptides deficient in one or more amino acids in the amino acid sequence reported herein for naturally occurring CelE, or polypeptides in which one or more amino acids in the amino acid sequence of natural CelE are replaced by other amino acids are within the scope of the invention and have "equivalent" sequences to that given in SEQ ID NO:2, provided that they exhibit the functional (enzymatic) activity of CelE. This invention is intended to embrace all the allelic variations of CelE. Moreover, as noted above, derivatives obtained by simple modification of the amino acid sequence of the naturally-occurring product, e.g., by way of site-directed mutagenesis or other standard procedures, are included within the scope of the present invention. Forms of CelE produced by proteolysis of host cells that exhibit similar biological activities to mature, naturally-occurring CelE are also encompassed by the present invention. The present specification provides guidance to the skilled worker for preparing a large number of equivalent sequences which preferably do not alter areas of homology shared with other cellulases. This invention also provides for genomic DNA and cDNA and non-naturally occurring recombinant DNA molecules encoding an Orpinomyces CelE protein or polypeptide. The gene encoding CelE is termed celE herein. The cDNA sequence of this gene from Orpinomyces is given in Table 3, SEQ ID NO:1, from nucleotide 39 to 1472. The celE gene is useful for recombinantly expressing the CelE mature protein in S. cerevisiae or other host cells. Of course, it is recognized by those skilled in the art that the DNA sequences may vary due to the degeneracy of the genetic code and codon usage. All DNA sequences which encode the CelE polypeptide are included in this invention, including DNA sequences (as given in SEQ ID NO:1 from 39 to 1472) having an ATG preceding the coding region for the mature protein. Additionally, it will be recognized by those skilled in the art that allelic variations may occur in the DNA sequences which will not significantly change activity of the amino acid sequences of the peptides which the DNA sequences encode. All such equivalent DNA sequences are included within the scope of this invention and the definition of the CelE mature protein coding region and CelE signal sequence coding region. The skilled artisan will understand that the amino acid sequence of the exemplified CelE polypeptide and signal peptide can be used to identify and isolate additional, nonexemplified nucleotide sequences which will encode functional equivalents to the polypeptides defined by the amino acid sequences given in SEQ ID NO:1, or an amino acid sequence of greater than 90% identity thereto and having equivalent biological activity. DNA sequences having at least about 85% homology to the DNA sequences of SEQ ID NO:1 and encoding polypeptides with the same function are considered equivalent to the sequences of SEQ ID NO:1 and are included in the definition of "DNA encoding the CelE protein" and the celE gene." Following the teachings herein, the skilled worker will be able to make a large number of operative embodiments having equivalent DNA sequences to those listed herein. The CelE coding sequences, including or excluding that encoding a signal peptide of this invention can be used to express the cellulase of the present invention in fungal host cells as well as in bacteria, including without limitation, Bacillus spp. Any host cell in which the signal sequence is expressed and processed may be used. Preferred host cells are Aureobasidium species, Aspergillus species, Trichoderma species and Saccharomyces cerevisiae, as well as other yeasts known to the art for fermentation, including Pichia pastoris (Sreekrishna, K. (1993) in Baltz, R. H., et al. (eds.) Industrial Microorganisms: Basic and Applied Molecular Genetics, ASM Press, Washington, D.C. 119-126; Glick, B. R. and Pasternak J. J. (1 994) ASM Press (1994) Washington, D.C. Filamentous fungi such as Aspergillus, Trichoderma, Penicillium, etc. are also useful host organisms for expression of the DNA of this invention. (Van den Handel, C. et al. (1991) In: Bennett, J. W. and Lasure, L. L. (eds.), More Gene Manipulations in Fungi, Academy Press, Inc., New York, 397-428). BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a comparison of the deduced amino acid sequences of Orpinomyces PC-2 CelE (top) (SEQ ID NO:2) and CelB (bottom)(SEQ ID NO:5). Amino acid residues with an identical match (.vertline.) and those with greater or lesser degrees of conservation (: or.) are indicated. The repeated peptides of the NCRPD and linker regions are underlined and double-underlined. FIGS. 2A-B provide the results of SDS-PAGE and zymogram analysis of the recombinant CelE and CelB. FIG. 2A is a photograph of a Coomassie brilliant blue stained SDS-polyacrylamide gel. FIG. 2B is a photograph of a zymogram gel. Lane S, protein molecular mass standards; lane 1, CelE (50 .mu.g); lane 2, CelB (55 .mu.g). DETAILED DESCRIPTION OF THE INVENTION The amino acids which occur in the various amino acid sequences referred to in the specification have their usual three- and one-letter abbreviations routinely used in the art: A, Ala, Alanine; C, Cys, Cysteine; D, Asp, Aspartic Acid; E, Glu, Glutamic Acid; F, Phe, Phenylalanine; G, Gly, Glycine; H, His, Histidine; I, Ile, Isoleucine; K, Lys, Lysine; L, Leu, Leucine; M, Met, Methionine; N, Asn, Asparagine; P, Pro, Proline; Q, Gln, Glutamine; R, Arg, Arginine; S, Ser, Serine; T, Thr, Threonine; V, Val, Valine; W, Trp, Tryptophan; and Y, Tyr, Tyrosine. Additional abbreviations used in the present specification include the following: aa, amino acid(s); bp, base pair(s); CD, catalytic domain(s); cDNA, DNA complementary to RNA; GCG, Genetics Computer Group, Madison, Wis; CMC, carboxymethyl cellulose; CMCase, carboxymethyl cellulase; FPase, filter paper-ase; HMWC, high-molecular weight complex(es); IPTG, isopropyl-.beta.-D-thiogalactoside; OSX, oat spelt xylan; ORF, open reading frame; RBB, remazol brilliant blue; RP, repeated peptide(s); pfu, plaque forming units. Screening of the Orpinomyces PC-2 cDNA library constructed in .lambda.ZAPII yielded twenty cellulase-producing plaques when 2.times.10.sup.5 plaque forming units (PFU) were plated. The positive plaques were further enriched and purified. PCR, restriction enzyme digestion, and sequencing analyses revealed that sixteen out of these plaques represented cDNAs of celA and celB which were isolated in our previous work [Li et al.(1997) Appl. Environ. Microbiol. 63, 628-635; Li et al. (1997) Appl. Environ. Microbiol. 63, 628-635]. The other four plaques represented four different new cellulase cDNAs (pCEL2, pCEL3, pCEL5 and pCEL8). Sequencing of the inserts in the plasmids obtained from the plaques by in vivo excision revealed that pCEL3 contained a 1,532 bp (celE) insert (SEQ ID NO:1) with a complete open reading frame (ORF) encoding a polypeptide (CelE) of 477 amino acids with a calculated mass of 53,635 Da (SEQ ID NO:2). Its size is similar to those of CelBs of Orpinomyces PC-2 [Li et al.(1997) Appl. Environ. Microbiol. 63, 628-635] and N. patriciarum [Durand et al. (1996) Curr. Genet. 30, 531-540]. A typical 9 residue poly(A) tail was found at its 3' end. The translation start codon (ATG) for celE was assigned based on the fact that there were stop codons in all three frames preceding the ORF, there was no ATG codon upstream of the proposed ORF, and a typical signal peptide [Von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690] was located at the N-terminus of the polypeptide. The G+C content of the ORF of celE was 38.6% and that of the 5' and 3' noncoding regions was extremely low (6.4%). High A+T contents have also been found in other cDNAs of the anaerobic fungi [Zhou et al. (1994) Biochem. J. 297, 359-364; Li et al.(1997) Appl. Environ. Microbiol. 63, 628-635; Chen et al. (1997) J. Bacteriol. 179, 6028-6034]. The ORF region of celE genomic DNA was amplified by PCR, and its size was compared with that amplified from cDNA. DNA bands of the same size, 1.4 kb, were visualized using genomic DNA or the cDNA library as templates, indicating that celE is devoid of introns in its ORF region. The Orpinomyces PC-2 and N. patriciarum celBs are also devoid of introns [Zhou et al. (1994) Biochem. J. 297, 359-364; Li et al. (1997) Appl. Environ. Microbiol. 63, 628-635]. The lack of introns in the endoglucanase genes of the anaerobic fungi is in contrast with at least some hydrolase genes of aerobic fungi, which have introns [Li., X.-L. and Ljungdahl, L.G. (1994) Appl. Environ. Microbiol. 60, 3160-3166; Knowles et al. (1 987) Trends Biotechnol. 5, 255-261]. Table 3 provides the nucleotide and deduced amino acid sequences of celE (Cellulase CelE) from Orpinomyces sp. strain PC-2. The repeated peptides of the NCRPD and linker regions are underlined and double-underlined. The asterisk indicates the stop codon. See also SEQ ID NO:1, SEQ ID NO:2. The nucleotide sequence of celE of Orpinomyces PC-2 has been assigned Genbank accession number U97153. The sequence of CelE of Orpinomyces PC-2, when compared with other protein sequences in the GP data bank, shared some homology with several endoglucanases of anaerobic bacteria and with CelBs of Orpinomyces and Neocallimastix. No homology with sequences of aerobic organisms was found. The highest identity was with CelBs of Orpinomyces PC-2 (72.3%) [Li et al. (1997) Appl. Environ. Microbiol. 63, 628-635] and N. patriciarum (67.9%) [Zhou et al. (1994) Biochem. J. 297, 359-364]. The CelBs have been assigned to glycosyl hydrolase Family 5 (formerly family A) [Henrissat, B. and Bairoch, A. (1993) Biochem, J. 293, 781-788], which contains endoglucanases from anaerobic bacteria, including the rumen bacteria. A comparison between the Orpinomyces PC-2 CelE and CelB amino acid sequences (SEQ ID NO: 2 and SEQ ID NO:5, respectively) is given in FIG. 1. The Orpinomyces PC-2 celE gene sequence is 99.2% identical to that of celB29 of Orpinomyces joyonii (submitted to the GenBank on Jul. 20, 1997, Accession Number AF01 5248). The deduced amino acid sequences differ in 6 amino acid residues. CelE, like CelBs of Orpinomyces PC-2 and N. patriciarum, has the noncatalytic repeated peptide domain (NCRPD) and a linker sequence separating the catalytic domain from the NCRPD. Many hydrolytic enzymes of anaerobic fungi have a NCRPD [Gilbert et al. (1992) Mol. Microbiol. 6, 2065-2072; Dalrymple et al. (1997) Microbiology 143, 2605-2614; Durand et al. (1996) Curr. Genet. 30, 531-540; Fanutti et al. (1995) J. Biol. Chem. 270, 29314-29322; Li et al.(1997) Appl. Environ. Microbiol. 63, 628-635; Li et al. (1997) supra]. The NCRPD of different enzymes contain 2 or 3 repeats, which have high homology to each other and contain from 32 to 40 amino acid residues each. NCRPDs are not involved in catalysis or cellulose binding [Gilbert et al. (1992) Mol. Microbiol. 6, 2065-2072; Black et al. (1994) Biochem. J. 299, 381-387; Li et al. (1997) supra]. It has been suggested that they function as docking domains in a fashion similar to the dockerin domains of catalytic subunits of the cellulosome of Clostridium thermocellum [Fanutti et al. (1995) J. Biol. Chem. 270, 29314-29322; Choi, S.-K. and Ljungdahl, L. G. (1996) Biochemistry 35, 4906-4910]. Enzymes from anaerobic fungi with the NCRPDs associate as large multienzyme cellulosomal like complexes [Fanutti et al. (1995) J. Biol. Chem. 270, 29314-29322; Li et al. (1997) p. 424. Abstr. 97.sup.th Gen. Meet. Am. Soc. Microbiol., American Society for Microbiology, Washington, D.C.]. The presence of a NCRPD in CelE indicates that it is one of the catalytic components of Orpinomyces PC-2 cellulase/hemicellulase multienzyme complex. Carboxymethyl cellulase (CMCase) activities were detected in cell-free extracts of E. coli harboring the plasmids pCEL3 (celE) or pOC1 (celB) [Li et al.(1997) Appl. Environ. Microbiol. 63, 628-635]. Zymogram analysis showed that the apparent molecular masses of both CelE and CelB produced in E. coli were approximately 42 kDa (FIG. 2), which is consistent with the deduced molecular masses of the mature CelE and CelB lacking the signal peptides and NCRPDs. The linker region between the catalytic domain and NCRPD is unstable and undergoes cleavage [Gilbert et al. (1992) Mol. Microbiol. 6, 2065-2072; Li et al.(1997) Appl. Environ. Microbiol. 63, 628-635]. The activities of cell-free extracts of E. coli expressing Orpinomyces CelE and CelB on various substrates were determined (Table 1). Both enzymes present in the extracts hydrolyze CMC, barley .beta.-glucan, and lichenin. The cell-free extract containing CelE and CelB have specific activities of 3.2 and 2.9 U/mg protein with CMC as substrate. Cellobiose, cellotriose and glucose are the main products generated during prolonged hydrolysis of CMC (Table 2). No detectable hydrolysis is observed with Avicel, pNP-.beta.-D-glucoside, oat spelt xylan or pNP-.beta.-D-xyloside. CelE showed some activity towards pNP-.beta.-D-cellobioside. This is in contrast to CelB, which lacks activity against this substrate. CelB from N. patriciarum also has only a low level of activity against pNP-.beta.-D-cellobioside [Xue et al. (1992) J. Gen. Microbiol. 138, 1413-1420]. Hydrolysis products formed during the action of the recombinant CelE and CelB on cello-oligosaccharides were determined by HPLC (Table 2). Neither of the two enzymes hydrolyze cellobiose. CelE slowly hydrolyzes cellotriose to cellobiose and glucose, but CelB is unable to hydrolyze this substrate. This is consistent with the fact that CelE hydrolyzed pNP-.beta.-D-cellobioside (Table 1). Cellotetraose was degraded into cellobiose, cellotriose and glucose, whereas cellopentaose was largely converted into cellobiose and cellotriose and some glucose. In the case of CelB towards cellopentaose, some cellotetraose was present in the products and it was further converted into cellobiose, cellotriose, and glucose. These observations, together with the product profiles of the hydrolysis of CMC and the substrate specificity determinations (Table 1) indicate that CelE and CelB are endoglucanases. The genes of celE and celB [Li et al.(1997) Appl. Environ. Microbiol. 63, 628-635] of Orpinomyces PC-2 and of celB [Zhou et al. (1994) Biochem. J. 297, 359-364] of N. patriciarum are devoid of introns. They are highly homologous to each other (FIG. 1) and genes encoding rumen bacterial endoglucanases. This seems to indicate that the Orpinomyces PC-2 celE and celB probably were originally transferred from rumen bacteria and subsequently duplicated in the fungus. It seems that gene duplication is a common phenomenon in anaerobic fungi. It has been postulated for multiple mannanases [Millward-Sadler et al. (1996) FEMS Microbiol. Letts. 141, 183-188] and cellobiohydrolases [Li et al. (1997) Appl. Environ. Microbiol. 63, 4721-4728]. It will be understood by those skilled in the art that other nucleic acid sequences besides that disclosed herein for CelE will function as coding sequences synonymous with the exemplified coding sequences. Nucleic acid sequences are synonymous if the amino acid sequences encoded by those nucleic acid sequences are the same. The degeneracy of the genetic code is well known to the art. For many amino acids, there is more than one nucleotide triplet which serves as the codon for a particular amino acid, and one of ordinary skill in the art understands nucleotide or codon substitutions which do not affect the amino acid(s) encoded. It is further understood in the art that codon substitutions to conform to common codon usage in a particular recombinant host cell is sometimes desirable. Specifically included in this invention are sequences from other strains of Orpinomyces and from other anaerobic fungi which hybridize to the sequence disclosed for celE under stringent conditions. Stringent conditions refer to conditions understood in the art for a given probe length and nucleotide composition and capable of hybridizing under stringent conditions means annealing to a subject nucleotide sequence, or its complementary strand, under standard conditions (i.e., high temperature and/or low salt content) which tend to disfavor annealing of unrelated sequences, (indicating about 95-100% nucleotide sequence identity). Also specifically included in this invention are sequences from other strains of Orpinomyces species and other anaerobic fungi which hybridize to the sequences disclosed for celE under moderately stringent conditions. Moderately stringent conditions refer to conditions understood in the art for a given probe sequence and "conditions of medium stringency" means hybridization and wash conditions of 50.degree.-65.degree. C., 1.times.SSC and 0.1% SDS (indicating about 80-95% similarity). Also specifically included in this invention are sequences from other strains of Orpinomyces from other anaerobic fungi, and from other organisms, including humans, which hybridize to the sequences disclosed for celE under highly stringent conditions. Highly stringent conditions refer to conditions understood in the art for a given probe sequence and "conditions of high stringency" means hybridization and wash conditions of 65-68.degree. C, 0.1.times.SSC and 0.1% SDS (indicating about 95-100% similarity). Hybridization assays and conditions are further described in Sambrook et al. (1989). A method for identifying other nucleic acids encoding celE-homologous enzymes is also provided wherein nucleic acid molecules encoding cellulases are isolated from an anaerobic fungus, and nucleic acid hybridization is performed with the nucleic acid molecules and a labeled probe having a nucleotide sequence that includes all or part of nucleotide sequence SEQ ID NO:1. By this method, silencing genes similar to the exemplified celE gene may be identified and isolated from other strains of Orpinomyces or other anaerobic fungi. All or part of a nucleotide sequence refers specifically to all continuous nucleotides of a nucleotide sequence, or e.g. 1000 continuous nucleotides, 500 continuous nucleotides, 100 continuous nucleotides, 25 continuous nucleotides, and 15 continuous nucleotides. Sequences included in this invention are those amino acid sequences which are 75% similar to the amino acid sequences encoded by the exemplified Orpinomyces strain PC-2 celE. Sequences included in this invention are also those amino acid sequences which are 80, 85, 90, 95 to 100%, and all integers between 75% and 100%, similar to the amino acid sequences encoded by exemplified Orpinomyces celE. It is well-known in the biological arts that certain amino acid substitutions may be made in protein sequences without affecting the function of the protein. Generally, conservative amino acid substitutions or substitutions of similar amino acids are tolerated without affecting protein function. Similar amino acids can be those that are similar in size and/or charge properties, for example, aspartate and glutamate, and isoleucine and valine, are both pairs of similar amino acids. Similarity between amino acid pairs has been assessed in the art in a number of ways. For example, Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement 3, Chapter 22, pp. 345-352, which is incorporated by reference herein provides frequency tables for amino acid substitutions which can be employed as a measure of amino acid similarity. Dayhoff et al.'s frequency tables are based on comparisons of amino acid sequences for proteins having the same function from a variety of evolutionarily different sources. Monoclonal or polyclonal antibodies, preferably monoclonal, specifically reacting with the particular cellulase (CelE) of the present invention may be made by methods known in the art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories; Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press, New York. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Harnes and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein. Each reference cited in the present application is incorporated by reference herein. The following examples are provided for illustrative purposes, and is not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified articles which occur to the skilled artisan are intended to fall within the scope of the present invention. |
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