WO1999035274A2

WO1999035274A2 - Nucleic acids encoding thermostable pullulanases

Info

Publication number: WO1999035274A2
Application number: PCT/IB1999/000069
Authority: WO
Inventors: Per Linå JØRGENSEN; Fiona Duffner
Original assignee: Novo Nordisk A/S
Priority date: 1998-01-07
Filing date: 1999-01-05
Publication date: 1999-07-15
Also published as: WO1999035274A3; AU1779199A

Abstract

The present invention relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the thermostable pullulanases from Fervidobacterium sp. Ven 5.

Description

NUCLEIC ACIDS ENCODING THERMOSTABLE PULLULANASES

Background of the Invention

Field of the Invention

The present invention relates to isolated nucleic acid sequences encoding polypeptides having pullulanase activity. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides.

Description of the Related Art

Thermostable puUulanases have been isolated from, for example, Bacillus acidopullulyticus, and their use in industrial saccharification processes has been described, vide EP patent publication No. 63,909. Thermostable puUulanases from Fervidobacterium are described in U.S. Patent No. 5,486,469.

It is an object of the present invention to provide DNA sequences encoding improved polypeptides having pullulanase activity.

Summary of the Invention

The present invention relates to isolated nucleic acid sequences encoding polypeptides having pullulanase activity selected from the group consisting of:

(a) a nucleic acid sequence comprising the sequence of SEQ ID NO:l; (b) a nucleic acid sequence which hybridizes under low stringency conditions with

(i) the nucleic acid sequence of SEQ ID NO:l, (ii) its com.plement.ary strand, or (iii) a subsequence of SEQ ID NO:l which encodes a polypeptide fragment which has pullulanase activity;

(c) an allelic variant of (a) or (b); and (d) a subsequence of (a), (b) or (e), which encodes a polypeptide fragment having pullulanase activity. The invention encompasses nucleic acid sequences which hybridize under medium and high stringency conditions to the nucleic acid sequence of SEQ ID NO:l.

The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides.

Brief Description of the Figures

Fig. 1 is a schematic of the pullulanase sequence strategy (not to scale). The full length of the inert is 8.1 kb.

Fig. 2 is the nucleic acid sequence of pulA .and the upstream region from

Fervidobacterium pennavorans Nen5 (SEQ ID ΝO:l). The predicted start site, ATG, is highlighted in bold. A proposed Shine-Dalgarno sequence is underlined.

Fig. 3A-C is the CLUSTAL W(1.5) multiple alignment of the amino acid sequences encoded by pulA from Thermo toga maritima Genbank accession number

AJ001087 (SEQ ID NO:2) and Fervidobacterium pennavorans Ven5 (SEQ ID NO:3). The degree of identity between the two amino acid sequences is determined by the GAP method (Higgins, 1989, CABIOS 5: 151-153) with an identity table, a gap penalty of 10, .and a gap length penalty of 10.

Detailed Description of the Invention

Nucleic Acid Sequences Encoding Polypeptides Having Pullulanase Activity

The present invention relates to isolated nucleic acid sequences which encode a polypeptide having pullulanase activity. The term "pullulanase activity" is defined herein as a pullulanase activity which catalyzes the degradation of pullulan endoglycolytically only at its α-l,6-linkages. The action pattern of the pullulanase activity is typical of random endo-attack. Starch and amylose are essentially not attacked by the pullulanase activity. Two relevant references are Antranikian, G. and Jørgensen, P.L. (1992)

Thermostable pullulanase. European Patent EP 0578672 Bl, and Koch, R., Canganella, F., Hippe, H., Jahnke, K.D., Antranikian, G. (1997) Purification and properties of a thermostable pullulanase from a newly isolated thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5. Appl. Environ. Microbiol. 63:1088-1094, both of which references are herein specifically incorporated by reference. In a preferred embodiment, the nucleic acid sequence encodes a polypeptide obtained from Fervidobacterium, e.g., Fervidobacterium pennavorans, and in a more preferred embodiment, the nucleic acid sequence is obtained from Fervidobacterium pennavorans Ven5 DSM 6204, e.g., the nucleic acid sequence set forth in SEQ ID NO:l . The present invention also encompasses nucleic acid sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO:3, which differ from SEQ ID NO:l by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO:l which encode fragments of SEQ ID NO:3 which has pullulanase activity. A subsequence of SEQ ID NO:l is a nucleic acid sequence encompassed by SEQ ID NO:l except that one or more nucleotides from the 5' and/or 3' end have been deleted. Preferably, a subsequence contains at least 15 nucleotides. The nucleic acid sequences may be obtained from microorganisms which are taxonomic equivalents of Fervidobacterium, regardless of the species name by which they are known. In another preferred embodiment, a nucleic acid sequence of the present invention encodes a polypeptide having pullulanase activity which is active at temperatures from below 45°C to above 100°C with a temperature optimum in the r.ange of from 80-90°C; active in the pH range of about 5-7, and has a residual activity, after 24 hours of incubation at 70°C and pH 6.0, of more than 60% relative.

In another preferred embodiment, the present invention relates to isolated nucleic acid sequences encoding polypeptides having pullulanase activity which hybridize under low stringency conditions, more preferably medium stringency conditions, and most preferably high stringency conditions, with an oligonucleotide probe which hybridizes under the same conditions with the nucleic acid sequence of SEQ ID NO:l or its complementary strand (Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York); or allelic variants and subsequences of SEQ ID NO:l, which encode polypeptide fragments which have pullulanase activity. Hybridization indicates that the nucleic acid sequence hybridizes to the oligonucleotide probe corresponding to the polypeptide encoding part of the nucleic acid sequence shown in SEQ ID NO:l, under low to high stringency conditions (i.e., prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25, 35 or 50% formamide for low, medium and high stringencies, respectively), following standard Southern blotting procedures.

The amino acid sequence of SEQ ID NO:3 or a partial sequence thereof may be used to design an oligonucleotide probe, or a nucleic acid sequence encoding a polypeptide of the present invention, such as the nucleic acid sequence of SEQ ID NO:l, or a subsequence thereof, may be used to identify and clone DNA encoding polypeptides having pullulanase activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 40 nucleotides in length. Longer probes can also be used. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).

Thus, a genomic, cDNA or combinatorial chemical library prepared from such other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having pullulanase activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA which is homologous with SEQ ID NO:l, the carrier material is used in a Southern blot in which the carrier material is finally washed three times for 30 minutes each using 2 x SSC, 0.2% SDS preferably at least 50°C, more preferably at least 55°C, more preferably at least 60°C, more preferably at least 65°C, even more preferably at least 70°C, and most preferably at least 75°C. Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using X-ray film. Polypeptides encoded by nucleic acid sequences which hybridize with an oligonucleotide probe which hybridizes with the nucleic acid sequence of SEQ ID NO:l, its complementary strand, or allelic variants and subsequences of SEQ ID NO.l; or allelic variants and fragments of the polypeptides may be obtained from microorganisms of any genus. The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The term "isolated nucleic acid sequence" as used herein refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40%) pure, more preferably at least about 60%) pure, even more preferably at least about 80%> pure, most preferably at least about 90%> pure as determined by agarose electrophoresis. For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

The present invention also relates to nucleic acid sequences which have a degree of homology to the nucleic acid sequence of SEQ ID NO:l of at least about 65%o, preferably about 70%), preferably about 80%>, more preferably about 90%>, even more preferably about 95%, and most preferably about 97% homology, which encode an active polypeptide. For purposes of the present invention, the degree of homology between two nucleic acid sequences is determined by the Clustal method (Higgins, 1989, supra) with an identity table, a gap penalty of 10, and a gap length penalty of 10.

Modification of a nucleic acid sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source. For example, it may be of interest to synthesize variants of the polypeptide where the variants differ in specific activity, thermostability, pH optimum, or the like using, e.g., site-directed mutagenesis. The analogous sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of SEQ ID NO:l, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al, 1991, Protein Expression and Purification 2: 95-107, herein specifically incorporated by reference.

It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, .and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for pullulanase activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., de Vos et αl, 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899- 904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

Nucleic Acid Constructs The present invention also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. "Nucleic acid construct" is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term "coding sequence" as defined herein is a sequence which is transcribed into mRNA and translated into a polypeptide of the present invention. The boundaries of the coding sequence are generally determined by a translation start codon ATG at the 5 '-terminus and a translation stop codon at the 3 '-terminus. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleic acid sequence encoding a polypeptide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing cloning methods are well known in the art.

The term "control sequences" is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide of the present invention.

Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. The term "operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the production of a polypeptide. The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquejaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al, 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al, 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook et al, 1989, supra.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a suitable leader sequence, a nontranslated region of a mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of the polypeptide which can direct the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the polypeptide. The signal peptide coding region may be obtained from an amylase or a protease gene from a Bacillus species.. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.

An effective signal peptide coding region for bacterial host cells is the signal peptide coding region obtained from the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilis PrsA gene. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

The control^' sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE) or the Bacillus subtilis neutral protease gene (nprT). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

The nucleic acid constructs of the present invention may also comprise one or more nucleic acid sequences which encode one or more factors that are advantageous for directing the expression of the polypeptide, e.g., an activator (e.g., a tr m-acting factor), a chaperone, and a processing protease. Any factor that is functional in the host cell of choice may be used in the present invention. The nucleic acids encoding one or more of these factors are not necessarily in tandem with the nucleic acid sequence encoding the polypeptide.

An activator is a protein which activates transcription of a nucleic acid sequence encoding a polypeptide (Kudla et al, 1990, EMBO Journal 9: 1355-1364; Jarai and Buxton, 1994, Current Genetics 26: 2238-244; Verdier, 1990, Yeast 6: 271-297). The nucleic acid sequence encoding an activator may be obtained from the gene encoding Bacillus stearothermophilus NprA (nprA). For further examples, see Verdier, 1990, supra and MacKenzie et al, 1993, Journal of General Microbiology 139: 2295-2307.

A chaperone is a protein which assists another polypeptide in folding properly (Hartl et al, 1994, TIBS 19: 20-25; Bergeron et al, 1994, TIBS 19: 124-128; Demolder et al, 1994, Journal of Biotechnology 32: 179-189; Craig, 1993, Science 260: 1902-1903; Gething and Sambrook, 1992, Nature 355: 33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry 269: 7764-7771; Wang and Tsou, 1993, The FASEB Journal 7: 1515-11157; Robinson et al, 1994, Bio/Technology 1 : 381-384; Jacobs et al, 1993, Molecular Microbiology 8: 957-966). The nucleic acid sequence encoding a chaperone may be obtained from the genes encoding Bacillus subtilis GroE proteins and Bacillus subtilis PrsA. For further examples, see Gething and Sambrook, 1992, supra, and Hartl et al, 1994, supra. A processing protease is a protease that cleaves a propeptide to generate a mature biochemically active polypeptide (Enderlin and Ogrydziak, 1994, Yeast 10: 67-79; Fuller et al, 1989, Proceedings of the National Academy of Sciences USA 86: 1434-1438; Julius et al, 1984, Cell 37: 1075-1089; Julius et al, 1983, Cell 32: 839-852). It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Ex.amples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems would include the lac, tac, and trp operator systems. Other examples of regulatory sequences are those which allow for gene amplification. In these cases, the nucleic acid sequence encoding the polypeptide would be operably linked with the regulatory sequence.

Expression Vectors The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i. e. , a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The vector system may be a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resist.ance. Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO 91/17243, where the selectable marker is on a separate vector.

The vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell. For integration into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination. These nucleic acid sequences may be any sequence that is homologous with a target sequence in the genome of the host cell, and furthermore, may be non-encoding or encoding sequences. For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUBl 10, pE194, pTA1060, and pAMBl permitting replication in Bacillus. The origin of replication may be one having a mutation which makes its functioning temperature- sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).

More than one copy of a nucleic acid sequence encoding a polypeptide of the present invention may be inserted into the host cell to amplify expression of the nucleic acid sequence. Stable amplification of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by culturing the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al, 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. The term "host cell" encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur during replication. A vector comprising a nucleic acid sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self- replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the host chromosome may occur by homologous or non- homologous recombination as described above. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-5278).

Methods of Production

The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a strain, which in its wild-type form is capable of producing the polypeptide, to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide. Preferably, the strain is of the genus Fervobacterium.

The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell ly sates. The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide. Procedures for determining pullul.anase activity .are known in the art and include, e.g., Antraniki.an, G. and Jørgensen, P.L. (1992). Thermostable pullulanase. European Patent EP 0578672 Bl and Koch, R., Canganella, F., Hippe, H., Jahnke, K.D., Antranikian, G. (1997). Purification and properties of a thermostable pullulanase from a newly isolated thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5. Appl. Environ. Microbiol. 63:1088-1094. The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Examples

EXAMPLE 1. Cultivation Example

The strain DSM 6204 was cultivated at 60°C under continuous gassing with

N₂/C0₂ (80:20) in a medium (starch complex medium) of the following composition (per litre):

K₂HP0₄ 1.6 g

NaH₂P0₄ 1.0 g

(NH₄)₂S0₄ 0.8 g

NH₄C1 0.6 g Tryptone 1.0 g

Yeast extract 1.0 g

Peptone 1.0 g

MgS0₄ X 7H₂0 0.4 g

CaCl₂ X 2H₂0 0.2 g Trace Element Solution SL 11 1.0 ml

Vitamine Solution* (10-fold) 2.0 ml

Soluble starch** 2.5 g

Resazurin 1.0 mg

Na-S X 9 H₂0 0.5 g NaHC0₃ 1.0 g pH: 6.8-7.0

* Vitamin solution according to WOLIN et al., 1964, J. Bacteriol. 87, p. 993-998.

** Soluble starch from Fluka

The medium was prepared anaerobically under N₂/C0₂ (80:20). CaCl₂;2H₂0, Na₂S;9H₂0 and NaHC0₃ were autoclaved separately under nitrogen. The Na₂S-solution was adjusted to pH 7.0 before it was added to the medium. During growth amylolytic and pullulytic activity, optical density, pH, and residual starch concentration were measured. After 20 hours of growth 20 U/l amylase and 20 U/l pullulanase were detected in the cell free supernatant.

Method of Analysis for Pullulanase Activity. Pullulanase activity was measured by determining the amount of reducing sugars liberated during incubation with pullulan at 85°C and pH 6.0. The activity of 1 U of pullulanase was defined as that amount of enzyme which liberates 1 μmol of reducing sugar per minute using maltose as a standard. The enzymatic reactions were conducted in 50 mM sodium-phosphate buffer, pH 6.0, the concentration of the substrate was 0.5%) (w/v). The reaction mixture was prepared by adding 50 μl enzyme solution to 200 μl substrate solution. The mixture was incubated at 85°C for 5 minutes, cooled and diluted with 2.5 ml water. After incubation 250 μl dinitrosalicylic acid reagent (i.e. dinitrosalicylic acid 1 g, 2 N NaOH 20 ml, K-Na-tartrate x 4 H₂0 30 g, and distilled water 100 ml) were added. The optical density was measured at 546 nm against a mixture which was not in- cubated.

EXAMPLE 2. Purification Example

40 1 of cell free supernatant obtained as described in Example 1 were concentrated 200 fold, dialysed against 50 mM sodium phosphate pH 7.5, and separated by ion exchange chromatography with Q-Seph.arose. Pullulanase did not bind to the column but was eluted during washing with buffer. The fractions containing pullulytic activity were pooled, concentrated and separated by gel filtration on a Hi Load 16/60 Supadex 200 column. After this procedure the pullulanase was free of amylase and only very low amylolytic activity which was due to unspecifϊc reactions of the pullulanase could be detected. Further purification was achieved by hydrophobic chromatography on Phenyl- Superose with a decreasing (NH₄)₂S0₄-gradient (1.5 M - 0 M). After this procedure the specific activity of the pullulanase reached approximately 110 U/mg and most of the experiments were performed with enzyme which was purified in the above mentioned manner. The enzyme purified to homogeneity by preparative gel electrophoresis and SDS- gel electrophoresis revealed only one protein band with a molecular mass of 93 kDa. Due to the low stability of the pullulanase during this separation the specific activity decreased to 8 U/mg.

EXAMPLE 3. Pullulanase Characterization

5 The purified pullulanase obtained as described in Example 2 was subjected to characterization using the method for analyzing pullulanase activity described in Example 1. pH and Temperature Optima. The purified pullulanase is active at temperatures in the range 80-90°C, around 85°C (at pH 6.0), and at a pH range of 5-7, at 85°C. 0 Substrate Specifity. The pullulanase was incubated with different glucose polymers such as starch, pullulan, branched oligosaccharides, amylose and glycogen. The enzyme showed the highest activity with pullulan as a substrate and 50%) of the pullulytic activity were detected if it was incubated with branched oligosaccharides. Starch and amylose remained almost unattacked, only 5»10%o of the pullulytic activity were found with these s substrates. The highly branched saccharide glycogen was not degraded.

Analysis of the sugars released during the degradation of pullul-an, branched oligosaccharides and amylose showed that pullulan was degraded endoglycolytically and cleaved only at its α-l,6-linkages. Also the branched oligosaccharides were attacked only on their branching points, and the released linear oligomers were easily degraded by an 0 added oglucosidase to glucose. Amylose remained almost unattacked.

Influence of Metal Ions, Cvclodextrins and Glucose Polymers on the Pullulytic Activity. To investigate the influence of metal ions, cyclodextrins and various glucose polymers on the activity of the pullulanase the purified enzyme was incubated with a 0.5% pullulan solution (pH 6.0) in the presence of up to 5 mM of metal ions and cyclodextrins. 5 Amylose, starch, glycogen, pullulan and branched oligosaccharides were added in concentrations of up to 0.05%). Results showed that calcium, magnesium, molybdenum ions and EDTA did not influence the pullulytic activity. The addition of cobalt and nickel ions as well as α-cyclodextrins decreased the activity of the enzyme slightly and in the presence of 5 mM -of copper, zinc, and chromate ions or β-cyclodextrin the pullulanase o was inhibited completely. Surprisingly, the addition of amylose and starch did also influence the pullulanase and about 20 to 30% of the initial activity were found if 0.05%) of this polymer was added.

Thermal Stability. Investigation of the thermal stability showed that the enzyme could be incubated in the absence of substrate and metal ions at 70°C for 24 hours at a pH of 6.0 without any substantial loss of activity. During incubation at 80°C only approximately 10% of the initial activity could be detected after the same period, while at

90°C the enzyme was inactivated within 15 minutes.

In order to investigate the thermal stability under the conditions prevailing in the starch saccharification process the enzyme was also incubated in the presence of starch and calcium ions at 80°C and pH 4.5. Both the addition of calcium ions and starch increased the thermal stability significantly although the activity of the pure enzyme was reduced.

In the absence of starch and metal ions the enzyme was completely inactivated during 10 minutes of incubation. In the presence of 5 mM calcium the inactivation was slower, 5%> of the initial activity were detected after 30 min. The addition of starch increased the thermal stability significantly although the initial pullulytic activity was reduced to 20%> of the starch free sample. The highest stability was observed in the presence of both calcium ions and starch. Under these conditions the pullulanase was stable for 24 hours.

EXAMPLE 4. Cloning of a Pullulanase Gene from Fervidobacterium Fervidobacterium sp. Ven 5 (DSM 6204) chromosomal DNA was isolated according to Pitcher et al. (1989) Lett. Appl. Microbiol. 8:151-156 .and partially digested with Sau3A. 100 μg of Fervidobacterium DNA were digested with 20 units of Sau3A for 10 min. at 37°C. The digestion was terminated by phenolxhloroform extraction and the DNA was ethanol precipitated. Ligation was performed by using chromosomal DNA: pSJ933 (Digested by BamHI and the larger fragment of 5.8 kb was isolated) with a ratio of 1:3 using 4 μg of DNA/ 10 μl and adding 2 units of T4 ligase and incubating at room temperature (25°C) for 4 hours. The plasmid pSJ933 has been deposited in the E. coli strain SJ989 on 26 September 1990 according to the Budapest Treaty at National Collections of Industrial & Marine Bacteria Ltd., 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, UK, under Accession No. NCIMB 40320. E. coli strain MCI 000 was transformed with the ligated DNA and plated on Luria broth plus 2% agar containing 10 μg/ml chloramphenicol and incubated at 37°C. After 16 hours of incubation approximately 14,000 chloramphenicol resistant colonies were observed on the plates. These colonies were replica plated onto a new set of Luria broth plates containing 2%> agar, 6 μg/ml chloramphenicol and 0,1 %> dyed pullulan and grown overnight.

Dyed pullulan (50 g pullulan; Hayashibara Biochemical laboratories) and 5 g of Cibachrom Rot B (Ciba Geigy) were suspended in 500 ml 0.5 M NaOH and the mixture incubated under constant stirring at room temperature for 16 hours. pH was adjusted to 7.0 with 4N H2S0₄. The dyed pullulan was thereafter harvested by centrifugation and the pullulan is washed 3 times with distilled water and resuspended in an appropriate volume of distilled water.

The plates were then incubated at 60°C for 4 hours and around one of the colonies a halo appeared resulting from degradation of the dyed pullulan. The corresponding colony on the first set of Luria broth plates was isolated and analyzed for plasmid content. The isolated colony PL2125 was grown in 10 ml Luria broth, the plasmid was isolated by the method described by Kieser et al. (1984) Plasmid 12:19. The plasmid was analyzed by restriction mapping and showed an insert of Fervidobacterium DNA of approximately 8 kb.

EXAMPLE 5. Subcloning and Sequencing of the Pullulanase from Fervobacterium pennavorans Ven5. The entire 8.128 kb insert in pPL2125 conferring pullulanase activity on the E. coli clone PL2125 (described in patent EP 0578672 Bl) was sequenced as described below, and three large open reading frames (ORF's) were identified. ORF1 identified experimentally and on the basis of amino acid homologies as encoding the pullulanase is 2550 bp long and encodes a protein of 849 amino acids with a predicted molecular mass of 96.6 kDa before processing. This fragment is processed in E.coli to yield a protein of 732 amino acids and molecular mass of 83 kDa. The N-terminal sequence, QGIEQIYTTKPDTSPRVL (SEQ ID NO:4), of the pullulanase purified from the E.coli clone PL2125 was identified 108 amino acids downstream from the proposed ATG start site. A perfect SD sequence of AGGAGG is present at postions -10 to -15 from the ATG site. This gene is referred to .as pulA. The N-terminal sequence of the enzyme isolated from Fervidobacterium pennavorans has been deterimned to be ETELIIHYHRW. A signal sequence is therefore present at the N-terminal end of the protein and signal peptide 5 cleavage takes place 28 amino acids upstream from the initial methionine between the amino acids Ala and Glu.

On comparison with the sequence databases pulA shared 61.5% pairwise amino acid identity identity (using pairwise alignment in Clustal W, gap penalty 3 and PAM250 residue weight table) and 62%> pairwise DNA identity (using pairwise alignment in Clustal 0 W, gap penalty 5) with j?w/-4 from Thermotoga maritima. Using the GAP programmme of GCG (Version 8, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin USA 53711; Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48:443- 453) pulA from Fervidobacterium pennavorans shared 62.6%) amino acid identity and 80%) amino acid similarity with pulA from Thermotoga maritima. The conditions were 5 Gap weight, 30; length weight, 1. Using the GAP programme from GCG (see above) with Gap weight, 50 and length weight, 3 the percent identity and similarity between the DNA sequence of pulA from Fervidobacterium pennavorans .and pulA from Thermotoga maritima was 66%>.

In addition, the presence of an ORF of 2694 bp encoding a protein of 897 amino 0 acids with a predicted molecular mass of 103.5 kDa and having 68.3% overall amino acid homology (deterined with Clustal W) with isoleucyl-tRNA synthase (ileS) of Thermotoga maritima was also found. The G+C%o content of ORF2 is 41.79%). The construction of a phylogenetic tree based on isoleucyl-tRNA synthase sequences from Aquifex pyrophilus, Thermotoga maritima, Staphylococcus aureus, human T-lymphocyte. Tetrahymena 5 thermophila .and Campylobacter jejuni placed ORF2 firmly in the T. maritima group. This is a very useful finding as the isoleucyl-tRNA synthase genes are used for taxonomic identification (Brown, J.R. and Doolittle, W.F., Root of the universal tree of life based on ancient aminoacyl-tRNA synthase gene duplications, Proc. Natl. Acad. Sci. U.S.A. 92 (7) 2442-2445, 1995). This confirms that the insert containing the pullulanase is from a o bacterium of the Thermotoga group which includes Fervidobacterium sp. 0FR3 is 1272 bp in length encoding a protein of 423 amino acids with a predicted molecular mass of 46.1 kDa. No significant homologies to database sequences were shown to exist with FASTA and BLAST searches.

The plasmid pPL2125 containing the Sau3A fragment of genomic DNA cloned into pSJ933 and encoding pullulanase activity as detected on pullulan red agar plates was obtained as described in European Patent EP 0578 672 Bl, herein specifically incorporated by reference. Isolation of plasmid DNA from E. coli pPL2125 (CMLR ) was carried out using the Qiagen Miniprep kit, cat# 27106. Extraction of the ~ 8kb insert from a Pstϊ digest of pPL2125 was carried out following electrophoresis using the Qiagen Gel Extraction kit, cat # 28704. Sequencing reactions contained 7-1 lμl of the plasmid DNA isolated using the Qiagen mini prep, and 5-10pMol primer. The dye termination method was used and the sequencing was carried out on an ABI automatic DNA sequencer. The list of primers is given below (Table 1). PCR amplification was performed using the ExpandTM Long template PCR system from Boehringer Mannheim. Cloning the PCR fragments into pCR2.1 was carried out using the TA cloning kit from Invitrogen. Sequence alignments were carried out using Clustal W.

Subcloning of the pullulanase into pUC18. Restriction analysis revealed that the insert contained 4 EcoRI, 4 Hindlϊl, 1 BamHI, 3-4 Clal, 3 Sail, 2 Ncol, 2 Ndel, 1 Sphl .and no Pstl, Smal, BgHl, Kpήl sites. The Pstl fragment containing the pullulanase gene was purified and cloned into pUC18 using standard procedures. The recombinant E. coli clone which displayed pullulanase activity on pullulan red agar plates was called E. coli pUCPULL. The insert could then be sequenced in both directions.

PCR amplification using the N-terminal sequence of the pullulanase. The first good N-terminal sequence of the pullulanase purified from E.coli PL2125 was obtained and a degenerate primer was made. A series of PCR reactions established the orientation of the gene with respect to the Ml 3 sequencing primers of pUCPULL. Using the N- terminal primer (Npull) and seq5 a fragment of approximately 3kb was amplified. The size of this fragment increased according to calculations when using primers Npull with seq 4 and seq3. The smallest fragment (N5) was cloned into the vector pCR2.1 (TA cloning kit, Invitrogen) however, transformants displayed no pullulanase activity at 37°C on pullulan red agar. The transformants were identified by PCR amplification of the N5 fragment. The E.coli clone, confirmed by restriction analysis and sequencing to have homology to the partial sequence of pPL2125 and pUCPULL and containing the primers Npull and seq5 was named pCRN5.

Following restriction and confirmation by sequencing the pCR2.1 vector used in the construction of pCRN5 was found to have a mutated EcoRI site on the T7 promoter side which prevents the cutting out of the insert using this enzyme alone.

Sequencing of pPL2125. pUCPULL and pCRN5. Sequencing could proceed in both directions using the plasmid pUCPULL and for all further reactions both pPL2125 and pUCPULL plasmids were used. The primers used were named seq 1,2,3 etc. in one direction and seqla, 2a etc. for the complementary strand direction (Fig.l). The primers used to sequence downstream from primer Npull were named lb,2b,3b etc. Based on the sequence obtained from pCRN5 primers were made to sequence upstream from the Npull sequence (primers lc, 3c, 4c, Ob).

The correct N-terminal amino acid sequence was not found in pPL2125 and pUCPULL upstream from the Npull primer. However, a "Npull-like" sequence was found at the correct position but there were some significant differences leading to the conclusion that the primer bound non-specifically and amplified a fragment not containing the pullulanase gene. The deduced amino acid sequence of the largest ORF of this fragment (total 4.57kb) which was 556 aa displayed very high homology to the isoleucyl- tRNA synthase gene from Thermotoga maritima and so it was concluded that the pullul.anase must be found in the first half of the fragment. The isoleucyl-tRNA synthase . genes are used in taxonomical classification of bacteria and archaea and have been used to show that the Archaea and Eucarya are sister groups. This unexpected finding proves that the DNA fragment containing the pullulanase does in fact come from a bacteria closely related to Thermotoga (ie., Fervidobacterium).

Repeated PCR amplification of the pullulanase gene. Using the primers la,21a,3a, Npull in combination with lc and 3c, fragments of approximately 3Jkb were amplified. These were purified, pooled together, cloned into E.coli using the vector pCR2.1 (TA cloning kit, Invitrogen) and the transformants were screened for activity. Plasmid DNA from 2 pullulanase positive clones and 3 pullulanase negative clones with inserts were sequenced and the presence of the primers confirmed. The 2 pullulanase positive clones contained identical sequences. The pullulanase positive clones were ampicillin and kanamycin resistant and chloramphencol sensitive as expected. From this result we conclude that the pullulanase activity can be localized to a gene fragment of about 3kb which is flanked by primers la and 3c. The remaining part of the 8.1kb insert was therefore sequenced and the complete insert was analyzed for ORF's. The largest ORF in this fragment was identified and analyzed for homology to previously published glycosyl hydrolases.

Sequence analysis of pulA and identification of the start site. PulA demonstrated most amino acid identity (63%) and DNA identity/simil.arity (66%o) calculated using the GAP programme of the GCG package version 8 to the pullulanase gene of Thermotoga maritima. There are 2 potential start sites present in the Fervidobacterium pennavorans DNA sequence, at positions 651 (ATG) and 1002 (TTG) which both have an identical Shine Dalgarno sequence (E.coli sequence, there is no full 16s data available for Fervidobacterium pennavorans) placed ideally upstream from the translational start and both translate in frame with the N-terminal sequence (Fig. 2).

On alignment with the same gene from T maritima, (Fig. 3A-C) a possible signal peptide cleavage site was identified 28 amino acids from the initial methionine between .amino acids 28 and 29 (A and E). This was confirmed by N-terminal sequencing of the pullulanase purified from Fervidobacterium pennavorans. This confirmed that ATG is the translational start site of the pullulanase gene and that a signal peptide of 28 amino acids is present at the N-terminal of the protein.

The N-terminal sequence of the pullulanase purified from E.coli PL2125 is identical to the amino acid sequence translated from the TTG site. No signal sequence was detected downstream from the TTG site.

TABLE 1. LIST OF PRIMERS USED FOR SEQUENCING AND PCR

Sequence (5' to 3 ' direction) max length

ID NO

5 seq2 TTG TTT TGG TTA CGG TCT TTA TGA 1-570

6 seq3 TAC AAC CCC CCT CGA AAC TAC TA 1-451

7 seq4 CCT TTA GAC AGC TGT AAG TCT GT 60-400

8 seq5 TTT TGA ATT TCA AGT TCT TC 86-705

9 seqδ TTG AAC CGT CTA CAA AAA TC 81-627

10 seqδl TAT CAA TC CGG GAC CAA TGG CAC TAA C 20-219

11 seq^'7 CGG CAT TGA GAG CGG TGA TGA TT 143-308

12 seq8 CAT CCG TTA CAT TAT CAA AAT C 34-713

13 seq9 GCT CTT ACC GTT GGG TCG TAA AAT G 30-652

14 seqlO CCA TCG AGA AAT GTG AAA GGA AAG 40-462

15 seqlOl CAT CAA CTT TGA GTT CTT TTC CCA G no sequence, 2x

16 seqll TTG ACT TCT GCT CTT TGG ACT 22-452

17 pullN ACN ACN AAR CCN GAY AC no sequence, 4x

18 seqla CCC CGT TGA ACT ACC TGC CC 36-575

19 seq2 CAC CCC GGG AAA ACC GTT no sequence, 3x

20 seq21a GAT GTC TGG ATT CTG TTT GTT GA 81-419

21 seq3a CCT TTT ATA ACC AAC GCA GTC G 57-577

22 seq4a TTA AAA TGA ACT TCC TCA TAC CT 54-335

23 seq5a TGC TCG TCT CTT TTT TCA CTG G 34-538

24 seq6a TTG TAG AAA AAA TCT GGG ATA ATG 52-700

25 seq7a GCG GAA ACA GAG CTG ATT ATC C 63-690

26 seq8a GGC GGA GGT GTG GCT CTT GC 1-687

27 seq9a TAC AAA CCG GCA AGG GTC ATC 1-675

28 seqlOa AGG TCC TAA TGG TGT AAC AAC 50-479

29 seqlla CCA TTC GAC CAG GCA GTT CCA 50-564

30 seqOb GAA CAC AGA GTT ACA ACA TCC TTG no sequence, 2x

31 seqlb GAC TTT GAA GTT GTT GAA AAA TTC G 38-700

32 seq2b CAA ACG GGT TTA AAA TAC AAT CTT CC 3-725

33 seq3b GAA AAG ACA CAT GAT ACA TTA GA 2-479

34 seq4b GGT AAG AAC ATA ATA GAA CAG C 45-738

35 seq5b GAA AGC GTG CAT CTT GAA TAC TGG; 1-555

36 seqOc TGG AAG ATT GTA TTT TAA ACC CGT TTG 16-354

37 seqlc AAC ATT TGC TGG GAT AGT CCA AGG 1-480

38 seq3c GGA TGT TGT AAC TCT GTG TTC A 10-432

39 seq4c TGA AAG TTT GTT TGT GGT AAG TTC 65-452

40 seq4cl TTG AAA GTT TGT TTG TGG TAA GTT CA 46-700

41 seq5c GCG CTT CGT CAG CAT TTT CAG 61-748

42 seq6c TTG GAC TTG GTA AGA ATG TCA G 59-675

Claims

ClaimsWhat is claimed is:

1. An isolated nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence comprising the sequence of SEQ ID NO:l;

(b) a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: l, (ii) its complementary strand, or (iii) a subsequence of SEQ ID NO: l which encodes a polypeptide fragment which has pullulanase activity;

(c) an allelic variant of (a) or (b); and

(d) a fragment of (a), (b) or (e), wherein the fragment has pullulanase activity.

2. The nucleic acid sequence of claim 1 (b) which hybridizes under medium stringency conditions to the nucleic acid sequence of SEQ ID NO: l .

3. The nucleic acid sequence of claim 1 (b) which hybridizes under high stringency conditions to the nucleic acid sequence of SEQ ID NO: l.

4. The nucleic acid sequence of claim 1 obtained from Fervidobacterium sp. Ven 5.

5. The nucleic acid sequence of claim 4, obtained from Fervidobacterium sp. Ven 5. DSM 6204.

6. An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes a polypeptide having the amino acid sequence of SEQ ID NO:3.

7. A nucleic acid construct comprising the nucleic acid sequence of claim 1 operably linked to one or more control sequences which direct the expression of the polypeptide in a suitable expression host.

8. A recombinant expression vector comprising the nucleic acid construct of claim 7, a promoter, and transcriptional and translational stop signals.

9. A recombinant host cell comprising the nucleic acid construct of claim 7.

10. A method for producing a pullulanase comprising (a) cultivating a host cell comprising a nucleic acid construct comprising the nucleic acid sequence of claim 1 , and (b) recovering the pullulanase.