CN1209842A - Nucleic acids encoding polypeptides having absidia lipase activity - Google Patents

Abstract

The present invention relates to isolated nucleic acid sequences encoding polypeptides having Absidia lipase 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. The invention further relates to compositions comprising the polypeptides.

Description

Coding has the nucleic acid of the polypeptide of absidia lipase activity

The isolated nucleic acid sequences of polypeptide with absidia lipase activity the present invention relates to encode.The invention still further relates to the nucleic acid construct, carrier and the host cell that comprise described nucleotide sequence, and the recombination method that is used to prepare described polypeptide.

The known washing composition made from lipolytic enzyme has improved characteristic of removing fatty spot.For example, will be available from a kind of microbial lipase LIPOLASE that dredges cotton shape high temperature mould (Thermomyces lanuginosus) (be otherwise known as and dredge cotton shape humicola lanuginosa (Humicola lanuginosa)) fungi ^TM(Novo Nordisk A/S, Bagsvaerd Denmark) introduce in the washing composition of multiple commercial brand.

The somebody proposes will be available from pseudomonas cepacia (Pseudomonas cepacia) (US4,876,024), (US 3 for streptomyces (Streptomycetes) (WO 94/14940) and avette sorosphere holder mould (Gongronella butleri) bacterial strain NRRL 3521,634,195 these bacterial strains were known as avette spore colter mould (Absidia butderi) in the past, referring to K.H.Domsch etc., Compendinm of Soil Fungi, Academic Press 1980, other microbial lipase p.381) is used for washing composition.

U.S. Pat 3,634,195 is disclosed with post spore colter mould shape mutation (Absidiacylindrospora var.rhizomorpha) NRRL 2815 and Absidia blakes leeanaNRRL 1305 preparation lipase.Koritala etc. disclose (1987, Journal of theAmerican Oil Chemists Society 64:509-513), soybean oil is can be by partial hydrolysis when NRRL1309 cultivates with mould (Absidia coerula) NRRL 5926 of blue colter and absidia rasmosa (Absidia ramosa).Satyanarayana (1981, Current Science50:680-682) discloses, and absidia corymbifera (Absidia corymbifera) bacterial strain can be secreted lipase.Aisaka etc. (1979, Agricultural Biological Chemistry 43:2125-2129) disclose, and can produce lipoprotein lipase by Absidia halospora (Absidia hyalospora) bacterial strain KY 303 (now being known as Absidiablakesleeana).

A lot of washing composition are alkalescence (for example, about pH10) in solution, and contain can be in conjunction with Ca ⁺⁺The ionic auxiliary agent.Need a kind ofly there be Ca ⁺⁺, have highly active novel lipolytic enzyme under the high pH.The lipase of absidia has above-mentioned characteristic, therefore, uses it in the detergent formulation very good.But, up to now, still there is not the method for the above-mentioned enzyme of recombinant production.

An object of the present invention is to provide the method for the above-mentioned valuable enzyme of reorganization preparation.

The present invention relates to the encode isolated nucleic acid sequences of polypeptide with lipase activity, this nucleotide sequence is selected from:

(a) nucleotide sequence of the endogenous polypeptide of a kind of absidia bacterial strain of coding, this polypeptide has the aminoacid sequence shown in sequence 2, sequence 4, sequence 6, sequence 8 or the sequence 10;

(b) a kind of endogenous nucleic acid sequence of absidia bacterial strain, it can be under the condition of medium strictness and (ⅰ) nucleotide sequence shown in sequence 1, sequence 3, sequence 5, sequence 7 or the sequence 9 or (ⅱ) its any complementary strand hybridization;

(c) a kind of nucleotide sequence, it can be under the condition of medium strictness and (ⅰ) nucleotide sequence shown in sequence 1, sequence 3, sequence 5, sequence 7 or the sequence 9 or (ⅱ) its any complementary strand hybridization;

(d) a kind of coding has the nucleotide sequence of the polypeptide of lipase activity, and the identity of aminoacid sequence is at least 65% shown in this amino acid sequence of polypeptide and sequence 2, sequence 4, sequence 6, sequence 8 or the sequence 10;

(e) (a) and (b), (c) or equipotential form (d); With

(f) (a) and (b), (c), (d) or fragment (e).

The invention still further relates to the nucleic acid construct, carrier and the host cell that contain described nucleotide sequence, and the recombination method for preparing described polypeptide.

Fig. 1 represents to come from the sepharose purification result of the lipase specific PCR product of Absidia griseola and Absidia griseola var.iguchii genomic dna.1 swimming lane: the φ X17RF-DNA size criteria thing of the λ DNA of Hind III-digestion and Hae III-digestion; 2 swimming lanes: Absidia griseola PCR product; 3 swimming lanes: the PCR product of Absidia griseola var.iguchii.As if the size of two kinds of PCR products all be roughly 870bp.

Fig. 2 represents to use the radioactive automatic developing figure of Southern hybridization analysis of the genomic dna digest of the kind of surveying from the radiolabeled lipase gene fragment of Absidia griseola that comes from several absidias.The size of the φ X17RF-DNA size criteria thing of the λ DNA of Hind III digestion and the digestion of Hae III is on the right side of this radioactive automatic developing figure.1-3 swimming lane: Absidia sporophoravariabilis DNA (respectively with EcoR I+Asp718 I, Asp 718 I and the digestion of EcoR I); 4-6 swimming lane: absidia corymbifera DNA (respectively with EcoR I+Asp718 I, Asp718 I and the digestion of EcoR I); 7-9 swimming lane: Absidiablakesleeana DNA (respectively with EcoR I+Asp718 I, Asp718 I and the digestion of EcoR I); Swimming lane 10-12:Absidia griseola var.iguchii DNA (respectively with EcoR I+Asp718 I and the digestion of EcoR I); 13-15 swimming lane: Absidia griseola DNA (respectively with EcoR I+Asp718 I, Asp718 I and the digestion of EcoR I).

Fig. 3 represents the dna sequence dna of Absidia griseola var.iguchii lipase and the aminoacid sequence of deduction (being respectively sequence 1 and sequence 2).Intron marks with solid line.Corresponding to before draw (---) below the fragment of the peptide sequence determined.

Fig. 4 represents the dna sequence dna of Absidia blakesleeana lipase and the aminoacid sequence of deduction (being respectively sequence 3 and 4).Intron marks with solid line.Corresponding to before draw (---) below the fragment of the peptide sequence determined.

Fig. 5 represents the dna sequence dna of absidia corymbifera lipase and the aminoacid sequence of deduction (being respectively sequence 5 and 6).Intron marks with solid line.Corresponding to before draw (---) below the fragment of the peptide sequence determined.

Fig. 6 represents the dna sequence dna of Absidia sporophoravariabilis lipase and the aminoacid sequence of deduction (being respectively sequence 7 and 8).Intron marks with solid line.

Fig. 7 represents to reflect mould (Absidia reflexa) dna sequence dna of lipase of colter and the aminoacid sequence of deduction (being respectively sequence 9 and 10).

Fig. 8 represents that the absidia lipase and the homology of the aminoacid sequence of rice black root Mucor (Rhizomucor miehei) lipase (sequence 15) and thin cotton shape humicola lanuginosa lipase (sequence 16) compare.Identical residue goes out with the lines frame.

Fig. 9 represents the restriction figure of pBANe6.

Figure 10 represents the restriction figure of pKB2.

Figure 11 represents the restriction figure of pRamB19.

Nucleotide sequence

In the first embodiment, the nucleotide sequence of the separation of the polypeptide with lipase activity of the present invention relates to encode, described polypeptide has the amino acid sequence shown in sequence 2, sequence 4, sequence 6, sequence 8 or the sequence 10. In a kind of specific embodiments, described nucleotide sequence is shown in sequence 1, sequence 3, sequence 5, sequence 7 and the sequence 9. Nucleotide sequence of the present invention comprises that also coding has the nucleotide sequence of polypeptide of the amino acid sequence shown in sequence 2, sequence 4, sequence 6, sequence 8 or the sequence 10, but is different from respectively sequence 1, sequence 3, sequence 5, sequence 7 or sequence 9 because of the degeneracy of genetic code. The invention still further relates to the respectively fragment of coded sequence 2, sequence 4, sequence 6, sequence 8 or sequence 10, but still the subsequence with sequence 1, sequence 3, sequence 5, sequence 7 or sequence 9 of lipase activity. In a kind of preferred embodiment, nucleotide sequence of the present invention is the nucleotide sequence that is contained on plasmid pZL-NL1, pZL-NL61, pZL-ZL95 and the pZL-NL124, and above plasmid is included in respectively among Escherichia coli (Escherichia coli) NRRL B-21520, NRRL B-21521, NRRL B-21522 and the NRRL B-21523.

This paper employed " nucleotide sequence of separation " word refers to the nucleotide sequence of essentially no other nucleotide sequence, for example, measure its purity by agarose electrophoresis and be at least about 20%, preferably be at least about 40%, more preferably be at least about 60%, preferably be about 80% again, be most preferably 90 %. For example, can obtain with standard cloning process used in the genetic engineering nucleotide sequence of separation, and this nucleotide sequence is re-positioned at the different loci that it is replicated by its natural site. Described cloning process may comprise the nucleotide sequence that contains coding said polypeptide the purpose nucleic acid fragment shearing with separate, this fragment is inserted in the carrier molecule, and this recombinant vector is incorporated in a kind of host cell, in this host cell, copy a plurality of copies or the clone of described nucleotide sequence. Described nucleotide sequence can be genome, cDNA, RNA, semi-synthetic, synthetic source or its combination.

" lipase " word is defined as lipolytic enzyme in this article, according to the rule (1992) of international bio chemistry and molecular biology federation (IUBMB) it is returned under enzyme classification E.C.3.1.1.-(carboxylic ester hydrolases). Therefore, lipolytic enzyme has hydrolysing activity to one type the ester bond of mentioning at least in E.C.3.1.1. one joint, for example, be present in ester bond in monoglyceride, diglyceride and triglyceride, phosphatide (all types), thioesters, cholesteryl ester, wax ester, cutin, suberin, the synthetic ester etc. For instance, lipolytic enzyme of the present invention can have activity to triglycerides (lipase activity, E.C.3.1.1.3), for example, 1,3-position specificity lipase activity.

In the second embodiment, the nucleotide sequence of separation of polypeptide with lipase activity the present invention relates to encode, described nucleotide sequence can high, in or under the low stringency condition with a kind of can be under the same conditions and the Probe Hybridization (J.Sambrook of nucleotide sequence shown in sequence 1, sequence 3, sequence 5, sequence 7 or the sequence 9, its complementary strand or the hybridization of its subsequence, E.F.Fritsch, with T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, the 2nd edition, Cold Spring Harber, New York). Hybridization shows, similarly nucleotide sequence can (for example hybridized under low paramount stringent condition with the oligonucleotide probe of the peptide coding part that is equivalent to nucleotide sequence shown in sequence 1, sequence 3, sequence 5, sequence 7 or the sequence 9, prehybridization and hybridization are carried out under 42 ℃, for high, in and low stringency condition, respectively that sheared the solution with salmon sperm DNA sex change and 50,35 or 25% formamide carries out at 5 * SSPE, 0.3%SDS, 200 μ g/ml), hybridization is undertaken by standard Southern blotting. In a kind of preferred embodiment, described nucleotide sequence can under medium stringent condition, preferably be hybridized under the height stringent condition. In another embodiment, described nucleotide sequence can be hybridized with sequence shown in sequence 1, sequence 3, sequence 5, sequence 7 or the sequence 9 or its complementary strand.

Can be with sequence 1, sequence 3, sequence 5, sequence 7 or sequence 9, and sequence 2, sequence 4, sequence 6, sequence 8 or sequence 10 or its subsequence be used for designing a kind of oligonucleotide probe, in order to separate homologous gene in other bacterial strain that never belongs to together or plant according to known method in this area. Therefore, can with standard Southern blotting from by screening genome of described other biological preparation or the cDNA library can with the DNA of described Probe Hybridization, in order to identify and separate wherein related gene. Described probe can be significantly shorter than its whole sequence, but its length should be at least 15, preferably is at least 25, more preferably is at least 40 nucleotides. Also can use long probe, but its length preferably is no more than 1200 nucleotides. DNA and rna probe all can use. Usually probe is carried out mark, (for example, use in order to detect corresponding gene³²P、 ³H, biotin or Avidin mark). Also can obtain a kind of Absidia lipase specificity product with degeneracy probe as herein described with from genomic DNA or first strand of cDNA chain of Absidia bacterial strain, then, can be with this product as probe, to clone corresponding genome or cDNA.

Can pass through agarose or polyacrylamide gel electrophoresis or other separation method isolation of genomic DNA or other DNA from described other biology. Can will shift and be fixed on the long and slender element of nitric acid or other suitable support material from the DNA in described library or the DNA of separation. In order to identify clone or the DNA with sequence 1, sequence 3, sequence 5, sequence 7 or sequence 9 homologies, described carrier material is used for the Southern engram analysis, wherein, finally with 2 * SSC, 0.2%SDS this carrier material is washed 3 times, each 30 minutes, washing was preferably carried out not being higher than under 40 ℃ the temperature, more preferably is not higher than 45 ℃, more preferably no higher than 50 ℃, more preferably be not higher than 55 ℃, also further preferably be not higher than 60 ℃, particularly be not higher than 65 ℃. Detecting with X-ray can be under the described conditions and the molecule of described oligonucleotide probe hybridization.

The invention still further relates to the nucleotide sequence of separation, the homogeneity of nucleotide sequence is at least about 65% shown in it and sequence 1, sequence 3, sequence 5, sequence 7 or the sequence 9, preferably be about 70%, preferably be about 75%, preferably be about 80%, be more preferably 85%, further preferably be about 90%, be most preferably 95%, the most preferably be about 97%, its a kind of active peptides of encoding. Homogeneity degree between two nucleotide sequences can be used computer program known in the art, measures such as the GAP that provides in the GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48:443-453). In order to measure the homogeneity degree between two kinds of nucleotide sequences of the present invention, use Clustal method (DNASTAR take a homogeneity table, breach grade (gap penalty) and the notch length (gap length) as 10 as 10, Inc., Madison, WI).

In the third embodiment, the nucleotide sequence of separation of polypeptide with lipase activity the present invention relates to encode, the homogeneity of amino acid sequence is at least about 65% shown in the amino acid sequence of described polypeptide and sequence 2, sequence 4, sequence 6, sequence 8 or the sequence 10, preferably be about 70%, preferably be about 75%, preferably be about 80%, be more preferably 85%, further preferably be about 90 %, be most preferably 95%, the most preferably be about 97%, it is the activity that still keeps in nature described polypeptide (below be referred to as " homeopeptide "). In a kind of preferred embodiment, amino acid sequence shown in the amino acid sequence of described homeopeptide and sequence 2, sequence 4, sequence 6, sequence 8 or the sequence 10 has 5 amino acid whose differences, preferably have 4 amino acid whose differences, 3 amino acid whose differences are more preferably arranged, further preferably have 2 amino acid whose differences, 1 amino acid whose difference is most preferably arranged. The homogeneity degree of two or more amino acid sequences can be measured by computer program known in the art, as by GCG program package (Needleman and Wunsch, 1970, Journal of Molecular Biology 48:443-453) GAP that provides. In order to measure the homogeneity between the two seed amino acid sequences of the present invention, use Clustal method (DNASTAR, Inc.Madison, WI) take a homogeneity table, breach grade and the notch length as 10 as 10.

Can be different from the aminoacid sequence shown in sequence 2, sequence 4, sequence 6, sequence 8 or the sequence 10 because of the insertion of one or several amino-acid residue or disappearance and/or one or several amino-acid residue are replaced by different amino-acid residues by the aminoacid sequence of the coded homeopeptide of nucleotide sequence of the present invention.It is desirable to, amino acid whose variation is less important property, promptly can obviously not influence proteic folding and/or active conservative amino acid and replace; Little disappearance is generally 1～about 30 amino acid; Little amino or C-terminal extend, as the extension of a N-terminal methionine residues; A little joint peptide that is about 20-25 residue at the most; Or a little extension, it helps by changing net charge or other function, as a polyhistidyl segment, an antigenic epitopes or a purifying that carries out in conjunction with the territory.

The example of preservative replacement comprises basic aminoacids (as arginine, Methionin and Histidine), acidic amino acid (as L-glutamic acid and aspartic acid), polare Aminosaeren (as glutamine and l-asparagine), hydrophobic amino acid (as leucine, Isoleucine and Xie Ansuan), aromatic amino acid (as phenylalanine, tryptophane and tyrosine), and p1 amino acid (as glycine, L-Ala, Serine, Threonine and methionine(Met)).The aminoacid replacement that can not change specific activity substantially is known in the art, for example, can consult the book (1979, The Proteins, Academic Press, New York) of H.Neurath and R.L.Hill.Modal exchange has: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, and the reverse of above-mentioned exchange.

Isolated nucleic acid sequences of the present invention can with a kind of oligonucleotide probe hybridization, this probe can be hybridized with sequence 1, sequence 3, sequence 5, sequence 7 or sequence 9 described nucleotide sequences, its complementary strand or subsequence, described isolated nucleic acid sequences can be available from the microorganism of any genus, for example, can be available from bacterium or originated from fungus, but preferably from the fungal cell, more preferably from filamentous fungal cells or yeast cell.In the present invention, " available from " (coming from) speech is meant that when being used in combination with particular source described polypeptide is to be produced by this source or the cell that inserted from the gene in this source.Preferred homologous gene source is the bacterial strain of absidia and kind thereof, and these bacterial strains can obtain from public preservation mechanism.In addition, can identify from other source and obtain homologous gene that described source comprises from the isolating microorganism of occurring in nature (for example, from soil, compost, water etc.) with above mentioned probe.Be used for being widely known by the people in the art from the method for natural surroundings separate microorganism.Can obtain described nucleotide sequence by the cNDA library of screening another kind of microorganism with similar approach then.Particularly preferred bacterial strain is a filamentous fungal strains, as bacterial strain: little Acremonium (Acremonium), Aspergillus (Aspergillus) with the subordinate, aureobasidium genus (Aureobasidium), Cryptococcus (Cryptococcus), thread Basidiomycotina (Filibasidium), Fusarium (Fusarium), Humicola (Humicola), Magnaporthe, Mucor (Mucor), myceliophthora (Myceliophthora), Neocallimastix, Neurospora (Neurospora), paecilomyces (Paecilomyces), Penicillium (Penicillium), Piromyces, Schizophyllum (Schizophyllum), blue stain fungi (Talaromyces), Thermoascus, Thielavia (Thielavia), Tolypocladium or Trichoderma (Trichoderma); Or yeast strain, as mycocandida (Candida), genus kluyveromyces (Kluyveromyces), Pichia (Pichia), yeast belong (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces) or Yarrowia bacterial strain.

In a kind of preferred embodiment, a kind of nucleotide sequence of the present invention available from M.A.A.Schipper at Persoonia, Vol.14, Part 2, the bacterial strain of the absidia disclosed in the PP.133-148 (1990) is as the bacterial strain of Abisidia griseola, Absidia sporophora-variabilis, Abisidia griseola var.iguchii, absidia corymbifera or Absidia blakesleeana.In a kind of preferred embodiment, described nucleotide sequence is available from Absidia blakesleeanaNN 100826 (NRRL 1304), for example nucleotide sequence shown in the sequence 3; Absidia corymbifera NN 100062 (IFO 8084), for example nucleotide sequence shown in the sequence 5; Abisidiagriseola NN 000987 (ATCC 20430); Absidia griseola var.iguchiiNN000591 (ATCC20431), for example, the nucleotide sequence shown in the sequence 1; Absidiasporophora-variabilis NN 102 427 (ATCC 36019), for example, the nucleotide sequence shown in the sequence 7; With the reflection mould NN 102 427 of colter (ATCC 44896), for example, the nucleotide sequence shown in the sequence 9.

In absidia, preferred following subgenus, population, kind and bacterial strain.Also comprise modification and mutant that it can produce lipolytic enzyme.Should be noted that, Schipper reclassifies determined kind name before some in the book of being quoted, and, for convenience's sake, in following table, enumerated employed title before some bacterial strain, wherein, cited a plurality of numbers are represented the repeatedly preserving number of same bacterial strain in the same square frame.

Subgenus, population	Plant name	The preceding name of planting:	Contriver's bacterial strain number	Preserving number
					The mould subgenus of stem	A.blakesleeana	A. blakesleeana	NN100826	NRRL1304, ATCC10148a, CBS100.28,CMI 111736
A. blakesleeana	NN102406	?CBS100.36
			A. blakesleeana	NN102407			CBS102.36, NRRL2696
?A. blakesleeana	NN102408	CBA420.70
			A. blakesleeana	NN102413			NRRL1305
A.griseola	NN000987	ATCC20430
			A.griseola	NN102403			CBS519.71, ATCC22618,IFO 9472
A.griseola var.iguchii	NN000591	ATCC20431
			Absidia halospora	NN102432			CBS173.67, NRRL2916
A.blakesleeana var.atrospora	A.atrospora	NN102423						CBS518.71, ATCC22617,IFO 9471
			Absidia corymbifera	Absidia corymbifera		NN100060	CBS100.31,IFO 4009,NRRL2982
Absidia corymbifera	NN100062	IFO8084
				Absidia corymbifera		NN102404	CBS102.48
Absidia corymbifera	NN102405	CBS582.65, ATCC22574, NRRL1309
				A.hesseltinii		NN102426	CBS958.68, ATCC24263

The mould subgenus of colter, population B	Mould shape mutation of post spore colter	-	NN102422	CBS154.63, the mould NN102434ATCC 24169 of NRRL2815 feigned column spore colter, CBS 100.62, and NRRL 2770
					-	The reflection colter is mould	-	NN102424	ATCC44896,IFO 5874
-	A.sporophora- variabilis	-	NN102427	ATCC36919

The public is easy to obtain above-mentioned bacterial strains from how tame culture collection mechanism, as U.S. typical case culture collection institute (ATCC), DSM (Deutsche Sammlung vonMikroorganismen und ZellKulturen GmbH), CBS (Centraalbureauvoor Schimmelcultures) and NRRL (Agricultural Research ServicePatent Culture Collection, Northern Regional Research center).

In case to a kind of nucleotide sequence, just can separate or clone this sequence (for example,, the same) with method well-known in the art referring to the book of Sambrook etc. with described probe in detecting.Describedly be used for separating or the currently known methods of cloning nucleic acid sequences comprises: from genomic dna separate, the combination of preparation or these two kinds of methods from cDNA.For example, can realize like this from described genomic dna cloning nucleotide sequence of the present invention: adopt well-known polymerase chain reaction (PCR).For example, referring to Innis etc., 1990, A Guide to Methods and Application, Academic Press, New York.Can be by a kind of absidia bacterial strain that can produce described polypeptide or other or the relevant described nucleotide sequence of biological cloning, therefore, for instance, can be the equipotential of described polypeptid coding area of described nucleotide sequence or the modification of kind.

For synthetic similar to described polypeptide substantially polypeptide, may need the nucleotide sequence of coding said polypeptide is modified.Be meant the non-natural existence form of this polypeptide to described polypeptide " similar substantially ".Can make described polypeptide be different from isolating this polypeptide from its natural origin with certain engineering method.For example, the modification of synthetic described polypeptide may be interesting, wherein, uses the method such as site-directed mutagenesis that this variant is changed at aspects such as specific activity, thermostability, oxidative stability or best pH.Can make up described similar sequence according to the nucleotide sequence of the described polypeptid coding area of sequence 1, sequence 3, sequence 5, sequence 7 or sequence 9, its subsequence, and/or by introducing the another kind of aminoacid sequence that can not produce by the polypeptide of described nucleic acid sequence encoding, but the Nucleotide that the codon that meets the host living beings that is used to produce described enzyme is selected replaces, or replaces to make up described similar sequence by the Nucleotide that meeting produces different aminoacid sequences.The generality explanation that replaces about Nucleotide can be referring to the book (1991, Protein Expression andPurification 2:95-107) of Ford etc.

It will be appreciated by persons skilled in the art that this replacement can carry out in the zone outside the function of this molecule of decision, and still can produce activated polypeptide.Can use means known in the art, as site-directed mutagenesis or alanine scanning mutagenesis (for example, book referring to Cunningham and Wells, 1989, Science 244:1081-1085) determine by the active important amino-acid residue of isolated nucleic acid sequences encoded polypeptides of the present invention, and therefore not to be substituted by suitable to these residues.In a kind of mutafacient system in back, each residue of corresponding molecule is introduced in sudden change, and tested the lipase activity of gained mutant molecules, to determine the amino-acid residue of this molecular activity of decision.Can also by the analyzing crystal structure determine substrate-enzyme do mutually for example, analyze (for example, referring to de Vos etc., 1992, Science 255:306-312 in the site by the technology such as nuclear magnetic resonance spectroscopy, crystallography or photoaffinity labeling; Smith etc., 1992, Journal of MolecularBiology 224:899-904; Wlodaver etc., 1992, FEBS Letters 309:59-64).

Also comprise fusion polypeptide by nucleotide sequence encoded polypeptide of the present invention, wherein, another kind of polypeptide merges at described polypeptide or its segmental N-end or C-end.Fusion polypeptide is to merge with a kind of nucleotide sequence of the present invention (or its part) by the nucleotide sequence of the another kind of polypeptide of will encoding (or its part) to produce.The technology that is used to produce fusion polypeptide is widely known by the people in this area, comprises the encoding sequence that connects coding said polypeptide, so that it meets frame, and the expression of described fusion polypeptide is to be subjected to the identical promotor and the control of terminator.

The feature that has by the polypeptide of the lipolytic activity of nucleic acid sequence encoding of the present invention is, under alkaline pH (pH is approximately 9-10), even without Free Ca ⁺⁺Also has high reactivity.

More particularly, there be not Free Ca ⁺⁺Condition under when testing, about pH9 or under the higher pH, described polypeptide has best lipolytic activity (activity under pH9 is higher than the activity under pH8).

By the polypeptide of some preferred nucleic acid sequence encoding, there be not Free Ca ⁺⁺Condition under, have lipase activity when under pH9, measuring.These lipolytic enzymes can be available from the mould genus of absidia subgenus stem (Mycocladus), for example, and above cited kind and bacterial strain.

By the polypeptide of another kind of preferred excellent nucleic acid sequence encoding, there be not Ca ⁺⁺Condition under, the lipolytic activity when lipolytic activity is higher than pH9 when pH10.This nucleotide sequence can be available from the reflection mould NN 102 427 of colter (ATCC 44896).

After cultivating 30 minutes down, pH10 and 45 ℃ still can keep residual activity more than 90% by another kind of preferred nucleic acid sequence encoded polypeptide.Described sequence can be available from a kind of bacterial strain of Absidiasporophora-variabilis, as Absidia sporophora-variabilis NN102 427 (ATCC 36019).Nucleic acid construct

The invention still further relates to the nucleic acid construct that contains the nucleotide sequence of the present invention that operably is connected in one or several control sequence, described control sequence can be suitable for instructing the expression of corresponding coding sequence in proper host cell under the condition of this control sequence.

" nucleic acid construct " is defined as strand or double chain acid molecule in this article, and this molecule is isolating or it is modified from naturally occurring gene, so that it contains with non-existent mode combination of occurring in nature and nucleic acid fragment arranged side by side.When described nucleic acid construct contains when expressing the necessary whole control sequence of encoding sequence of the present invention, " nucleic acid construct " speech is a synonym with " expression cassette " speech." encoding sequence " speech defined herein is meant a kind of sequence, when this sequence is placed under the control of suitable control sequence, can be transcribed into mRNA, and be translated into polypeptide of the present invention.The border of described encoding sequence generally be by be positioned at 5 '-terminal translation initiation codon ATG and be positioned at 3 '-terminal translation stop codon determines.An encoding sequence can include, but are not limited to DNA, cDNA and recombinant nucleic acid sequence.

Can operate the nucleotide sequence of a separated coding peptide species of the present invention with several different methods, so that this polypeptide is expressed.According to the expression vector situation, it may be that wish or necessary operating on it before the nucleotide sequence with described coding one peptide species inserts this carrier.Technology with the cloning process modification of nucleic acids is widely known by the people in the art.

" control sequence " speech is defined as in this article comprising that the expression of encoding sequence of described nucleotide sequence is necessary or helps all factors of its expression.The nucleotide sequence of the described polypeptide of relative coding, each control sequence can be natural or external sources.Described control sequence includes, but are not limited to a leader sequence, a polyadenylation sequence, a propeptide sequence, a promotor, a signal sequence and a transcription terminator.This control sequence minimally comprises a promotor and transcribes and the translation termination signal.This control sequence can have joint, to be used to introduce the special restriction site that helps this control sequence to be connected with the coding region of the nucleotide sequence of coding one peptide species.

Described control sequence can be a suitable promoter sequence, i.e. the nucleotide sequence that is used to express described nucleotide sequence that energy is discerned by host cell.This promoter sequence contains the transcriptional control sequence that can mediate described expression of polypeptides.This promotor can be any nucleotide sequence that can show transcriptional activity in selected host cell, and can available from coding with this host cell homology or allogenic born of the same parents the gene of polypeptide in the outer or born of the same parents.

Be suitable for instructing nucleic acid construct of the present invention to transcribe, the example of the promotor of especially transcribing in bacterial host cell has available from following promotor: the intestinal bacteria operon, streptomyces coelicolor (Streptomyces coelicolor) gelase gene (dagA), subtilis (Bacillus subtilis) levansucrase gene (sacB), bacillus licheniformis (Bacillus licheniformis) alpha-amylase gene (amyL), bacstearothermophilus (Bacillus stearothermophilus) gives birth to wheat starch enzyme gene (amyM), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) alpha-amylase gene (amyQ), bacillus licheniformis penicillinase gene (penP), subtilis xylA and xylB gene, with protokaryon beta lactose enzyme gene (Villa-Kamaroff etc., 1978, Proceedings ofthe National Academy of Science USA 75,3727-3731), and tac promotor (DeBoer etc., 1983, Proceedings of the National Academy of ScienceUSA 80:21-25).Other promotor is disclosed in Scientific American, and 1980, " available from the useful proteins of the recombinant bacteria " literary composition among the 242:74-94; And the book (1989, the same) of Sambrook etc.

The example that is used for instructing the promotor that nucleic acid construct of the present invention transcribes at filamentous fungal host cell is the promotor available from the gene of the following enzyme of coding: aspergillus oryzae (Aspergillus oryzae) TAKA amylase, rice black root Mucor (Rhizomucor miehei) aspartate protease, the neutral α-Dian Fenmei of aspergillus niger (Aspergillus niger), aspergillus niger (Aspergillusniger) acid acceptance α-Dian Fenmei, aspergillus niger or Aspergillus awamori (Aspergillusawamori) glucoamylase (glaA), rice black root Mucor lipase, the aspergillus oryzae Sumizyme MP, the aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans (Aspergillus nidulans) acetamidase, point sickle spore (Fusarium oxysporum) trypsin-like proteolytic enzyme is (as U.S. Pat 4,288, disclosed in 627, this patent is done this paper reference by receipts), and heterozygote.The particularly preferred promotor that is used for filamentous fungal host cell has TAKA amylase, NA2-tpi (coming the heterozygote of promotor of the gene of neutral α-Dian Fenmei of own coding aspergillus niger and aspergillus oryzae triosephosphate isomerase) and glaA promotor.

In yeast host, useful promotor is available from yeast saccharomyces cerevisiae (Saccharomycescerevisiae) Hydratase, phosphoenolpyruvate (ENO-1) gene, yeast saccharomyces cerevisiae galactokinase gene (GAL1), yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase gene (ADH2/GAP) and yeast saccharomyces cerevisiae 3-phoshoglyceric acid kinase gene.Other promotor that can be used for yeast host is disclosed in the book of Romanos etc. (1992, Yeast 8:423-488).In mammalian host cell, useful promotor comprises viral promotors, as the promotor available from simian virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus and bovine papilloma virus (BPV).

Described control sequence can also be suitable transcription termination sequence, can be discerned the sequence of transcribing to stop by host cell.This terminator sequence operably is connected in 3 ' end of the nucleotide sequence of coding said polypeptide.Any terminator that can work in selected host cell all can be used among the present invention.

Be used for the gene of the preferred terminator of filamentous fungal host cell: aspergillus oryzae TAKA amylase, black mold glucoamylase, Aspergillus nidulans anthranilic acid synthetic enzyme, aspergillus niger alpha-glucosidase and sharp sickle spore trypsin-like proteolytic enzyme available from the following enzyme of coding.

Be used for the gene of the preferred terminator of yeast host cell available from coding yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate, brewing yeast cell pigment C (CYC1) or yeast saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.Other terminator that can be used for yeast host cell is disclosed in the book of Romanos etc. (1992, the same).The terminator sequence that is applicable to mammalian host cell is widely known by the people in the art.

Described control sequence can also be a suitable leader sequence, i.e. mRNA non-translational region, but this district is important for the translation of host cell.Described leader sequence operably is connected in 5 ' end of the nucleotide sequence of coding said polypeptide.Any leader sequence that can work in selected host cell all can be used among the present invention.

Be used for the gene of the preferred leader sequence of filamentous fungal host cell available from coding aspergillus oryzae TAKA amylase and aspergillus oryzae triosephosphate isomerase.

The suitable leader sequence that is used for yeast host cell is available from yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate (ENO-1) gene, yeast saccharomyces cerevisiae 3-phoshoglyceric acid kinase gene, yeast saccharomyces cerevisiae α-factor and yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase gene (ADH2/GAP).

Described control sequence can also be a polyadenylation sequence, this sequence operably be connected in 3 of described nucleotide sequence '-end, and, can be when transcribing by the identification of described host cell, as a signal that adds poly-adenosine residue to the mRNA that transcribes.

Be used for the gene of the preferred polyadenylation sequence of filamentous fungal host cell: aspergillus oryzae TAKA amylase, aspergillus niger glucoamylase, Aspergillus nidulans anthranilic acid synthase, aspergillus niger α-Dian Fenmei available from the following enzyme of coding.

The useful polyadenylation sequence that is used for yeast host cell is disclosed in the book of Guo and Sherman (1995, Molecular Cellular Biology 15:5983-5990).The polyadenylation sequence that is applicable to mammalian host cell is widely known by the people in the art.

Described control sequence can also be a signal peptide coding region, and being encoded by its is connected in the aminoterminal aminoacid sequence of described polypeptide, and it can guide to polypeptide expressed the Secretory Pathway of corresponding cell.5 of the encoding sequence of described nucleotide sequence '-end can naturally contain a signal peptide coding region, the encode coding region fragment of described secrete polypeptide of this district natural connection in the translation frame.In addition, can to contain an encoding sequence with respect to described coding secrete polypeptide partly be the signal peptide coding region of external source to 5 ' end of described encoding sequence.When described coding region is undesired when containing signal peptide coding region, may need this external source signal peptide coding region.In addition, this external source signal peptide coding region can replace described natural signals peptide-coding region simply, to obtain the lipase secretion stronger than the natural signals peptide-coding region that links to each other with described encoding sequence under the normal circumstances.Described signal peptide coding region can be available from gene, the amylase that comes from the bacillus bacterial classification or proteinase gene or the ox renninogen protogene of the α-factor of the glucoamylase that comes from the Aspergillus kind or amylase gene, the lipase that comes from the Rhizomucor kind or proteinase gene, coding yeast saccharomyces cerevisiae.But, the signal peptide coding region in any Secretory Pathway that the lipase of expressing can be guided to selected host cell all can be used among the present invention.

The useful signal peptide-coding region of bacterial host cell is the signal peptide coding region available from following gene: the living wheat starch enzyme gene, bacstearothermophilus alpha-amylase gene, bacillus licheniformis subtilisin gene, bacillus licheniformis β-Nei Xiananmei gene, bacstearothermophilus neutral protease gene (nprT, nprS, nprM) and the subtilis prsA gene that come from bacillus NCIB 11837.Other signal peptide is disclosed in the book of Simonen and Palva (1993, Microbiological Reviews 57:109-137).

The useful signal peptide-coding region of filamentous fungal host cell is the signal peptide coding region available from following gene: aspergillus oryzae TAKA amylase gene, aspergillus niger neutral starch enzyme base, rhizomucor miehei aspartic protease gene, thin cotton shape humicola lanuginosa cellulose enzyme gene or rice black root Mucor lipase gene.

Can be used for the gene of the signal peptide of yeast host cell available from coding yeast saccharomyces cerevisiae α-factor and yeast saccharomyces cerevisiae saccharase.Other useful signal peptide coding region is disclosed in the book of Romanos etc. (1992, the same).

Described control sequence can also be preceding peptide-coding region, and its coding is positioned at a kind of aminoterminal aminoacid sequence of polypeptide.Resulting polypeptide is called as proenzyme or propolypeptide.Propolypeptide is non-activity normally, but can be by catalysis or autocatalysis cracking from the propetide of described propolypeptide are changed into sophisticated active polypeptide.Peptide-coding region can be available from bacillus subtilis alkali proteinase gene (aprE), subtilis neutral protease gene (nprT), yeast saccharomyces cerevisiae α-factor gene or thermophilic rMtL gene (WO 95/33836) before described.

Nucleic acid construct of the present invention can also comprise encodes one or several one or more helps the nucleotide sequence of the factor of described expression of polypeptides, and the described factor for example has activator (for example, trans-acting factor), chaperonins and processing protease.Any factor that can work in selected host cell all can be used among the present invention.The nucleotide sequence that the nucleic acid of above-mentioned one or more factors of coding needs not to be with coding said polypeptide is series relationship.

Activator is albumen (Kudla etc., 1990, the EMBO Journal 9:1355-1364 that the nucleotide sequence of energy activated code one peptide species is transcribed; Jarai and Buxton, 1994, CurrentGenetics 26:2238-244; Verdier, 1990, Yeast 6:271-297).The nucleotide sequence of described coding activator can be regulated the gene of albumen (areA) available from coding bacstearothermophilus NprA (nprA), yeast saccharomyces cerevisiae protoheme activator 1 (hap1), yeast saccharomyces cerevisiae semi-lactosi metabolism protein 4 (gal4) and Aspergillus nidulans ammonia.As its example of base, referring to the book (1993, Journal ofGeneral Microbiology 139:2295-2307) of the book (1990, the same) of Verdier and MacKenzie etc.

Chaperonins is a kind of another kind of polypeptide correctly folding albumen (Hartl etc., 1994, TIBS 19:20-25 of helping; Bergeron etc., 1994, TIBS 19:124-128; Demolder etc., 1994, Journal of Biotechnology 32:179-189; Craig, 1993, Science260:1902-1903; Gething and Sambrook, 1992, Nature 355:33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry 269:7764-7771; Wang and Tseu, 1993, The FASEB Journal 7:1515-11157; Robinson etc., 1994, Bio/Technology 1:381-384).The nucleotide sequence of described coding chaperonins can connect the gene of albumen, yeast saccharomyces cerevisiae BiP/GRP78 and yeast saccharomyces cerevisiae Hsp70 available from coding subtilis GroE albumen, aspergillus oryzae protein disulfide bond isomerase, yeast saccharomyces cerevisiae calcium.

Processing protease is proteolytic enzyme (Enderlin and Ogrydziak, 1994, the Yeast 10:67-79 of energy a kind of propetide of cracking to form a kind of sophisticated biochemical active polypeptide; Fuller etc., 1989, Proceedings of the National Academy of Sciences USA 86:1434-1438; Julius etc., 1984, Cell 37:1075-1089; Julius etc., 1983, Cell 32:839-852).The nucleotide sequence of described coding processing protease can be available from the gene of coding yeast saccharomyces cerevisiae dipeptidylaminopeptidase, yeast saccharomyces cerevisiae Kex 2 and Yarrowia lipolytica divalence processing endo-protease (xpr6).

Also wish to increase the adjusting sequence that to regulate described polypeptide expression (host cell growth relatively).The example of regulation system has the expression that can cause described base corresponding to chemistry or physical stimulation, comprises the existence of regulating compound and starts or the terminated system.The regulator control system of prokaryotic system comprises lac, tac and trp operator gene system.In yeast, can use ADH2 system or GAL1 system.In filamentous fungus, can be with described TAKA α-Dian Fenmei promotor, aspergillus niger glucoamylase promotor and aspergillus oryzae glucoamylase promotor as regulating sequence.Other example of regulating sequence has the sequence that allows gene amplification.In eukaryotic system, described adjusting sequence comprises dihydrofolate reductase gene, and this gene is to increase having under the condition of methotrexate, also comprises metallothionein gene, and this gene is increased by heavy metal.Under said circumstances, the nucleotide sequence of coding said polypeptide is the form of series connection with described adjusting sequence.Expression vector

The invention still further relates to and contain a nucleotide sequence of the present invention, a promotor and transcribe recombinant expression vector with the translation termination signal.Above-mentioned various nucleotide sequences and control sequence can be linked together, to produce a kind of recombinant expression vector, this carrier can comprise the restriction site that one or several is common, so that implement the insertion or the replacement of the nucleotide sequence of coding said polypeptide in described site.In addition, nucleotide sequence of the present invention can be expressed like this: this nucleotide sequence or the nucleic acid construct that contains this nucleotide sequence are inserted in the suitable carriers express.When making up described expression vector, described encoding sequence is positioned on this carrier, and this encoding sequence operably is connected with suitable control sequence, so that express, and might secrete.

Described recombinant expression vector can be any carrier (for example, plasmid or virus), and this carrier can be convenient to it is implemented recombinant DNA method, and may cause the expression of described nucleotide sequence.The carrier and the consistency of the host cell of this carrier to be imported are wherein depended in the selection of carrier usually.Described carrier can be linear or closed cyclic plasmid.Described carrier can be the carrier of self-replicating, and promptly as the carrier of the outer entity existence of karyomit(e), it duplicates and is independent of chromosome duplication, and for example, this carrier can be plasmid, extrachromosomal element, minichromosome or artificial chromosome.This carrier can contain and is useful on all key elements of guaranteeing self-replicating.In addition, described carrier can be such: when it is imported host cell, be integrated on the genome, and duplicate jointly with its karyomit(e) of integrating.Described carrier system can be a carrier or plasmid, or contains the two or more carriers or the plasmid of all DNA in the genome of host cell to be imported jointly, or a transposon.

Carrier of the present invention can be incorporated in the genome of host cell when importing host cell.In order to integrate, described carrier can depend on any other factor of nucleotide sequence or this carrier of coding said polypeptide, so as by homology or non-homogeneous reorganization with described carrier stable integration in described genome.In addition, described carrier can contain other nucleotide sequence, is integrated in the genome of host cell by homologous recombination so that instruct.Described other nucleotide sequence makes described carrier can be incorporated in the host cell gene group on the corresponding chromosomal exact position.Be incorporated into a possibility on the accurate site in order to improve, described conformity gene should preferably contain the nucleic acid of sufficient amount, as 100-1,500bp, preferred 400-1,500bp, 800-1 most preferably, 500bp, described nucleic acid is the height homologous with relevant target sequence, to increase the possibility of homologous recombination.Conformity gene can be with the host cell gene group in any sequence of target sequence homologous.And this conformity gene can be a nucleotide sequence non-coding or coding.On the other hand, can by non-homogeneous reorganization with described vector integration in the genome of host cell.Described nucleotide sequence can be with the host cell gene group in any sequence of target sequence homologous, and, can be non-coding or encoding sequence.

In order to carry out self-replicating, described carrier can comprise one make this carrier can be in relevant host cell the replication orgin of self-replicating.The example of bacterium replication orgin has: the replication orgin of plasmid pBR322, the pUC19 that can duplicate in intestinal bacteria, the replication orgin of pACYC177 and the pUB110 that can duplicate in bacillus, pE194, pTA1060 and pAM β 1.The example that is used for the replication orgin of yeast host cell has: the combination of 2 μ m replication orgin, CEN6 and ARS4, and the combination of CEN3 and ARS1.This replication orgin can be the replication orgin with a kind of sudden change, this sudden change (for example can be regulated its temperature sensitivity in host cell, referring to Ehrlich, 1978, Proceedings of the National Academyof Sciences USA 75:1433).In a kind of specific embodiments, this expression vector can be pZL-NL1, pZL-NL61, pZL-NL95, or pZL-NL124.

Carrier of the present invention preferably contains one or several selective marker of being convenient to select cell transformed.Selective marker is a kind of gene, and its product can produce biocide or virus resistance, to the resistance of heavy metal with to auxotrophic prototroph etc.The example of bacterium selective marker has the dal gene that comes from subtilis or bacillus licheniformis, maybe can give antibiotics resistance, as the marker gene of amicillin resistance, kalamycin resistance, chlorampenicol resistant or tsiklomitsin antibiosis.A kind of Mammals marker gene commonly used is a dihydrofolate reductase gene.What be applicable to yeast host cell is marked with ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.The selective marker that is used for filamentous fungal host cell can be selected from a following group echo, comprise, but be not limited to amdS (acetamidase), argB (ornithine carbamyl transferase, bar (phosphinothricin (phosphinothricin) Transacetylase), hygB (hygromix phosphotransferase), niaD (nitrate reductase), pyrD (orotidine-5), sC (vitriol adenylyl transferase), trpC (anthranilic acid synthase) and glufosinate resistance marker, and available from the equivalent of other kind.The amdS that preferably is labeled as Aspergillus nidulans or aspergillus oryzae and the pyrG mark that are used for the Aspergillus cell, and the bar mark of streptomyces hygroscopicus (Streptomyces hygroscopicus).In addition, selection can also realize by cotransformation, and for example, disclosed in the WO 91/17243, wherein, described selective marker is positioned at one independently on the carrier.

The more than one copy of a kind of nucleotide sequence of code book invention polypeptide can be inserted in the described host cell, with the expression of this nucleotide sequence that increases.The stable amplification of described nucleotide sequence can realize like this: the copy with method well known in the art this sequence that at least one is extra inserts in the genome of host cell, and selects transformant.

Be used to connect the above-mentioned factor, be well known to those skilled in the art (for example,, 1989, the same) referring to the book of Sambrook etc. with the method that makes up recombinant expression vector of the present invention.Host cell

The invention still further relates to the recombinant host cell that contains a nucleotide sequence of the present invention, the recombinant production that this cell is used for described polypeptide is useful." host cell " speech comprises all filial generations of a kind of parental cell, and these filial generations are different from its parental cell because of the sudden change that is taken place between replicative phase.

Preferably transform described cell with the carrier that contains a kind of nucleotide sequence of the present invention, then with described vector integration to described host chromosome." conversion " is meant that a kind of carrier that will contain nucleotide sequence of the present invention imports in a kind of host cell, so that keep described carrier with the outer carrier format of the karyomit(e) of chromosomal integration body or self-replicating.It is generally acknowledged that it is favourable integrating, because so more described nucleotide sequence stably may be remained in the cell.The integration of described carrier on host chromosome carried out with above-mentioned homology or non-homogeneous recombinant forms.

Host cell be chosen in gene and the source thereof of depending on coding said polypeptide to a great extent.Described host cell can be a unicellular microorganism, for example, and prokaryotic organism, or non-unicellular microorganism, for example, eukaryote.Useful monocyte is a bacterial cell, as gram positive bacterium, comprise, but be not limited to bacillus cell, for example, basophilia genus bacillus (Bacillus alkalophilus), bacillus amyloliquefaciens, bacillus brevis (Bacillusbrecis), Bacillus circulans (Bacillus circulans), Bacillus coagulans (Bacilluscoagulans), bacillus firmus (Bacillus firmus), Bacillus lautus, bacillus lentus (Bacillus lentus), bacillus licheniformis, bacillus megaterium (Bacillusmegaterinm), bacillus pumilus (Bacillus pumilus), bacstearothermophilus, subtilis, bacillus thuringiensis (Bacillus thuringiensis); Or the streptocin cell, for example, shallow Streptomyces glaucoviolaceus (Streptomyces lividans) or mouse ash streptomycete (Streptomyces murinus) or gram negative bacterium are as intestinal bacteria and pseudomonas (Pseudomonas sp.).In a kind of preferred embodiment, described bacterial host cell is bacillus lentus, bacillus licheniformis, bacstearothermophilus or bacillus subtilis mycetocyte.For instance, the conversion of bacterial host cell can be by protoplast transformation (for example, referring to Chang and Cohen, 1979, Molecular.General Geuetics 168:111-115), by use experience attitude cell (for example, referring to Young and Spizizin, 1961, Journal ofBacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (for example, referring to Shigekawa and Dower, 1988, Biotechniques 6:742-751) or by puting together (for example, referring to Koehler and Thorne, 1987, Journal of Bacteriology 169:5771-5278) and realize.

Described host cell can be an eukaryotic cells, as mammalian cell, insect cell, vegetable cell or fungal cell.Useful mammalian cell comprises Chinese hamster ovary cell, HeLa cell, young hamster kidney (BHK) cell, COS cell, other immortalized cell of any amount that can obtain system maybe, for example cell that can be obtained from U.S.'s typical case's culture collection.

In a kind of preferred embodiment, described host cell is a kind of fungal cell." fungi " used herein comprised that river rattan Ascomycota, Basidiomycota, chytrid door and Zygomycota were (defined as Haw-ksworth etc., see Ainsworth and Bisby ' s Dictionary of theFungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) and the oomycetes door (referring to the book of Hawksworth etc., 1995, the same, the 171st page) and all mitospore fungi (Hawksworth etc., 1995, the same).For example, the representative population of Ascomycota comprises Neurospora (Neurospora), Eupenicillium sp (Eupenicillium=Penicillium), Emericella (=Aspergillus), Eurotium (Eurotium=Aspergillus), and above-mentioned true yeat.The example of Basidiomycota comprises mushroom, rest fungus and ustilago.For example, the representative population of chytrid door comprises Allomyces (Allomyces), little Blastocladia (Blastocladiella), Coelomomyces (Coelomomyces) and aquatic fungi.For example, the representative population of oomycetes door comprises water mold fungoid aquatic fungi (water mold), as Achyla (Achlya).The example of mitospore fungi comprises Aspergillus, Penicillium (Penicillium), mycocandida and Alternaria (Alternaria).For example, the representative population of Zygomycota comprises Rhizopus (Rhizopus) and Mucor (Mucor).

In a kind of preferred embodiment, described fungal host cells is a yeast cell." yeast " used herein speech comprises ascosporogenous yeast (Endomycetale), product sporidium yeast and belongs to the yeast (gemma steel) of imperfect fungi.Described ascosporogenous yeast can be divided into Spermophthoraceae and Saccharomycetaceae.The latter comprises four subfamilies: Schizosaccharomycoideae (for example, Schizosaccharomyces), Nadsonioideae, Lipomycetoideae (Lipomycoideae) and yeast subfamily (for example, Pichia, genus kluyveromyces and yeast belong).Described product sporidium yeast comprises Leucosporidium (Leucosporidium), Rhodosporidium (Rhodosporidium), locks and throw yeast belong (Sporidiobolus), thread basidiomycetes (Filobasidium) and Filobasidiella (Filobasidiella).The yeast of imperfect fungi can be divided into two sections, ballistospore Saccharomycetaceae (for example, Sorobolomyces and Bu Le bullet spore yeast belong (Bullera)) and Cryptococcaceae (for example, mycocandida).Because the zymic classification may change in the future to some extent, for the present invention, zymic definition as at Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., work, Soc.App Bacteriol SymposiumSeries No.9,1980) disclosed in.The operation of biology of yeast and yeast genetics be widely known by the people in the art (for example, referring to Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J., and Stopans, AO.M., work, the 2nd edition, 1987; The Yeasts, Rose, A.H., and Harrison, J.S., work, the 2nd edition, 1987; With The MolecularBiology of the Yeast Saccharomyces, work such as Strathern, 1981).

In a kind of preferred embodiment, described yeast host cell is the cell of the kind of mycocandida, genus kluyveromyces, yeast belong, Schizosaccharomyces, Pichia or Yarrowia genus.

In a kind of optimum implementation, described yeast host cell is saccharomyces carlsbergensis (Saccharomyces carlsbergensis), yeast saccharomyces cerevisiae, saccharomyces diastaticus (Saccharomyces diastaticus), Douglas yeast (Saccharomycesdouglasii), Crewe not yeast (Saccharomyces kluyveri), promise ground yeast (Saccharomyces norbensis) or ellipsoideus yeast (Saccharomyces oviformis) cell.In another kind of optimum implementation, described yeast host cell is lactic acid Crewe dimension (Kluyveromyces lactis) cell.In another optimum implementation, described yeast host cell is a Yarrowia lipolytica cell.

In a kind of preferred embodiment, described fungal host cells is a filamentous fungal cells." filamentous fungus " comprises the group (determined by Hawksworth etc., 1995, the same) of whole thread forms of Mycophyta and oomycetes door.The trophicity mycelium that the feature of described filamentous fungus is made up of chitin, Mierocrystalline cellulose, dextran, chitosan, mannosans and other complex polysaccharide.Nourish and grow and undertaken, and the katabolism of carbon is obligate aerobic by the mycelia elongation.On the contrary, nourish and grow to sprout such as the zymic of yeast saccharomyces cerevisiae and carry out, and the katabolism of carbon is undertaken by fermentation by unicellular thalline.In a kind of preferred embodiment, described filamentous fungal host cell is the cell with subordinate's kind, but is not limited to these genus: little Acremonium, Aspergillus, fusarium, Humicola, Mucor, myceliophthora, Neurospora, Penicillium (Penicillium), Thielavia (Thielavia), Tolypocladium and Trichoderma (Trichoderma).

In a kind of better embodiment, described filamentous fungal host cell is the Aspergillus cell.In another kind of further preferred embodiment, described filamentous fungal host cell is little Acremonium cell.In another kind of further preferred embodiment, described filamentous fungal host cell is the fusarium cell.In another kind of further preferred embodiment, described filamentous fungal host cell is the Humicola cell.In another kind of further preferred embodiment, described filamentous fungal host cell is the Mucor cell.In another kind of further preferred embodiment, described filamentous fungal host cell is the myceliophthora cell.In another kind of further preferred embodiment, described filamentous fungal host cell is the Neurospora cell.In another kind of further preferred embodiment, described filamentous fungal host cell is the Penicillium cell.In another kind of further preferred embodiment, described filamentous fungal host cell is the Thielavia cell.In another kind of further preferred embodiment, described filamentous fungal host cell is that Tolypocladium belongs to cell.In another further preferred embodiment, described filamentous fungal host cell is the Trichoderma cell.

In a kind of optimum implementation, described filamentous fungal host cell is Aspergillus awamori, smelly aspergillus (Aspergillus foetidus), aspergillus japonicus (Aspergillus japonicus), Aspergillus nidulans, aspergillus niger or aspergillus oryzae cell.In another kind of optimum implementation, described filamentous fungal host cell is cereal sickle spore (Fusarium cerealis), Fusariumcrookwellense, fusarium graminaria (Fusarium graminearum), sharp sickle spore (Fusarium oxysporum), Williams Elder Twig sickle spore (Fusarium sambucinum) or Fusarium sulphureum cell.In another kind of optimum implementation, described fungal host cells is Humicola insolens or dredges cotton shape humicola lanuginosa cell.In another kind of optimum implementation, described filamentous fungal host cell is the mould cell of meter black wool.In another kind of optimum implementation, described filamentous fungal host cell is the thermophilic mould cell of silk of ruining.In another kind of optimum implementation, described filamentous fungal host cell is the Neuraspora crassa cell.In another kind of optimum implementation, described filamentous fungus Mu chief cell is penicillium purpurogenum (Penicillium purpurogenum) cell.In another kind of optimum implementation, described filamentous fungal host cell is a Thielavia terrestris cell.In another optimum implementation, described Trichoderma cell is Trichodermaharzianum, healthy and free from worry wood mould (Trichoderma koningii), Trichodermalongibrachiatum, Trichoderma reesei or viride (Trichoderme viride) cell.

Can be by a kind of known method transformed eukaryotic mycetocyte own, this method comprises protoplastis formation, protoplast transformation and cell walls regeneration.Be applicable to that the appropriate method that transforms the Aspergillus host cell is disclosed in the book of EP 238 023 and Yelton etc. (1984, Proceedings ofthe National Academy of Sciences USA 81:1470-1474).Be applicable to that the method that transforms sickle spore bacterial classification is disclosed in the book (1989, Gene 78:147-156) of Malardier etc. or in the Application No. 08/269,449 that awaits the reply, above document is done this paper reference by receipts.Zymic transforms and can carry out with the method that is disclosed in the following document: Becker and Guarente, see Abelson, J.N. and Simon, M.I. work, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito etc., 1983, Journal of Bacteriology153:163; With Hinnen etc., 1978, Proceedings of the National Academy ofSciences USA 75:1920.The conversion of mammalian cell can realize by the method for directly absorbing with the calcium phosphate precipitation method (1978, Virology 52:546) of Graham and Van derEb.Production method

The invention still further relates to the method for production polypeptide of the present invention, comprise that (a) cultivates a kind of host cell under the condition that can induce described expression of polypeptides; (b) reclaim described polypeptide.

In aforesaid method, be in being suitable for producing the nutritional medium of described polypeptide, to cultivate described cell with methods known in the art.For example; can pass through shaking culture, the laboratory with or industrial fermentor tank in suitable medium described polypeptide can be expressed and/or isolating condition under carry out small-scale or large scale fermentation (comprise continuously ferment, batch fermentation, fed-batch fermentation or solid state fermentation) and cultivate described cell.Cultivation is to use approach well known, is containing carbon and nitrogenous source, and carry out in the suitable nutrient medium of inorganic salt (for example, referring to bacterium and zymic reference; Bennett, J.W. and LaSure, L., work, More Gene Manipulationsin Fungi, Academic Press, CA, 1991).Suitable medium can from the supplier locate to buy or according to disclosed formulation (for example, can referring to U.S. typical case culture collection products catalogue).If described polypeptide is secreted in the nutritional medium, then can directly from substratum, reclaim this peptide species, if described polypeptide is not a secretion property, then from cell pyrolysis liquid, reclaim it.

Described polypeptide can detect with means known in the art, and this method is exclusively used in this polypeptide.Described detection method can comprise the formation of the use of specific antibody, a kind of enzyme product or a kind of disappearance of enzyme substrates.For example, enzyme testing method can be used to measure the activity of this polypeptide.Can measure the generation of lipase activity with any currently known methods in this area.In one approach, a lipase unit (LU) is under 30 ℃ and pH7.0 (phosphoric acid buffer), is substrate when being emulsifying agent with the Sudan Gum-arabic with the tributyrin, and per minute disengages the required enzyme amount of the titratable lipid acid of 1.0 μ mol.In the scope of pH7-10, in no Free Ca ⁺⁺Condition under survey the activity of lipolytic enzyme, mensuration is to use the substrate olive oil emulsion: 2%PVA solution (1: 3) carried out 10 minutes under specific pH and 40 ℃.When reaction finished, use chloroform under acidic conditions: methyl alcohol (1: 1) extracted this reaction mixture, and analyzed the lipid acid that is discharged during (Iatroscan) assaying reaction by TLC-FID.An OPID unit (OPDIU) is meant that per minute disengages 1.0 μ mol lipid acid.When testing each time, all adopt 10mM EDTA and 200mM damping fluid (Tris-HCl damping fluid, pH7 and 8, pH8,9 and 10 diethanolamine buffer) simultaneously.

Can reclaim resulting polypeptide with methods known in the art.For example, can reclaim described polypeptide from nutritional medium with ordinary method, that described method includes, but are not limited to is centrifugal, filtration, extraction, spraying drying, evaporation or precipitation.Can reclaim described polypeptide with various chromatography methods then, for example, ion exchange chromatography, gel permeation chromatography or affinity chromatography etc.

Can be with various methods known in the art purifying polypeptide of the present invention, described method (for example includes, but are not limited to chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatofocusing and size exclusion chromatography), electrophoresis method (for example, preparation isoelectrofocusing (IEF)), differential solvability (for example, ammonium sulfate precipitation), or extraction is (for example, referring to Protein Purification, J.-C.Janson and Lars Ryden, work, VCH Publishers, New York, 1989).Purposes

Recombinant polypeptide by nucleic acid sequence encoding of the present invention can be used for the most common use of lipolytic enzyme, especially under high pH, use, for example, be used for laundry and wash the dish washing composition, specialty and industry washing and leather processing.

Also lipolytic enzyme of the present invention can be used for transesterify, so that comprehensive hydrolysed fat and oil, and be used for the rotational isomerism method for splitting.

The invention will be further described by the following examples, these embodiment should be considered as the qualification to protection domain of the present invention.

Embodiment example 1: extracting genome DNA

Under 32 ℃, in 25ml 0.5% yeast extract-2% glucose (YEG) substratum, allow Absidia griseola NN000987 (ATCC20430), Absidia sporophora-variabilis NN102 427 (ATCC 36019), Absidia grisieola var.iguchii NN 000591 (ATCC 20431), absidia corymbifera NN 100062 (IFO 8084) and Absidia blakesleeana NN 100826 (NRRL 1304) growth 24 hours respectively with the rotating speed of 250rpm.Then by (CA) filtration is collected mycelium from every kind of culture, and washs once with 25ml 10mM Tris-0.1MEDTA (TE) damping fluid for Calbiochem, LaJolla with Miracloth.Unnecessary damping fluid is siphoned away from the mycelium preparation, then freezing said preparation in liquid chlorine.In coffee grinder, described refrigerated mycelium preparation is worn into fine powder, these fine powders are added respectively in the disposable plastic centrifuge tube that 20ml TE damping fluid and 5ml 20%w/v sodium lauryl sulphate (SDS) are housed.This mixture is reversed several times gently, and mix guaranteeing, and use isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1 v/v/v) extracts 2 times.Sodium acetate (3M solution) is added in the stripped sample, make its final concentration reach 0.3M, add 2.5 times of ice-cold ethanol of volume subsequently, to precipitate described DNA.15, under the 000xg with centrifugal 30 minutes of described pipe, with deposit D NA.Allow this DNA precipitate air-dry 30 minutes, be suspended in again then in the 0.5ml TE damping fluid.The ribonuclease A of no DNase is added in the DNA precipitation that suspends again, and making its concentration is 100 μ g/ml, under 37 ℃ this mixture is cultivated 30 minutes then.Proteinase K is added in each centrifuge tube, and under 37 ℃, cultivate 1 hour again.At last, use phenol: chloroform: primary isoamyl alcohol (25: 24: 1 v/v/v) is used sodium acetate and ethanol sedimentation DNA then with every kind of sample extraction 2 times.Dry this DNA precipitation is suspended in again in the TE damping fluid, and preserves down at 4 ℃ under vacuum condition.Example 2:Absidia griseola NN000987 and the segmental pcr amplification of Absidia griseola var.iguchiiNN000591 lipase gene

According to Gormsen etc. patent application DK 95/00424 (content of this application by receipts do this paper with reference to) the disclosed Absidia griseola NN000987 lipase and the aminoacid sequence of Absidia griseolavar.iguchii NN000591 lipase, with an Applied BiosystemsModel 394 DNA/RNA synthesizers, the specification sheets that provides according to the manufacturer synthesizes following Oligonucleolide primers, the lipase gene fragment that comes from Absidia griseola NN 000987 and Absidia griseolavar.iguchii NN000591 with pcr amplification (is annotated: R=A or G, the I=inosine, Y=T or C, H=A or T or C, and N=A or T or C or G).1. forward primer

Aminoacid sequence:

GluThrGluIleGlnAlaHisThrPhe (sequence 11)

Oligonucleotide (+chain):

5 '-GARACIGARATHCARGCICAYACITT-3 ' (sequence 12) is reverse primer 2.

Aminoacid sequence:

ProProGlyAlaPheGlyPheLeu (sequence 13)

Oligonucleotide (chain):

5 '-ARRAANCCRAAIGCNCCIGGNGG-3 ' (sequence 14)

Absidia griseola NN 000987 or Absidia griseola var.iguchii NN 000591 genomic dna with about 1 μ g are that template prepares amplified reaction thing (100ml).Contain following composition in each reactant: 1 μ g genomic dna, 40pmol forward primer, 40pmol reverse primer, dATP, dCTP, dGTP and each 200mM of dTTP, 1 * Taq polymerase buffer (Perkin-Elmer Corp., Branchburg, NJ) and Taq polysaccharase (the Perkin-Elmer corp. of 5 units, Branchburg, NJ).Sterile mineral oil (100 μ l) shop layer in each reaction mixture top, is cultivated described reaction mixture on a Perkin-Elmer Model480 thermal cycler, its program is: circulation 1-95 ℃ following 5 minutes, 45 ℃ of following 2 minutes and 67 ℃ are following 5 minutes; Circulation 2-30-95 ℃ following 2 minutes, 45 ℃ of following 2 minutes and 67 ℃ are following 2 minutes; And 4 ℃ of following infusion.(Sigma Chemical Co., St.Louis MO) go up the described reaction product of separation at 1% low melting-point agarose gel, and from the main PCR product band of this gel excision, and (New England Biolabs, Beverly MA) carry out purifying to use β-gelase according to manufacturer's specification sheets.Then with the PCR product cloning of purifying to pCR II carrier (Ivitrogen, San Diego, CA) on, and with lac forward and reverse primer (New England Biolabs, Beverly MA) measure this dna sequence dna.

With above-mentioned lipase specific PCR primer, the lipase gene fragment of the about 870bp that comes from Absidia griseolaNN 000987 and Absidia griseola var.iguchii NN 000591 shown in Figure 1 that increases.Dna sequence analysis shows that the gene fragment coding that is increased is equivalent to absidia lipase gene part.In addition, described dna sequence dna data confirms that these two kinds of gene products may be identical, and total and rice black root Mucor lipase homologous part.Example 3: the hybridization analysis of genomic dna

By Southern hybridization (Maniatis etc., 1982, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York) the full cell DNA sample available from five absidia bacterial strains described in the example 1 is analyzed.Digest every kind of DNA sample of about 2-5 μ g with EcoR I, Asp 718 I or EcoR I+Asp 718 I, and on 1% sepharose electrophoresis.Under short wavelength UV light, this gel is taken pictures, and in 0.5M NaOH-1.5M NaCl, soaked 15 minutes, in 1.5MNaCl-1M Tris-HCl pH8, soaked 15 minutes more subsequently.According to disclosed method such as Davis (1980, Advanced Bacterial Genetics, A Manual for GeneticEngineering, Cold Spring Harbon Press, Cold Spring Harbor, NewYork), by at 20 * SSPE (3M NaCl-0.2M NaH ₂PO ₄-0.02M Na ₂EDTA) carry out the capillary trace in, the DNA in the described gel is transferred to NytranO Hybond membrane (Schleicher﹠amp; Schuell, Keene, NH) on.With UV Stratulinker (Stratagene, La Jolla, CA) this DNA is crosslinked on described film, under 45 ℃, in following hybridization buffer, soaked this film 2 hours, jog: 5 * SSPE, 35% methane amide (v/v), 0.3%SDS and 200mg/ml sex change simultaneously and the salmon test dna of shearing.By with ( ³²P) dCTP (Amersham, Arlington Heights, IL) carry out nick translation (Maniatis etc., 1982, the same) the isolating lipase specific probe fragment in Absidia griseola NN000987 PCR-clone described in the example 2 is carried out radio-labeled, final concentration adding NaOH with 0.1M carries out sex change, and to be approximately 1 * 10 ⁶The activity of cpm/ml damping fluid adds in the hybridization buffer.Under 45 ℃, in shaking water-bath, cultivate this mixture overnight.After the cultivation, under 45 ℃, the described Hybond membrane of washing is 1 time in containing 0.2 * SSPE of 0.1%SDS, washs 2 times in 0.2 * SSPE (no SDS) under uniform temp then.On thieving paper, allow dry 15 minutes of this film, be encapsulated in Saran-Wrap then ^TMIn, and under-70 ℃, X-ray film exposure is spent the night, use intensifying screen (Kodak, Rochester, NY).

The lipase gene fragment probe that comes from Absidia griseola var.iguchii NN 000591 with the PCR generation, under medium stringent condition, the full cell DNA from each absidia orchidi is analyzed by the Southern blotting, the result shows, proof all absidia orchidis lipase gene all can with described probe cross hybridization (Fig. 2).Proof all bacterial classifications also have a hybridization signal that is approximately 2.1kb in its EcoR I digestion product, have only Absidiasporophora-variabilis NN 102 427 exceptions, and what it produced is the hybridization signal band of a 4kb.In addition, seeming proof all bacterial classifications all contains the relevant lipase gene of single copy in its genome.Example 4:DNA library and lipase clone's evaluation

(Life Technologies, Gaithersburg MD) go up the genome dna library that makes up enrichment at phage clone carrier 1ZipLox.At first, digest full cell DNA, and on 1% sepharose, carry out size separation with the EcoR I.Excision size is corresponding to the migration dna fragmentation of previous observed hybridization signal on the Southern trace described in the example 3, and with Prep-a-Gene reagent (BroRad Laboratories, Hercules, CA) wash-out from gel.The approximate size of dna fragmentation is as follows in above-mentioned fraction: 1.9-2.5kb (Absidiagriseola var.iguchii NN000591), (1.9-2.5kb Absidia blakesleeana NN100826), 1.9-2.5kb (absidia corymbifera NN 100062) and 2.5-4.3kb (Absidiasporophora-variabilis NN 102427).With the dna fragmentation of wash-out and the dephosphorylation λ ZipLox vector arms of EcoR I-cracking (Life Technologies, Gaithersburg, MD), and with the commercially available back extract (Stratagene, La Jolla CA) pack this connection mixture.Plate is paved in the DNA library of packing, and in intestinal bacteria Y1090ZL cell (Life Technologies, Gaithersburg, MD) middle amplification.Tiring of recombinant phage is 1.3-5.4 * 10 in each library ⁵Pfu/ml (tire and be 1.7-2.0 * 10 by the background of no DNA ⁴Pfu/ml).Make probe with the lipase specific PCR fragment that comes from Absidia griseola NN000987, to each about 15 without the library of amplification, 000-30,000 plaque screen (Davis etc., 1980, the same) by plaque hybridization.In intestinal bacteria Y1090ZL cell,, excise lipase gene with the pZL1-derivative form from described λ ZipLox carrier then to producing the plaque purification 2 times of strong hybridization signal with described probe.This recombinant dna fragment is inserted the phasmid pZL1 part of described carrier, and reclaim the segmental phasmid of insertion that has obtained described clone with the pZL1 form of self-replicating, by ehec infection DH 10Bzip cell (Life Technologies, Gaithersburg MD) reclaims with the method for shearing in the body.Prepare in this way isolating lipase clone, (Promega Madison WI) carries out dna sequence analysis with Wizard 373 DNA purification kits.Example 5: the dna sequence analysis of lipase gene

With an Applied Biosystems Model 373A automated DNA sequenator (Applied Biosystem, Inc.Foster City, CA) to carrying out dna sequencing in the clone's of the lipase disclosed in the example 4 two strands, stop chemistry by the primer walking technology with dyestuff and carry out (Giesecke etc. (1992, Journal of Virol.Methods 38:47-60)).The oligonucleotide sequencing primer is the specification sheets synthetic on an Applied Biosystems Model 394DNA/RNA synthesizer according to the manufacturer.

The nucleotide sequence of the gene of coding absidia lipase is shown in Fig. 3-6 ( sequence 1,3,5 and 7).Intron determines to be based on the known amino acid sequence (Boel etc. that (a) comes from peptide fragment in each gene, 1988, Lipids 23:701-706), (b) with meter comparison (the sequence 15) (Gurr etc. of the deduction aminoacid sequence of black root Mucor lipase, see Kinghorn, J.R., work, Gene Structure in Eukaryotic Microbes, pp.93-139, IRL Press, Oxford), (c) the known consensus sequence of filamentous fungus intron (Von Heijne, 1984, Jorunal of Molecular Biology 173:243-251).

In order to separate the gene of the mould lipase of coding reflection colter, allow reflection mould NN 102427 bacterial strains of colter (ATCC 44896) be grown in George Simond oil (jojoba oil) to induce on the optimal medium of composition.Prepare a kind of cDNA library by above-mentioned bacterial strains, and make probe by colony hybridization identification of cdna clone with above-mentioned absidia corymbifera gene.The sequence of this absidia corymbifera gene is (sequence 9) as shown in Figure 7.The homology of this lipase and Absidia sporophora-variabilis lipase is approximately 99%.Example 6: from the comparison of the lipase gene of various absidia orchidis

Intron-the exons structure of these absidia lipase genes is quite similar, promptly forms by 7 exons and 6 introns, and is as shown in table 1.The size of exon 2-6 is high conservatives.The size of first exon shows slight mobility because of certain variation in the zone of the preceding peptide moiety of described signal peptide of coding and described enzyme.In addition, homology of nucleotide sequence is also very high between the corresponding exon, and its identity is 85-97%.On the contrary, the length of its intron is variable, has only umbrella branch colter enzyme NN 100062 and Absidia sporophora-variabilis NN 102 427 lipase genes exception, and the sequence homology between its relevant intron is very low.5 of described lipase gene ' also show sequence divergence with 3 ' flanking sequence.

Table 1. comes from the intron-exons structure of the lipase gene of several absidia orchidis

Bacterial classification	Exon (length is in bp)							Intron (length is in bp)
															??1 489 492 ? 495 ? 495	??2	??3	??4	??5	??6	??7	1 82 75 61 61	?2	?3	?4	?5	?6
A.griseola?var.iguchii	?58	?97	?173	?81	?79	?31	?64	?56	?61	?64	?66
												A.blakesleeana	?58	?97		?173	?81	?79	?31	?52	?57		?60	?64	?67
Absidia corymbifera	?58	?97	?173	?81	?79	?31	?62	?54	?58	?53	?59
												A.sporophora-variabilis	?58	?97		?173	?81	?79	?31	?62	?54		?57	?53	?59

Based on the aminoacid sequence ( sequence 2,4,6,8 and 10) of inferring, biochemistry and biophysical properties to absidia lipase in table 2 compare.All 4 kinds of lipase are all quite similar, show all synthetic with 337-338 amino acid whose prozymogen form, contain 17-21 amino acid whose signal peptide, a 52-56 amino acid whose propetide and 264-265 amino acid whose maturing enzyme, the molecular weight of its non-glycosylated protein is approximately 29,000.The iso-electric point that calculates is 6.10～6.94 fluctuations, and what alkalescence was the strongest is the lipase that comes from Absidia sporophora-variabilis.The iso-electric point value that aforementioned calculation goes out is far below observing iso-electric point value (referring to the patent application DK95/00424 of Gormsen etc.) on the IEF gel in the experiment, this may be because be not that all charged residues all appear on the three-dimensional surface of this proteic exposure and cause.

Table 2. comes from the biochemistry and the biophysical properties of the prediction of several absidia orchidis

Bacterial classification	Preceding former form	The signal peptide of inferring	The propetide of inferring	Sophisticated lipase	Molecular weight	The iso-electric point that calculates	Molar extinction coefficient	1 A280 unit (mg/ml)
									A.griseola var.iguchii	?337aa	?18aa	?54aa	?264aa	?29,028	?6.45	?27,580	????1.05
A.blakesleeana	?337aa	?18aa	?55aa	?264aa	?28,959	?6.64	?24,900	????1.16
									Absidia corymbifera	?338aa	?21aa	?52aa	?265aa	?29,057	?6.10	?27,460	????1.06
A.sporophora- variabilis	?338aa	?17aa	?56aa	?265aa	?28,952	?6.94	?27,460	????1.05

As table 3 and shown in Figure 8, the amino acid sequence homology of the mould total extension of absidia fat, identity each other is 87-96%, and with the limited homology of rice black root Mucor lipase (sequence 15) be 53-55%, with the limited homology of dredging cotton shape humicola lanuginosa lipase (sequence 16) be 22-24%.

Table 3. comes from absidia orchidi kind, rice black root Mucor and dredges cotton shape detritus

Amino acid sequence similarity similarity per-cent between the mould lipase

		???1	????2	????3	????4	????5	????6
										Divergent per-cent	??1	???-	??87.2	??87.5	??88.1	??53.7	??22.3	??1	?A.blakesleeana
??2	??11.9	???-	??86.3	??87.2	??54.8	??22.7	??2	?A.griseola?var. ????iguchii
									??3		??11.6	??11.9	???-	??96.4	??53.3	??23.7	??3	Absidia corymbifera
??4	??11.0	??11.0	??3.6	???-	??53.3	??24.1	??4	?A.sporophora-var ????iabilis
									??5		??41.6	??40.5	??41.9	??41.9	???-	??23.7	??5	Rice black root Mucor
??6	??70.6	??70.9	??70.0	??70.0	??68.8	????-	??6	Dredge cotton shape humicola lanuginosa
											????1	????2	????3	????4	????5	????6

From the phylogeny angle, the lipase of absidia is the most approaching with other member's of the zygomycetes that comes from fungi lipase.The correlation it very of yeast, bacterium and Mammals lipase and fungi at low (if any).Example 7: the expression of absidia corymbifera lipase gene

The subclone that the clone and the nucleotide sequence of above-mentioned absidia corymbifera lipase gene is used for this gene, and in the Aspergillus host, express.Come from the lipase gene (promotor that does not have himself) of isolating genomic clone NL95A with the PCR method subclone, use primer by described nucleotide sequence design.For the ease of with this gene fragment subclone to the expression vector that is named as pBANe6 (Fig. 9), respectively Swa I and Pac I restriction enzyme sites are introduced 5 of this gene ' and 3 ' end.Carrier pBANE6 contains TAKA promotor, NA2-tpi leader sequence and AMG terminator as regulating sequence.This plasmid also contains the selective marker that Aspergillus nidulans amdS gene transforms as fungi.Following primer is used for pcr amplification:

Forward primer:

5 '-CCCATTTAAATATGCGTTTTTATTCAGTAGTATCAT-3 ' (sequence 17)

Reverse primer: 5 '-CTCGGCTTAATTAAAATGGGTTATAAGCAGAGACCAGTG-3 ' (sequence 18)

According to manufacturer's specification sheets, (Boehringer Mannheim, Indianapolis IN) carry out PCR with the Pwo polysaccharase.Pcr amplification product is carried out gel separation,, and carry out gel-purified with Swa I and Pac I enzymatic lysis.The fragment of purifying is connected to pBANe6 carrier (with Swa I and the cracking of Pac I), to obtain plasmid pKB2 (Figure 10), wherein, lipase gene transcribe the control that is subjected to the TAKA promotor.This pKB2 plasmid is transformed into the bacillus coli DH 5 cell.Separate the intestinal bacteria transformant that contains the pKB2 plasmid, and the preparation plasmid DNA is used for transforming and expressing in Aspergillus.

BANe3 prepares protoplastis by aspergillus oryzae strain, wherein, and the amdS genetically deficient of this host strain.The preparation of protoplastis and conversion are carried out (Christensen etc., the same) with previous disclosed method.Based on it ethanamide is selected to express the aspergillus oryzae transformant of acetamidase as the ability in unique ammonia source.Obtain 42 transformant altogether, and on the selectivity flat board with spore purifying 2 times.The transformant of spore purifying is studied as further.

Transformant (the 25ml substratum is housed in the flask of 125ml) by shake-flask culture screening lipase is expressed contains 50g maltose, 2.0gMgSO in per 1 liter of volume of used substratum ₄-7H ₂O, 10g KH ₂PO ₄, 2.0g K ₂SO ₄, 2.0g citric acid, 10g yeast extract, 0.5ml trace metal solution and 2.0g urea.This trace metal solution contains 14.3gZnSO for every liter ₄-7H ₂O, 2.5g CuSO ₄-5H ₂O, 0.5g NiCl ₂-6H ₂O, 13.8g FeSO ₄-7H ₂O, 8.5g MnSO ₄-H ₂O and 3.0g citric acid.Before carrying out autoclaving, the pH of this substratum is transferred to 6.5.With the spore inoculating flask of new collection, and the rotating speed with 200rpm is cultivated in incubator under 34 ℃.After cultivating 48 hours, measure the activity of lipase in the culture every day.Because the situation for the suitable substrates of this lipase is known nothing, measure enzymic activity by following three kinds of methods: ⅰ) carry out the dull and stereotyped mensuration of lipase as substrate with sweet oil, ⅱ) with right-the oil of mirbane butyric ester carries out colorimetric analysis as substrate, and ⅲ) carry out titration with tributyrin (tributyrin).

Carry out the lipase Analysis of Plate with plate culture medium, contain following composition in this substratum: 0.1MTris pH9.0,0.1M CaCl ₂, 1%Triton X-100,0.5% sweet oil and 2.0% agarose.Substratum is carried out autoclaving, and inject the plate of 150mm, every ware injects 50-60ml.After agarose solidifies, on each flat board, make a call to 15 holes, and in each hole, add the 25ml nutrient solution.Will be from the nutrient solution of unconverted aspergillus oryzae strain BANe3 with comparing.Cultivating described flat board down at 37 ℃ spends the night.Determine the existence of lipase activity in the transformant according to the existence of transparent region around the hole.This clarification phenomenon can not appear in the control wells that has added from the nutrient solution of unconverted bacterial strain, shows that lipase activity exists only in the transformant.

Also make substrate and measure lipase activity with right-oil of mirbane butyric ester.By this compound of 10ml being added the right-oil of mirbane butyric ester of preparation in 1.0ml dimethyl sulfoxide (DMSO) (DMSO) and the 4.0ml 0.1M Tris pH9.0 damping fluid.The nutrient solution that 100 μ l are suitably diluted adds in each hole.Add 100ml right-oil of mirbane butyric ester substrate starts reaction, and surveyed absorption value 3-5 minute under the 405nm wavelength.Activity according to the curve calculation enzyme of doing the standard substance drafting with the thin cotton shape humicola lanuginosa of known quantity.Unconverted bacterial strain can produce very little or not produce activity, and different transformant produces lipase after cultivating 48 hours.

By based on further measuring lipase activity by the volumetry of the catalytic tributyrin hydrolysis of lipase.By following the tracks of butyro-release with alkalimetric titration stablizing under the pH.To measuring from the nutrient solution of selected transformant with from the nutrient solution of unconverted control strain.The result shows that unconverted control strain does not produce lipase activity, produces detectable lipase activity and transform physical efficiency.The expression of example 8:Absidia sporophora-variabilis lipase gene in aspergillus oryzae

Described original gene group is cloned as Pwo polysaccharase (Boehringer-MannheimBiochemicals, Indianapolis, IN) in the PCR reactant, the increase coding region of A.sporophora-variabilis of template, contain in the reactant: the 61ml sterilized water, the template DNA of 10ml dilution (approximately 5ng/ml), 1ml primer 1 (about 30pmol:dATGATGCATTCTCATTTTGTAGTCTTATTG, sequence 19), 1ml primer 2 (about 25pmol:dGCTTAATTAATTATAAACAGAGACCAGTGTTCATGTCAAG, sequence 20), 16ml dATP, dCTP, dGTP and dTTP mixture (200mmol final concentration), 10ml Pwo damping fluid (10 * solution; Boehringer-Mannheim Biochemicals) and 1ml Pwo polysaccharase (5 unit).Amplification condition is as follows: first round was carried out under 95 ℃ 5 minutes, and 45 ℃ of following 2 minutes and 67 ℃ are following 5 minutes; The 2-30 round was carried out under 95 ℃ 2 minutes, and 45 ℃ of following 1 minute and 67 ℃ are following 2 minutes; An and infusion of under 4 ℃, carrying out., and separate excision and with Prep-a-Gene reagent (BioRad Laboratories, Hercules, CA) purifying with the lipase gene fragment of Pac I digest amplification by the preparation agarose gel electrophoresis.With the fragment of purifying be connected with the pBANe6 of Swa I with the cracking of Pac I, to obtain lipase expression vector pRaMB19 (Figure 11).

Use standard method (Christensen etc., 1988, Bio/Technology 1419-1422) then, transform alkaline protease deficient aspergillus oryzae host JaL142 with pRaMB19.To transform the bacterium colony purifying 2 times by condiospores, and, contain the 130g Star Dri 5 in the per 1 peaceful plate, 3g MgSO by expressing at tributyrin agar lining out test lipase ₄-7H ₂O, 5g KH ₂PO ₄, 4g citric acid, 6g K ₂SO ₄, 0.5ml trace metal (described in example 7), 5g yeast extract, 160ml 1M urea, 35.3ml 1M NaNO ₃, 25g Noble agar and 10g tributyrin pH4.5.30 ℃ down cultivate 48 hours after, 80 in 84 transformant show and occur unique clarifying areas on the tributyrin agar plate, show to have produced the lyolipase activity.In the shake-flask culture thing of MY50 substratum, 10 in these transformant are further tested, contain 50g Star Dri 5,2g MgSO in per 1 liter of substratum ₄-7H ₂O, 10gKH ₂PO ₄, 2g citric acid, 2g K ₂SO ₄, 0.5ml trace metal solution, 10g yeast extract, 2g urea pH6.0.Under 37 ℃, described shake-flask culture thing was cultivated 48 hours, made substrate is measured the culturing filtrate of every kind of culture by example 7 described methods lyolipase activity with right-oil of mirbane butyric ester then.The culture of unconverted control cells can not produce detectable lipase activity, and pRaMB19 conversion physical efficiency produces detectable lipase activity.

The preservation of biomaterial

Stipulate according to budapest treaty, on January 18th, 1996 following biomaterial is transferred to NRRL (Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, 1815 University Street, Peoria, Illinois, 61604) preservation, given go into to hide number as follows.

The preservation thing	Go into to hide number
		Intestinal bacteria DH10B (pZL-NL1)-Absidia blakesleeana	????NRRLB-21520
Intestinal bacteria DH10B (pZL-NL61)-absidia corymbifera	????NRRLB-21521
		Intestinal bacteria DH10B (pZL-NL95)-Abisidia griseola-iguchii	????NRRLB-21522
Intestinal bacteria DH10B (pZL-NL124)-Absidia sporophora-variablilis	????NRRLB-21523

Claims (56)

1. One kind the coding a kind of isolated nucleic acid sequences with polypeptide of lipase activity, this sequence is selected from:

   (a) nucleotide sequence of the endogenous polypeptide of a kind of absidia bacterial strain of coding, this polypeptide has the aminoacid sequence shown in sequence 2, sequence 4, sequence 6, sequence 8 or the sequence 10;

   (b) a kind of endogenous nucleic acid sequence of absidia bacterial strain, it can be under the condition of medium strictness and (ⅰ) nucleotide sequence shown in sequence 1, sequence 3, sequence 5, sequence 7 or the sequence 9 or (ⅱ) its any complementary strand hybridization;

   (c) a kind of nucleotide sequence, it can be under the condition of medium strictness and (ⅰ) nucleotide sequence shown in sequence 1, sequence 3, sequence 5, sequence 7 or the sequence 9 or (ⅱ) its any complementary strand hybridization;

   (d) a kind of coding has the nucleotide sequence of the polypeptide of lipase activity, and the identity of aminoacid sequence is at least 65% shown in this amino acid sequence of polypeptide and sequence 2, sequence 4, sequence 6, sequence 8 or the sequence 10;

   (e) (a) and (b), (c) or equipotential form (d); With

   (f) (a) and (b), (c) or fragment (d).
2. 2. nucleotide sequence as claimed in claim 1, wherein, described nucleotide sequence can be under the condition of medium strictness and (ⅰ) nucleotide sequence shown in sequence 1, sequence 3, sequence 5, sequence 7 or the sequence 9 or (ⅱ) its any complementary strand or fragment hybridization.
3. 3. nucleotide sequence as claimed in claim 2, wherein, described nucleotide sequence can be under the condition of medium strictness and (ⅰ) nucleotide sequence shown in sequence 3, sequence 5, sequence 7 or the sequence 9 or (ⅱ) its any complementary strand hybridization.
4. 4. nucleotide sequence as claimed in claim 3, wherein, described nucleotide sequence can be hybridized with nucleotide sequence shown in the sequence 1 or its complementary strand under the condition of medium strictness.
5. 5. nucleotide sequence as claimed in claim 3, wherein, described nucleotide sequence can be hybridized with nucleotide sequence shown in the sequence 3 or its complementary strand under the condition of medium strictness.
6. 6. nucleotide sequence as claimed in claim 3, wherein, described nucleotide sequence can be hybridized with the nucleotide sequence shown in the sequence 5 or its complementary strand under the condition of medium strictness.
7. 7. nucleotide sequence as claimed in claim 3, wherein, described nucleotide sequence can be hybridized with the nucleotide sequence shown in the sequence 7 or its complementary strand under the condition of medium strictness.
8. 8. nucleotide sequence as claimed in claim 3, wherein, described nucleotide sequence can be hybridized with the nucleotide sequence shown in the sequence 9 or its complementary strand under the condition of medium strictness.
9. 9. nucleotide sequence as claimed in claim 2, wherein, a kind of polypeptide that has available from the lipase activity of absidia of described nucleic acid sequence encoding.
10. 10. nucleotide sequence as claimed in claim 9, wherein, a kind of polypeptide that has available from the lipase activity of Absidia blakesleeala of described nucleic acid sequence encoding.
11. 11. nucleotide sequence as claimed in claim 9, wherein, a kind of polypeptide that has available from the lipase activity of absidia corymbifera of described nucleic acid sequence encoding.
12. 12. nucleotide sequence as claimed in claim 9, wherein, a kind of polypeptide that has available from the lipase activity of Absidia griseola of described nucleic acid sequence encoding.
13. 13. nucleotide sequence as claimed in claim 9, wherein, a kind of polypeptide that has available from the lipase activity of Absidia griseola var.iguchii of described nucleic acid sequence encoding.
14. 14. nucleotide sequence as claimed in claim 9, wherein, a kind of polypeptide that has available from the mould lipase activity of reflection colter of described nucleic acid sequence encoding.
15. 15. nucleotide sequence as claimed in claim 9, wherein, a kind of polypeptide that has available from the lipase activity of Absidia sporophora-variabilis of described nucleic acid sequence encoding.
16. 16. nucleotide sequence as claimed in claim 1, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, any or its segmental identity of aminoacid sequence shown in this aminoacid sequence and sequence 2, sequence 4, sequence 6, sequence 8 and the sequence 10 is at least 65%.
17. 17. as the nucleotide sequence of claim 16, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of any of aminoacid sequence shown in this aminoacid sequence and sequence 2, sequence 4, sequence 6, sequence 8 and the sequence 10 is at least 70%.
18. 18. as the nucleotide sequence of claim 17, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of any of aminoacid sequence shown in this aminoacid sequence and sequence 2, sequence 4, sequence 6, sequence 8 and the sequence 10 is at least 75%.
19. 19. as the nucleotide sequence of claim 18, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of any of aminoacid sequence shown in this aminoacid sequence and sequence 2, sequence 4, sequence 6, sequence 8 and the sequence 10 is at least 80%.
20. 20. as the nucleotide sequence of claim 19, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of any of aminoacid sequence shown in this aminoacid sequence and sequence 2, sequence 4, sequence 6, sequence 8 and the sequence 10 is at least 85%.
21. 21. as the nucleotide sequence of claim 20, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of any of aminoacid sequence shown in this aminoacid sequence and sequence 2, sequence 4, sequence 6, sequence 8 and the sequence 10 is at least 90%.
22. 22. as the nucleotide sequence of claim 21, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of any of aminoacid sequence shown in this aminoacid sequence and sequence 2, sequence 4, sequence 6, sequence 8 and the sequence 10 is at least 95%.
23. 23. as the nucleotide sequence of claim 22, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of aminoacid sequence is at least 95% shown in this aminoacid sequence and the sequence 2.
24. 24. as the nucleotide sequence of claim 23, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, this aminoacid sequence has the aminoacid sequence shown in the sequence 2.
25. 25. as the nucleotide sequence of claim 24, wherein, described nucleotide sequence is shown in sequence 1.
26. 26. the nucleotide sequence shown in claim 22, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of aminoacid sequence is at least 95% shown in this aminoacid sequence and the sequence 4.
27. 27. as the nucleotide sequence of claim 26, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, this aminoacid sequence has aminoacid sequence shown in the sequence 4.
28. 28. as the nucleotide sequence of claim 27, wherein, described nucleotide sequence is shown in sequence 3.
29. 29. as the nucleotide sequence of claim 22, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of aminoacid sequence is at least 95% shown in this aminoacid sequence and the sequence 6.
30. 30. as the nucleotide sequence of claim 29, wherein, the aminoacid sequence shown in the described nucleic acid sequence encoding sequence 6.
31. 31. as the nucleotide sequence of claim 30, wherein, described nucleic acid is shown in sequence 5.
32. 32. as the nucleotide sequence of claim 22, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of aminoacid sequence is at least 95% shown in this aminoacid sequence and the sequence 8.
33. 33. as the nucleotide sequence of claim 32, wherein, the aminoacid sequence shown in the described nucleic acid sequence encoding sequence 8.
34. 34. as the nucleotide sequence of claim 33, wherein, described nucleotide sequence is shown in sequence 7.
35. 35. as the nucleotide sequence of claim 22, wherein, a kind of aminoacid sequence of described nucleic acid sequence encoding, the identity of aminoacid sequence is at least 95% shown in this aminoacid sequence and the sequence 10.
36. 36. as the nucleotide sequence of claim 35, wherein, the aminoacid sequence shown in the described nucleic acid sequence encoding sequence 10.
37. 37. as the nucleotide sequence of claim 36, wherein, described nucleotide sequence is shown in sequence 9.
38. 38. nucleotide sequence as claimed in claim 1, wherein, described nucleotide sequence is a kind of endogenous sequence of absidia bacterial strain, and can hybridize with nucleotide sequence shown in the sequence 1 or its mutual preface chain under medium stringent condition.
39. 39. nucleotide sequence as claimed in claim 1, it comprises the absidia lipase nucleic acid sequence encoding on the plasmid pZL-NL1 that is contained among the intestinal bacteria NRRL B-21520.
40. 40. nucleotide sequence as claimed in claim 1, it comprises the absidia lipase nucleic acid sequence encoding on the plasmid pZL-NL61 that is contained among the intestinal bacteria NRRL B-21521.
41. 41. nucleotide sequence as claimed in claim 1, it comprises the absidia lipase nucleic acid sequence encoding on the plasmid pZL-NL95 that is contained among the intestinal bacteria NRRL B-21522.
42. 42. nucleotide sequence as claimed in claim 1, it comprises the absidia lipase nucleic acid sequence encoding on the plasmid pZL-NL124 that is contained among the intestinal bacteria NRRL B-21523.
43. 43. a nucleic acid construct comprises the described nucleotide sequence of claim 1, this sequence operably is connected in and can instructs on one or several control sequence that described polypeptide expresses in suitable expressive host.
44. 44. a recombinant expression vector comprises the described nucleic acid construct of claim 43, a promotor and transcribes and the translation termination signal.
45. 45., also comprise a selective marker as the carrier of claim 44.
46. 46. a recombinant host cell comprises the nucleic acid construct of claim 43.
47. 47. as the cell of claim 46, wherein, described nucleic acid construct is contained on a kind of carrier.
48. 48. as the cell of claim 46, wherein, described nucleic acid construct is integrated in the described host cell gene group.
49. 49. as the cell of claim 46, wherein, described host cell is a bacterial cell.
50. 50. as the cell of claim 49, wherein, described bacterial cell is bacillus, streptomyces or Rhodopseudomonas cell.
51. 51. as the cell of claim 46, wherein, described host cell is a kind of fungal cell.
52. 52. as the cell of claim 51, wherein, described fungal cell is a filamentous fungal cells.
53. 53. cell as claim 52, wherein, described filamentous fungal cells is little Acremonium, Aspergillus, fusarium, Humicola, Mucor, myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium belongs to or the cell of Trichoderma bacterial classification.
54. 54. as the cell of claim 46, wherein, described fungal cell is a yeast cell.
55. 55. as the cell of claim 54, wherein said yeast cell is the cell that mycocandida, genus kluyveromyces, Pichia, yeast belong, Schizosaccharomyces or Yarrowia belong to bacterial classification.
56. 56. a production has the method for the polypeptide of lipase activity, comprises that (a) cultivates the described host cell of claim 46 under the condition that can induce described expression of polypeptides; (b) reclaim described polypeptide.

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