JP3029435B2 - Molecular cloning and expression of a gene encoding a lipolytic enzyme - Google Patents

Abstract

Novel microbial host strains are provided which are transformed by a vector molecule comprising a DNA fragment encoding a lipolytic enzyme and a marker for selection, capable of producing active lipase. Said DNA fragment is preferably derived from a Pseudomonas species.

Description

Description: TECHNICAL FIELD The present invention relates to the preparation of enzymes used for the enzymatic degradation of fats by recombinant DNA technology, in particular lipolytic enzymes having properties suitable for use as detergent additives.

Background One problem with cleaning lies in the removal of fatty stains. At present, dirt containing fat is emulsified and removed by a combination of high temperature and high alkali. However, recently, washing at a relatively low temperature that is not particularly suitable for removing fatty spots, that is, at about 40 ° C. or less has become mainstream.
Therefore, there is a need for detergent additives that are effective at lower wash temperatures, are stable in highly alkaline detergent solutions, and are stable under storage conditions in solid and liquid detergent compositions. One group of enzymes that hydrolyze triglycerides are lipases (EC 3.1.1.3.). Lipases are widely distributed in eukaryotic cells and many other prokaryotes. Depending on the origin of the enzyme, there are various properties including substrate specificity and stability under various conditions. Lipases have hitherto been used in detergent compositions, but those used here exhibit only a low cleaning effect under laundering conditions and, furthermore, do not satisfy the stability suitable for detergents.

Lipid-degrading enzymes that can exhibit lipase activity under modern laundry conditions, ie, those that are effective at high detergent concentrations, high pH and low wash temperatures, are Pseudomonas pseudoalcaligenes, Pseudomonas stutzeri. ) And Acinetobacter calcoati
cus) species (European patent application EP-A
-0218272). However, these microbial species are potentially pathogenic to plants and animals, and there is little data on the fermentation conditions available for lipase production processes using these microorganisms.

It is therefore desirable to develop an effective and safe method for producing lipolytic enzymes with desirable properties for detergent additives. In addition, it is desirable that the enzyme be secreted from the host organism so that it can be recovered directly from the extracellular fluid of the fermentation mixture.

(Related literature) For lipases produced by Pseudomonas species, it is known that culture conditions strongly influence the final location of these enzymes (Sugiura, "Lipase" B. Borg Strom (Borg
strom) and HL Brockman (1984) 5
05-523, Elsbahia edition, Amsterdam). It addresses problems in the efficient expression of foreign genes in microorganisms, including erroneous folding of formed proteins, degradation of proteins, and incorrect locations of proteins. See Harris, Genetic Engineering, Vol. 4 (1983) Academic Press Edition, New York. The use of secretory cloning vectors in E. coli generally allows for the transfer of foreign gene products to the periplasmic space, and the products are sometimes found in culture media. Lun
n) et al., “Latest topics in microbiology and immunology” 125 (1986) 59-74. Gotz et al. (Nucleic Acids Res.) 13 (1985) 5895-5906, when Escherichia coli is used as a host cell.
Kugimiya, etc. (Biochem, Biofizz,
Research Communication (Biochem.Biophys.Res.
Comm.) 141 (1986) 185-190) and Odera et al. (J. Fermental. Technol. 64 (1986) 363-371). Microbial lipase is hardly secreted into the culture medium.

Wohlfarth and Winklar
kler) (J. Gen. Microbiol. 134 (1988) 433-440) is a Pseudomonas aeruginosa.
The physiological properties of a newly isolated lipase-deficient mutant from PAO2302 strain and the chromosomal map and cloning of the corresponding gene are reported.

Bacillus species, particularly Bacillus subtilis strains, have had varying success in using them as host strains for expression of foreign and endogenous genes and secretion of the encoded protein product. For a review, see, for example, Sarva
s), "Recent topics in microbiology and immunology", 125 (1986) 103-125, edited by HC Wu and PC Tai (Tai), Springer-Blaglag and Himeno (Him).
eno) et al., see FEMS Microbiol. Letters 35 (1986) 17-21.

U.S. Pat.No. 3,950,277 and British Patent Specification No.
No. 42,418 is an activator and calcium ion, respectively,
Alternatively, lipase enzymes have been disclosed in conjunction with magnesium ions, which are used to soak soiled fabrics and remove triglyceride stains and soils from polyester or polyester / cotton blend fabrics, respectively. Published useful microbial lipases include Pseudomonas
s), Aspergillus, Pneumococcus, Staphyloco
ccus), Mycobacterium suberculosis (Mycoba)
cterium tuberculosis), Mycotorula lipolytica (Mycotorula lipolytica) and Sclerotinia (Scler
otinia).

British Patent Specification No. 1,372,034 discloses a detergent composition comprising a bacterial lipase produced by Pseudomonas stutzeri strain ATCC 19154. The patent further states that this preferred lipolytic enzyme
It discloses that it has an optimum pH value of pH 6 to 10 and exhibits activity in this pH range, preferably pH 7 to 9. (Here, the strain presumed to be Pseudomonas stutzeri is a Pseudomonas stutzeri strain.
monas aeruginosa). British Patent Application (EP-A) 0130064 discloses an enzymatic detergent additive comprising lipase isolated from Fusarium oxysporum which has a higher lipolytic washing efficiency than conventional lipases. UK Patent Specification No. 1,293,613 and Canadian Patent 835,3
No. 43 also discloses lipolytic detergent additives.

European Patent Applications EP-A-0205208 and EP-A-020
No. 6396 discloses the use of lipases from Pseudomonas and chromovectors in detergents. For a comprehensive review of lipases as detergent additives, see Journal of Applied Biochemistry (J. Ap)
pl. Bicohem.) 2 (1980) 218-229.

SUMMARY OF THE INVENTION New compositions comprising transformed microbial cells that produce lipolytic enzymes suitable for use in detergent compositions and methods for their preparation are provided. Active at alkaline pH,
Transform the host microbial cells with an expression cassette containing a DNA sequence encoding a lipolytic enzyme that is stable under washing conditions. Methods for preparing lipolytic enzymes include cloning and expression in microbial systems and screening based on DNA homology.

Two DNA sequences are also provided, each of which is a pseudomonas pseudoalkagens (Pseudomona
s Peudoalcaligenes strain and Pseudomonas aeruginosa strain.

Lipase genes from certain Pseudomonas species are of particular interest for lipase production.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Strategy for lipolytic enzyme gene cloning.
See footnotes in Figure 2 for symbols.

FIG. 2; pAT1 restriction map. Many restriction enzyme recognition sites in plasmid pAT1 have been identified.

Symbols are used: -Ori E.coli; E. coli origin of replication -Ap ^r; of pUN121 encoding ampicillin resistance gene -Tc ^r; of pUN121 encoding tetracycline resistance gene -cI; cI bacteriophage encoding repressor Phage lambda gene Pseudomonas stutzeri Th
ai IV17-1 (CBS461.85), a partial digest of the chromosomal DNA of San3A was ligated to pUN121 at Bcl I / San3A
Indicated by The location of the gene encoding lipolytic activity is indicated by the dotted line.

FIG. 3; pAT3 restriction map. The symbols are the same as those used in FIG.

FIG. 4; restriction map of pET1. The symbols are the same as those used in FIG.

FIG. 5; pET3 restriction map. The symbols are the same as those used in FIG.

FIG. 6: pAM1 restriction map. The symbols are the same as those used in FIG.

FIG. 7: Restriction map of pM6-5. The symbols are the same as those used in FIG.

Fig. 8: Restriction map of pP5-4. The symbols are the same as those used in FIG. Acinetobacter calcoaceticus (Ac
inetobacter calcoaceticus) GR V-39 (CBS460.85)
The position where the EcoRI ^* partial digest of the chromosomal DNA was ligated to pUN121 is indicated by EcoRI / EcoRI ^* .

FIG. 9; SDS 10-15% gradient fast gel (Pharmacia) FIG. 9A; after staining with Comage Brilliant Blue FIG. 9B; after staining with β-naphthyl acetate / Fast Bull BB Lane 1: 95 ° C., 10 minutes with SDS Heated degradation product from E. coli JM101hsd S rec A harboring pUN121.

Lane 2: degradation product from Escherichia coli JM101hsd S rec A strain harboring PET3, heated at 95 ° C. for 10 minutes with SDS.

Lane 3: culture supernatant from P. stutzeri Thai IV17-1 strain, heated with SDS at 95 ° C for 10 minutes.

Lane 4: sample similar to lane 2, except heated at 95 ° C. for 5 minutes with SDS.

Lane 5: sample similar to lane 3 except heated at 95 ° C. for 5 minutes with SDS.

Lane 6: sample similar to lane 2, except incubated with SDS for 10 minutes at room temperature.

Lane 7: sample similar to lane 3, except incubated with SDS for 10 minutes at room temperature.

Lane 8: Pharmacia low molecular weight protein marker.

Figure 10: SDS13 after Comage Brilliant Blue staining
% Polyacrylamide gel.

Lane 1: purified M-lipase.

Lane 2: low molecular weight protein marker (Pharmacia).

 FIG. 11; pTMPv18A restriction map.

Symbols used: -Ori E.coli; replication origin -f 1 E. coli-derived pBR322 vector ori; filamentous bacteriophage f1 origin of replication origin -P _T7; promoter of bacteriophage T7 for preparing in vitro transcripts - Ap ^r ; gene encoding ampicillin resistance -Lip; gene encoding M-1 lipase FIG. 12: FIG. 12 shows the nucleotide sequence of the M-1 lipase gene (ie, the first 942 nucleotides) and M-1 derived therefrom 2 shows the amino acid sequence of lipase. The stop codon TGA is indicated by an asterisk. The A box represents the active center of the lipase protein. Arrows indicate putative signal peptidase cleavage sites. The amino terminal sequence of the mature lipase protein is underlined.

 FIG. 13; restriction map of pSW103.

Symbols are used; -Ori E.coli; pBR322 derived vectors E. coli replication origin -Plac; gene -Lip encoding ampicillin resistance; promoter -Ap ^r E. coli lac operon gene 14 encoding PAO1 lipase FIG The partial nucleotide sequence of the PAO lipase gene (ie from the internal Sal I site) and the P derived therefrom
Amino acid sequence of AO lipase. The stop codon TAG is indicated by an asterisk. The A box represents the active center of the lipase protein.

 FIG. 15: Construction of plasmids pBHAM1 and pBHCM1.

Symbols are used: -Bacillus ori; Bacillus origin of replication-Km ^r; of pUB110 encoding kanamycin resistance gene --Cm; black encoding ram phenyl call resistance Tn9 transposon gene -P _Hpa II; _Hpa II plasmid pUB110 The other symbols of the promoter are the same as in FIG.

FIG. 16 shows the construction of plasmid pBHAM1N1. The symbols are the same as those used in FIG.

FIG. 17 illustrates the construction of plasmid pTZN1M1. The symbols used are: -lac Zα; the N-terminal part of the Lac gene encoding the α-domain of β-galactosidase -P _Lac ; the promoter of E. coli lac operon The other symbols are the same as in FIG.

FIG. 18 shows the construction of plasmid pMCTM1. The symbols used are: -Cm ^r ; gene encoding chloramphenicol resistance -P _tac ; Escherichia coli hybrid trp-lac promoter The other symbols are the same as in FIGS. 11 and 15.

FIG. 19: Immunoblot detection of M-1 lipase protein produced from transformed E. coli cells.

Lanes AD contain the periplasmic space fraction of E. coli cells harboring the following constructs: Lane A; pTZ18RN lane B; pTZN1M1 lane C; pMCM1 lane D; from Pseudomonas pseudoalcaligens strain M-1 Purified M-1 rivase. The molecular weight of the marker protein (Amersham Rainbow) is shown in kDa on the right.

FIG. 20: Construction of plasmid pBHM1N1. The symbols are the same as those in FIG.

FIG. 21: Autoradiograph of ³⁵ S-labeled protein synthesized in vitro.

Lanes AD; immunoprecipitation of translated protein in vivo with monoclonal antibody to M-1 lipase.

Lanes EH; in vitro transcription / translation products of the following plasmids: Lanes A and E; pTZ18RN Lanes B and F; pTMPv18A Lanes C and G; pMCTM1 Lanes D and H; pMCTbliM1 FIGS. 22A and 22B; bacterial DNA Of the lipase-encoding sequence in the cell. Five nanograms of plasmid DNA and five micrograms of chromosomal DNA were digested with the indicated restriction enzymes, fractionated on a 0.8% agarose gel, blotted on nitrocellulose filters, and hybridized with nick-translated inserts of pET3 and pTMPv18A, respectively.

FIG. 22A; FIG. 22A is an autoradiograph after hybridization with the pET3EcoR I insert.

FIG. 22B; FIG. 22B shows autoradiographs after hybridization with the pTMPv18A XhoI-EcoRV insert.

Lane A; HindIII / SStI digest of plasmid pTMPv18A Lane B; EcoRI digest of plasmid pET3 BRL DNA gel marker MW; 0.5, 1.0, 1.6, 2.0, 3.0,
4.0, 5.0, 6.0, 7, 8, 9, 10, 11, 12 kb.

Lane C; P. pseudoalcalige
nes) Sal-1 digest of M-1 (CBS473.85) Lane D; P. pseudoalcalige
nes) Sal II digest of IN II-5 (CBS468.85) Lane E; P. alcaligenes DSM50342
Lane F; P. aeruginosa PAC 1R (CBS13
6.89) Sal I digest lane G; P. aeruginosa PAO2302 (6-
1) Sal I digest of lane H; P. stutzeri Thai IV17-1 (CBS
461.85) Sal I digest lane I; P. stutzeri PG-I-3 (CBS13
7.89) Sal I digest lane J; P. stutzeri PG-I-4 (CBS13
8.89) Sal I digest lane K; P. stutzeri PG-II-11.1 (CBS1
39.89) Sal I digest lane L; P. stutzeri PG-II-11.2 (CBS1
40.89) Sal I digest lane M; P. fragi Serm. DB1051 (= farm
BP1051) Sal I digested product Lane N; P. gladioli (CBS176.86) S
al I digested lane O; A. calcoaceticus Gr-
Sal I digest of V-39 (CBS460.85) Lane P; Sal of S. aureus (ATCC27661)
I Digests Description of specific embodiments In accordance with the present invention, there are provided new compositions comprising a new DNA construct and a microorganism that produces a lipolytic enzyme. The lipases of interest in the present invention have an optimum pH value of about 8 to 10.5 and a temperature below 60 ° C, preferably 30-40 ° C.
And exhibits an effective lipase activity in an aqueous solution containing the washing composition at a concentration of up to about 10 g / under washing conditions at a pH value of about 7-11, preferably a pH value of about 9-10.5. Plasmid constructs containing DNA sequences encoding lipase with the desired properties are used to transform host cells, either eukaryotic or prokaryotic. The transformed host cells are then propagated and the gene is expressed.

The techniques used to isolate the lipase gene are well known in the art and include synthesis, isolation from genomic DNA, preparation from cDNA or recombination thereof. Various techniques for working with this gene are well known, including restriction cleavage, digestion, cleavage, ligation, in vitro mutagenesis, primer repair, the use of linkers and adapters, and the like. Maniacs (Ma
See, Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.

Generally, the method involves the construction of a genomic library from an organism that expresses a lipase having the desired properties. Examples of these lipases include Pseudomonas (Ps
and Pseudomonas pseudoalcaligene, especially Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligene obtained from Acinetobacter.
s), Pseudomonas aerugin
osa), Pseudomonas stutz
eri) and Acinetobacter alcoaceticus (Acine
Some are obtained from strains belonging to the species tobacter calcoaceticus). These lipases and strains are fully described in EP-A-0218272, which is incorporated herein by reference. The genome of the donor microorganism is isolated and then cut with an appropriate restriction enzyme such as Sau3A. The obtained fragment is ligated to a vector molecule which has been cut with a suitable restriction enzyme in advance. Examples of suitable vectors include the restriction endonuclease Bc
There is a plasmid pUN121 that can be cut with lI. Further, the amino acid sequence may be a cDNA prepared from mRNA for the lipase gene or a genomic library or DN derived from a donor cell.
It can be used to design probes used to screen A.

Furthermore, by using the lipase DNA or a fragment thereof as a hybridization probe, cloning of a structurally related gene present in another microorganism can be easily performed. The invention is particularly applicable to EP-A-0218272.
The use of an oligonucleotide probe based on the nucleotide sequence of a lipase gene derived from an organism described in (1) above, the isolation of a gene from an organism having lipolytic activity. Alternatively, these oligonucleotides can be derived from the amino acid sequence of the lipase of interest. These probes can be significantly shorter than their entire sequence but should be at least 10, preferably at least 14, nucleotides in length. Also useful are longer oligonucleotides, preferably at most 500, more preferably at most 300 nucleotides in length up to the full length of the gene. Both RNA and DNA probes can be used.

In use, the probes are typically labeled according to the mode of detection (eg, with ³² P, ³⁵ S, ³ H, biotin or avidin), and are then labeled with single-stranded DNA and RNA from the organism for which the gene is being sought. Incubate. Single-stranded and double-stranded (hybridized) DNA (DNA / RNA)
After separation, hybridization is detected by a label (typically using nitrocellulose paper). Hybridization techniques suitable for the use of oligonucleotides are well known in the art.

Usually, probes are used with easily identifiable detectable labels, but unlabeled oligonucleotides can also be used as precursors to labeled probes, as well as double-stranded DNA (or D
Useful in methods that provide direct detection of NA / RNA). Thus, the phrase "oligonucleotide probe" refers to both labeled and unlabeled forms.

In one preferred embodiment of the invention, Pseudomonas
Pseudomonas pseudoalcalige
nes) is cloned from the CBS473.85 (M-1) strain. Surprisingly, once sequenced Pseudomonas aerugino
sa) PAO (ATCC15692) cloned lipase was M-
It showed a high sequence homology with one lipase gene sequence. Even more surprisingly, this high sequence homology
-1 lipase gene sequence and a number of Pseudomonas stutzeri isolates [PG-I-3
(CBS137.89), PG-I-4 (CBS138.89), PG-II-11.
1 (CBS139.89), PG-II-11.2 (CBS140.89)] and Pseudomonas alcaligene
s) It was also found between chromosomal DNAs of DSM50342. As used herein, "high hybridization" is defined as at least 300 bp of continuous DNA having at least 67% homology.

P. aruginosa and P. stutze
ri) The lipase enzyme was produced and the washing performance was tested by the SLM test. Surprisingly, all enzymes that show a high degree of homology with the M-1 lipase gene have been found to exhibit excellent stability, efficacy and performance under conditions that mimic modern washing processes. According to the method of Ausubel et al. (Current Protocols in Molecular B
iology), 1987-1988), Southern hybridization techniques can detect this level of homology. This discovered homology is described in EP-A-0205208 and EP-A-0
P. gladioli as described in 206390 or
It was not observed in the Humicola languinosa lipase described in EP-A-0305216.

The clone containing this DNA insert is E. coli (Kuh
n) et al., Gene 44 (1986) 253-263) and B. subtilis (Gryczan and Dubnau), Gene 20 (1982) 459-46.
It can be identified using the direct or positive selection procedure developed for 9). Examples of positive selection vectors for E. coli include pUN121 (Nucleic Acids Res.) Such as Nilsson 11 (19
83) 8019-8030).

In addition, clones expressing lipolytic enzymes include, for example, agar media containing tributyrin or olive oil along with rhodamine B (Kouker and Jaegar, Applied Environmental Microbiology (Appl. Env. Microbiol.) 53 (1987) 211) can be identified using a suitable indicator plate assay.
In addition, replicating colonies were developed using a soft agar technique based on the procedures described for detecting esterase activity (Hilgerd and Spizizen, Journal of Bacteriology (J. Bacteriol.) 114 (1978) 1184). The method can be used to screen. Separately, clones expressing lipolytic enzymes were
farth) and Winkler (Journal of General Microbiology (J.Gen. Microbio)
l.) 134 (1988) 433-440) can be identified by genetic complementation in a suitable lipase deficient recipient strain.

Once a complete gene has been identified, whether it is cDNA or chromosomal DNA, it can be handled in a variety of ways to express it. Microbial hosts include, for example, E. coli, Kluyveromyces, Aspergilas
llus), Bacillus and Pseudomonas (Ps
Bacteria, such as eudomonas) species, yeasts and fungi can be used. Therefore, since the gene is expressed in a host that recognizes the wild-type transcriptional and translational regulatory regions of the lipase, all genes having wild-type 5 'and 3' regulatory regions are introduced into an appropriate expression vector. There must be. Various expression vectors exist that have a prokaryotic cell-derived replication system. For example, Pouwel
s) et al., "Cloning Vectors, Laboratory Manual", Elsvia Edition 1985. These replication systems have been developed to provide markers that allow for the selection of transformants, and to provide convenient restriction sites into which genes can be inserted.

If the gene is to be expressed in a host that does not recognize the natural wild-type transcription and translation regulatory regions, additional manipulations will be required. Conveniently, various 3 'transcriptional regulatory regions are known and can be inserted downstream of the stop codon. The 5 'non-coding region upstream of the structural gene can be removed by endonuclease restriction treatment, Bal31 excision, or the like. Alternatively, if there is a convenient restriction site near the 5 'end of the structural gene, an adapter that restricts the structural gene and complements the deleted nucleotide of the structural gene may bind the structural gene to the promoter region. Used.

A lipolytic enzyme comprising a transcription regulatory region and a translation initiation region, which also include regulatory sequences that allow for induction of regulation in the 5'-3 'direction of transcription, preferably a secretory leader sequence recognized by a designated host cell. Various strategies are used to provide an expression cassette having an encoding reading frame and translation and transcription termination regions. Furthermore, the expression cassette contains at least one marker gene. The start and stop regions are functional in the host cell, whether they are homologous (derived from the host) or heterologous from the host, or heterologous and derived from another source or synthetic DNA sequence. Either. Thus, the expression cassette may be wholly or partially derived from a natural source, may be wholly or partially derived from a source homologous to the host cell, or may be derived from the host cell. They can also come from heterogeneous sources. Various DNA constructs (DNA
(Sequences, vectors, plasmids, expression cassettes) are isolated and / or purified or synthesized and therefore not "naturally occurring".

The following factors affecting expression are taken into consideration in selecting an appropriate regulatory sequence. For transcriptional regulation, the amount and stability of messenger RNA are important factors that influence the expression of gene products. The amount of mRNA is determined by the copy number of the particular gene, the relative efficiency of the promoter, and factors that control the promoter, such as enhancers or repressors. mRNA stability is governed by the sensitivity of the mRNA to ribonuclease. In general, exonuclease cleavage is inhibited by the structural palindromic structure at the end of the mRNA, the presence of modified nucleotides or specific nucleotide sequences. Endonuclease cleavage is thought to occur at specific recognition sites within the mRNA, and stable mRNAs may lack these sites. MRNA that translates at a high level
There is some evidence that the presence of ribosomes on the mRNA also protects it from degradation.

For translation, if mRNA is present, its expression is determined by the rate of initiation (ribosome binding to mRNA), the rate of elongation (mRNA
Ribosome migration), post-translational correction rate and the stability of the gene product. The extension rate is probably influenced by the codons used. That is, the use of codons for less tRNA reduces the translation rate. The start is thought to occur in the region immediately before the beginning of the coding sequence.
In most prokaryotes, this region contains the consensus nucleotide sequence of AGGA, which is called the Shine-Dalgarno sequence. While this sequence has the characteristics of being a ribosome binding site, it is clear that sequences upstream and downstream of this sequence can affect translation initiation.

Other evidence also points to the presence of nucleotide sequences within the coding region that may affect ribosome binding, possibly by forming a structural pattern in which the ribosome recognizes the start site. The location of the AGGA sequence relative to the start codon ATG can affect expression. Thus, the determination of a particular expression rate depends on the interaction of all these factors. However, the expressed gene has developed a combination of all these factors to produce a particular expression rate. The design of an expression system that produces high levels of gene product must take into account not only the specific regions that are determined to affect expression, but also how those regions (sequences) interact with each other.

Representative transcription regulatory regions or promoters include, for example, sequences from genes that are overexpressed in strains used for industrial production. Furthermore, the transcription regulatory region also includes a regulatory sequence that enables the expression of a structural gene that is regulated, for example, by the presence or absence of nutrients or expression products in the growth medium or by temperature. For example the expression of a structural gene in prokaryotes can be adjusted by the temperature when using control sequences which include bacteriophage lambda P _L promoter with the bacteriophage lambda O _L operator and the temperature-sensitive repressor. Regulation of this promoter is achieved through interaction between the repressor and the operator. Bacillus (Ba
Expression cassettes capable of expressing lipolytic enzymes using the regulatory sequences of the amylase and protease genes of Cillus) are of particular interest. The structural gene of interest is linked downstream of the ribosome binding site, thereby placing it under the regulatory control of the transcription regulatory region and the translation initiation region.

A fusion gene is prepared by adding a sequence encoding a secretory leader signal and a processing signal to the 5 'side of the structural gene. The signal sequence of the lipase gene itself is also used if it is functional in the host cell of choice. Representative heterologous secretory leader sequences include those of the alpha factor of penicillinase, amylase, protease and yeast. The mature lipolytic enzyme is secreted into the culture medium by fusing the structural gene of interest and the secretory leader sequence in the appropriate reading frame.

The expression cassette may be contained in a replication system that allows for episomal maintenance in a suitable host microorganism,
It may also be provided without a replication system and integrated into the host genome. The method of transformation of the various DNA constructs into the host microorganism is not critical to the invention. DNA is introduced into host cells according to conventional techniques such as calcium phosphate precipitated DNA, conjugation, electroporation, transfection by contacting cells with virus, and transformation using microinjection of DNA into cells. Is done. This host cell may be the cell itself or a protoplast.

The host organism may be any microorganism that is suitable for producing and extracting lipolytic enzymes. It is also desirable that the host organism be capable of secreting the produced enzyme, thereby recovering the enzyme from the cell-free fermentation broth. It is also desirable that the host microorganism be a non-pathogenic organism. Examples of host organisms satisfying the above conditions include Escherichia coli, Pseudomonas putida and Bacillus.
Strains, especially B. subtilis and B. licheniformis, Streptomyce
s) strains and fungi and yeast strains such as Aspergillus and Kluyveromyces, respectively.

The host strain may be a research strain or may include an industrial microbial strain. Industrial strains are characterized by resistance to genetic changes such as phage infection or transformation. This strain is stable, and some are capable of forming spores and others are not. They are autotrophic and
Also, endogenous protein products such as α-amylase and various proteases can be altered to provide high yields. The yield of endogenous protein product obtained in an industrial production process can be increased to at least 5 g / (0.5% w / v). Industrial strains also secrete DNase, which confers resistance to genetic changes by degrading DNA in the medium.

Once the structural gene has been introduced into a suitable host, the host cell can be grown and the structural gene expressed. The production level of lipolytic activity is similar to or higher than the original strain from which the gene is derived. The host cells are grown to high density in a suitable medium to produce a rich medium. If the promoter is inducible, inducing conditions such as temperature changes, oligotrophic or excess metabolites or nutrients are used.

If secretion is performed, the expression product is isolated from the growth medium by conventional methods. Release of the produced lipolytic enzyme can be enhanced with a dilute detergent solution. If no secretion occurs, the host cells are harvested and degraded according to conventional conditions. The desired product is then isolated and purified according to known techniques such as chromatography, electrophoresis, solvent extraction, phase separation and the like.

The composition may be used in a wide variety of ways. Transformed host microorganisms can be used to increase lipase production with properties that make them useful in detergent compositions. Also, the cloned lipase gene can be used for lipase gene screening, including identification of lipolytic genes as lipases rather than esterases.

Also, they are used in enzyme engineering using conventional techniques for random or site-directed mutagenesis to yield lipases with the required modified properties.

The lipolytic enzyme composition may be used in laundry compositions that generally include the detergents used in cleaning compositions and the separately added components. These components include at least one surfactant, complex phosphoric acid, softeners such as alkali metal silicates and bicarbonates, filers such as alkali metal sulfates, enzymes such as proteases and amylases, bleaching agents, And other compounds such as fragrances, fluorescent bleaches and the like.

In the enzymatic cleaning composition according to the present invention, the lipase activity is preferably in the range of 1 to 20,000 TLU / g (composition), while the proteolytic enzyme activity is 50 to 10,000.
00 Delft Units / g (cleaning composition)
Is preferably within the range. 1 TLU (true lipase unit)
Is defined as a fatty acid released from olive oil / gum arabic emulsion at pH 8.0 and 25 ° C. with a titration equivalent to 1 μmole NaOH / min. Telft units are defined in the Journal of American Oil Chemical Society (J. Amer. Oil Chem. Soc.) 60 (1983) 1672.

The detergents according to the invention are prepared in the customary manner, for example by mixing the components together or by preparing a premix and then mixing with the other components. In one formulation route, one or more lipase preparations are mixed with one or more other compounds to create a concentrate of the desired enzymatic activity, and then the concentrate is mixed with other desired ingredients.

The lipolytic enzyme of the present invention is preferably used in the form of an enzymatic detergent additive. The additives also include one or more other enzymes, such as proteases and / or amylases that may be used in current detergents, and are generally used in the art, for example, non-ionic, salts, stabilizers and / or coatings. One or more other components. Enzymatic detergent additives may include proteases and optionally α-amylases in addition to lipase. The proteolytic enzyme is well compatible with the lipolytic enzyme in the formulation. Generally, the enzymatic detergent additive is mixed with one or more detergents and other ingredients known in the art to formulate the detergent. Generally, enzymatic detergent additives are used in the range of 10 ² to 10 ⁷ TLU / g (additive), while the optional proteolytic activity is in the range of 5 × 10 ⁴ to 10 ⁶ delft units / g.

The enzymatic detergent additives of the present invention can be, for example, granules or granules prepared by methods generally known in the art.
l) has the shape. For example, British Patent Nos. 1,324,116 and 1,362,365 and U.S. Patents 3,519,570, 4,106,99
See No. 1 and 4,242,219.

The enzymatic detergent additive can also be in liquid form with an enzyme stabilizer such as, for example, propylene glycol. They can also be fixed on a soluble or insoluble support, or in the form of an organic or inorganic slurry, emulsion or capsule in aqueous or non-aqueous solution in the presence of one or more stabilizers. Such additives are desirably used in liquid detergents.

The examples described below are for illustration and do not limit the invention.

Experiment A common cloning technique is Maniatis.
(“Molecular Cloning, Laboratory Manual” Cold Spring Harbor Laboratory, 198
2, CSH, New York). All DNA modifying enzymes used were commercially available. They were used according to the manufacturer's instructions. Materials and equipment for DNA purification and separation were used according to the manufacturer's instructions.

Example 1 Molecular Cloning of Triacyl Chryseroyl Acyl Hydrolase A. Origin of DNA and Selection Vector EP-A-0218272 discloses several bacterial strains producing lipases suitable for use in detergents. Acinetobacter calcoaceticus (Acinetobacter ca.)
lcoaceticus) GrV-39 (CBS460.85), Pseudomonas stutzeri Thai IV 17-1 (CB
S461.85), Pseudomonas Psudoalcaligenes M-1 (CBS473.8)
5) was selected as the source of the lipolytic gene.

Plasmid vector pUN121 (Nilsso (Nilsso) having an ampicillin resistance gene, a tetracycline resistance gene and a bacteriophage lambda cI receptor gene.
n), Nukelic A. Research (Nacleic A)
cids Res.) 11 (1983) 8019) was obtained from Dr. M. Wooln (Royal Institute of Technology, Department of Biochemistry, Stockholm S-10044, Sweden, Teknikringen 10). Transcription of the tetracycline gene is blocked by the cI repressor. A single restriction site (Bcl
Insertion into I, Sma I, Hind III and EcoR I) activates the tetracycline gene. This allows direct (positive) selection of recombinant transformants on Lurla medium agar plates containing 8 μg / ml tetracycline and 50 μg / ml ampicillin.

B. Preparation of gene library (see Fig. 1) Plasmid and chromosomal DNA were prepared by Andreol
i) (Molecular and General Genetics (Mo
l. Gen. Genet.) 199 (1985) 372-380). Each chromosomal DNA isolated from Thai IV 17-1 and M-1 was partially digested with San3A. The DNA was then converted to Maniatis using T4 DNA lipase.
Ligated to pUN121 DNA digested with Bcl I according to the method reported by E. coli JM101hsd Sre
c A strains (Dagert and Ehrlic
h), transformed into competent cells of Gene 6 (1979) 23-28). E. coli JM101hsd S rec A
Was obtained from Utrecht, Holland, U.S.A. The transformants resistant to 8 μg / ml tetracycline in Luria medium agar plates were selected.

C. Screening of Transformants A replica plate of the gene library obtained as described above was prepared and screened for lipolytic activity using the following two procedures. In the first operation, replicated collories were screened for lipolytic activity on tributyrin-containing peptone agar. The lipolytic activity was detected as a clear area (halo) around the colony due to the decomposition of the cloudy lipid emulsion. In a second operation, replicating colonies were screened for esterase activity using the soft agar overlay technique. This method is based on the method of Hilgerd and Spizizen (Journal of the
Bacteriology (J. Bactoriol.) 114 (1987) 1184)
Based on Basically 0.4% low melting point agarose,
0.5M potassium phosphate (pH 7.5), 0.5 dissolved in acetone
When a mixture of mg / β-naphthyl acetate and 0.5 mg / Fast Blue BB (see Example 2) is poured into a transformant, colonies with esterase or lipase activity turn purple within minutes.

Three of the 1200 tetracycline resistant transformants obtained from the Thai IV 17-1 / pUN121 gene bank produced halos on tributyrin agar plates. M−1 /
Only one of the 12,000 recombinant transformants tested from the pUN121 gene bank showed weak lipolytic activity.

16 g of this tributyrin positive clone in 2TY medium)
/ Bactotryptone, 10 g / Bacto yeast extract, 5 g / NaCl, pH 7.0), and then grow the plasmid content (see 1D below) and various β-naphthyl substrates as indicators of lipolytic activity. Assays were performed for both conversion potential (see Example 2A).

D. Isolation of plasmid D.1 Inclusion of Thai IV 17-1 lipase gene Plasmids isolated from Thai IV 17-1 / pUN121 transformants were named pAT1 and pAT3. The structures are shown in FIGS. 2 and 3, respectively. A third clone, designated pAT2, harbored the same plasmid as the pAT1 construct.
The gene encoding lipolytic activity is the 2.7 kb EcoR I of pAT1.
Located within the fragment (FIG. 2, dotted line) and within the 3.2 kb EcoRI fragment of pAT3 (FIG. 3, dotted line). These two EcoRI fragments were subcloned into appropriate vectors for DNA sequencing and for obtaining high yield lipolytic activity.

Recombinant plasmid pE1 was created by cloning the 2.7 kb EcoRI fragment from pAT1 into the pUN121 vector (4th).
Figure). Escherichia coli JM101hsd S rec A harboring plasmid pET1 was registered with CBS on February 5, 1987 as CBS157.87.

Recombinant plasmid pET3 was created by cloning the 3.2 kb EcoRI fragment from pAT3 into the pUN121 vector (5th).
Figure). Escherichia coli JM101hsd S rec A harboring plasmid pET3 was registered in CBS on February 5, 1987 as CBS155.87.

D.2 Inclusion of the M-1 lipase gene The recombinant plasmid isolated from the M-1 / pUN121 transformant, designated pAM1, was isolated and characterized (FIG. 6). However, the biochemical properties of the lipolytic activity of pAM1 indicated that this plasmid did not encode the lipase of interest (see Examples 2 and 3). Therefore, other strategies, described below, had to be developed.

Plasmid pAM1 in E. coli JM101hsd S rec A is 154.
No. 87 was registered with CBS on February 5, 1987.

D.3 Incorporation of the IN II-5 lipase gene Pseudomonas pseudoalkagens (Pseudo
monas pseudoalcaligenes) E. coli K1
It was cloned into 2DH1 strain (ATCC33849). Ligation and Hanahan (Journal of Molecular Biology (J. Mol. Biol.) 166 (1983) 55
7-580), after transformation into DH1 competent cells prepared by the method reported by
Resistant to ampicillin and 8 μg / ml tetracycline
1500 transformants were obtained. Transformants capable of hydrolyzing tributyrin and β-naphthyl acetate were selected by the method described in 1C. Plasmid DNA was isolated from one positive colony and characterized by determination of several restriction enzyme recognition sites. The physical map of this plasmid, designated pM6-5, is shown in FIG.

Escherichia coli DH1 (pM6-5) against β-naphthyl ester
Was measured (Table 1). Escherichia coli DH1 harboring plasmid pM6-5 was designated as No. 153.87 by CBS on February 5, 1987.
Registered.

D.4 Gr V-39 lipase gene inclusion Partially digested with EcoR I ^* (Gardenr)
DNA1 (1982) 109-114) Acinetobacter calcoaceticus (Acinetobacter calcoaceticus).
s) Gr V-39 DNA was mixed with pUN121 DNA linearized with EcoRI, recircularized using T4 polynucleotide ligase, and the DNA mixture was transformed into E. coli using the transformation procedure described earlier in this example. DH1 (ATCC33849). A total of 1800 tetracycline resistant transformants obtained were screened for the lipolytic activity described in 1C.

Three colonies of this Gr V-39 ゜ / pUN121 gene library produced lipolytic enzymes. pP5-4
Plasmid DNA from one of the three clones, designated as, was isolated and characterized with restriction endonucleases. The physical map of this plasmid pP5-4 is shown in FIG. Then, the hydrolysis of β-naphthyl ester by the crude enzyme preparation derived from Escherichia coli DH1 (pP5-4) was measured (Table 1). Plasmid pP5-4 in E. coli DH1 is 151.
No. 87 was registered on February 5, 1987 with CBS.

Example 2 Properties of Cloned Lipolytic Enzyme Preparation A. Measurement of Lipolytic Activity Tributyrin-positive E. coli colonies were inoculated into 100 ml of 2TY medium containing ampicillin and tetracycline in a 500 ml conical flask. This E. coli culture is
Shake at 30 ° C. for 40 hours in a shaker at 0 rpm. 40 hours later,
The optical density at 575 nm was measured and the medium was then centrifuged at 6000 rpm with a Sorvall RC5B centrifuge using a GSA rotor.
Centrifugation was performed at pm for 10 minutes. Keep the supernatant at 4 ° C until enzyme assay
Saved in.

The cells were suspended in 4 ml of lysis buffer (25% sucrose, 50 mM Tris-HCl pH 7.5). After adding lysozyme and incubating at 21 ° C for 30 minutes, DNase (20 μg / m
l) was added and incubation was continued at 37 ° C. for 30 minutes. Triton X-100 (0.1% v / v) was added and the cell suspension was sonicated on ice using a Labsonic 1510 ultrasonic device (1 watt 30 sec treatment 5 minutes apart). Times). Cell debris was then removed by centrifugation at 12,000 rpm for 15 minutes using a Hettich Mikro Rapid / K centrifuge. The resulting supernatant lipolytic activity was assayed. This assay is based on the hydrolysis of β-naphthyl esters by lipolytic enzymes. The released β-naphthyl reacts with the diazonium salt Fast Blue BB to produce an azo dye having an absorption at 540 nm. This method is basically based on McKollar's method (J. Dairs Re
s.) 53 (1986) 117-127) and performed as follows.

Final volume in the reaction tube was 2.0 ml: 1.8 ml 55 mM TES
(N-tris (hydroxy-methyl) methyl-2-aminoethanesulfonic acid, Sigma); 0.02 ml 100 mM β-naphthyl ethyl dissolved in dimethyl sulfoxide (DMSO, Merck) or methyl cellosolve acetate (Merck), 0.1 ml 120 mM NaTC (Na-Torochlorate, Sigma) and 0.1 ml of enzyme preparation.

Control lacking enzymes and β-
Naphthyl (Sigma) standards were also used.

Place the Corning centrifuge tube (15 ml) containing the reaction mixture in 37
Incubated for 30 minutes at ° C or the specified temperature. 0.
Add 02 ml 100 mM MFB solution (Fast Blue BB salt (Sigma) dissolved in DMSO) and incubate for another 10 minutes. The reaction was stopped by adding 0.2 ml of 0.72 NTCA (trichloroacetic acid, Riedel De Haen), and the chromogenic complex was extracted by vigorous mixing with 2.5 ml of 1-butanol (Merck). The layer is 5000rpm using Herius Christo Minfuge (Heraeus Christ Minifuge) RF,
Separated by centrifugation for 5 minutes. The absorbance of the upper layer was measured at 540 nm using an LKB UltraSpec II spectrophotometer. After subtracting the control, the measured value was converted to a TLU value (true lipase unit) using Candida cylindracea lipase (L1754, Sigma) as a standard substance. 1T2U is defined as a fatty acid titration equivalent to 1 μmole NaOH / min (see EP-A-0218272). The results are shown in Table 1 below.

Comparison of the hydrolysis data of β-naphthyl esters with various acyl chain lengths varying from C _{4 to} C ₁₈ shows the following. Clones harboring the 1 ゜, pAT3 and pET3 plasmids produce true lipase. And 2 ゜, pAT1, pET
1, Clones harboring the pAM1, pP5-4 and pM6-5 plasmids produce enzymes having substantially esterase activity. Most lipolytic enzymes synthesized by E. coli with the recombinant plasmid were present in the cell lysate.

B. More detailed properties of the cloned lipolytic preparation The cloned lipolytic enzyme preparation was prepared by SDS gel on a fast gel system (Pharmacia) using a fast gel gradient 10-15% according to the manufacturer's instructions. Electrophoresis was performed and its properties were examined.

DH1 containing E. coli clone pET3 and pUN121 vectors
The cell-free extract derived from the strain was partially purified from a donor strain Thai IV 17-1 (Stuor et al., Journal of Bacteriology (J. Bacteriol.) 168 (1986) 1070).
-1079).

(Sample preparation) 10 parts in 4 parts of appropriate dilution of enzyme preparation
% SDS, 10% β-mercaptoethanol 0.5M Tris · H
Mix 1 part Cl (pH 6.8) solution. This solution was divided into three equal parts. One was stored at room temperature without any treatment until gel electrophoresis. The second and third were heated to 95 ° C. for 5 and 10 minutes, respectively, cooled in ice water and stored at room temperature until gel electrophoresis.

(Electrophoresis) The treated sample was electrophoresed twice using a fast gel system at 65 volts / hour. One gel was subjected to protein staining with Comage Brilliant Blue according to Pharmacia Development Technology File No. 200. The second gel was 50 mM Tris-HCl (pH 7.5), 0.1
% Triton X-100 was washed to remove SDS, and the enzyme activity was reactivated. The presence of lipolytic activity in the washed gel was visualized with the soft agar overlay technique based on the β-naphthyl acetate / fast blue BB salt method described in Example 1C. After incubation at 30 ° C. for 30 minutes, a purple band appeared on a transparent background. As shown in FIG. 9B, the lipase from the E. coli pET3 clone and the native P. Stutzeri Thai
IV 17-1 lipase (MW 40 kDa) showed the same mobility on SDS gel electrophoresis. Similar thermovariability has been reported for another outer membrane protein (Omp A gene product) of Escherichia coli K12 (Freudl et al., Journal.
Of Biological Chemistry (J. Biol. Chem.)
261 (1986) 11355-11361).

Example 3 Construction of Pseudomonas pseudoalcaligenes Gene Library in Broad Host Vector P. pseudoalcaligenes M
As an alternative strategy for cloning the lipase gene of the -1 strain, a two-element wide-area host cloning system was used. This allows direct screening of gene banks by complementation to various Pseudomonas mutants. Two broad host vectors pLAFR1
(Friedman et al., Gene 18 (19
82) 289-296) and pKT248 (Bagdasarian).
Gene 16 (1981) 237-247). Plasmid pLAFR1 is a broad host cosmid derived from RK2 that confers tetracycline resistance, which is capable of transfer but not capable of self-sustained transfer. Plasmid pKT2
48 is a transferable R300B-derived broad host plasmid that confers streptomycin resistance and chloramphenicol resistance. Pseudomonas from Escherichia coli
The transfer of these vectors into s) has the RK2 transfer function (Proc. Natl. Ac., Proc. in National Academy of Sciences, Jitter et al.).
ad.Sci.) USA 77 (1980) 7347-7351) With the help of pRK2013, Friedman et al. (Gene 18 )
(1982) 289-296). P.
A lipaseminum mutant of the aeruginosa PAO2302 strain (obtained from Wohlfarth and UK Winkler, (Lua University, Bocam, FRG)) was used as the recipient Pseudomonas (Journal. J. Gen. Microbiol. 134 (1988) 433-44
0).

 Lambda phage packing extract from in vitro
Preparation and packing of pLAFR1 DNA are basically
Vitz (Ish-Horowicz) and Burke (Nuk
Lake Acids Research (Nucleic Acids Res.)9
(1981) 2989-2998). Put simply,
All P. pseudoalcaligenes M
-1 DNA is partially digested with EcoRI or SalI, and EcoRI restricted.
Ligation to cut pLAFR1 DNA or SalI restriction cut pKT248 DNA
I did it. The ratio of insert to vector is 1: 5.
Reduces the possibility of vector and vector ligation
Was. M-1 / pLAFR1 DNA ligated in vitro
Into the head of lambda phage and transfer to E. coli DH1
Injection (Maniatis et al., 1982, supra).

P. pseudoalcaligenes M
About 2,500 tetracycline-resistant transductants were obtained per 11 μg. Assuming that P. pseudoalcaligenes has a genome size of 5000 kb and at least 50% of tetracycline resistant transductants have a 20 kb insert, a specific DNA sequence is found with a 99% probability 2 to guarantee that
300 different clones are required (Clarke
k) and carbon (Cell) 9 (1976) 9
1). Since this gene library contains more than 8,500 different recombinant colonies, it will probably contain the entire P. pseudoalcaligenes genome.

The ligated M-1 / pKT248 DNA was transformed into competent E. coli as described in Example 1. E. coli JM101hsd S rec A transformants were selected for streptomycin resistance (Sm ^R ) and counter-selected for chloramphenicol sensitivity (Cm ^S ). 5000 Sm ^R Cm ^S clones were obtained. Two M-1s obtained in E. coli host
Example 1 A replica of a gene library is placed on a plate.
Screened for lipolytic activity as described in. None of the 13,000 recombinant transformants showed lipase activity.

Therefore E. coli from P. aeruginosa PAO
Transfer of the clone to 2302 (6-1) was performed as follows. As for the recombinant plasmid, the donor strain was used as the recipient strain (PAO023
02 / 6-1) and a helper strain (Escherichia coli MC1061 or DH1 harboring the pRK2013 plasmid) were transferred to Pseudomonas receptors by taking replicas. The donor, recipient and helper strains are grown overnight on a heart infusion agar plate and then replicated on a minimal agar medium containing 0.2% citrate, methionine (10 μg / ml) and streptomycin or tetracycline. Exconjugant was selected.

Citrate is not metabolized by E. coli. The preservation of the lip phenotype of the lipase-deficient mutant having the recombinant plasmid was reported by Kouker and Jaeger (Applied and Environmental Microbiol (Appl. Env. Microbiol). .) 5
3 (1987) 211-213) The P. aruginosa
Was tested by taking a nutrient agar medium containing trioleoylglycerol and the fluorescent dye rhodamine B.

Four of the 13 000 screened conjugants were confirmed by expressing an orange fluorescent halo that emits visible light at 360 nm around bacterial colonies after incubation at 37 ° C for 40 hours. Lipase activity. One of these positive clones, pALM5, was selected for further characterization.

Finally, in order to confirm that the lipase produced by the PAO2302 / 6-1 exconjugant harboring pALM5 has the desired properties shown by the lipase from the P. pseudoalcaligenes M-1 strain, an enzyme sample was prepared. Prepared and performed biochemical analysis (see Example 2) and SLM test (described in Example 10 below). The results obtained in these tests showed that the lipolytic activity of the enzyme produced by the pALM5 clone had the same properties as the enzyme derived from the parent strain M-1.

Example 4 Pseudomonas Pseudomonas
Molecular cloning of the pseudoalcaligenes M-1 lipase gene A. Purification and sequencing of the protein Pseudomona
Fermentation and preparation of the lyophilized supernatant of S. pseudoalcaligenes) strain M-1 is described in EP-A-0218272. Lipid-degrading enzymes are basically described by Wingerder et al. (Applied Microbiol. Biotechnol.) 27 (1987) 139
According to the method of -145), the supernatant was purified. After purification, the protein preparation had a purity of greater than 80% as determined by SDS polyacrylamide gel electrophoresis followed by Commercial Brilliant Blue staining (see FIG. 10).

N-terminal sequence analysis was performed as described in Matudaira (Journal of Biological Chemistry (J. Bio).
l.Chem.) SD according to the method of 262 (1987) 10035-10038)
This was performed after S-gel electrophoresis and electroblotting on Immobilon transfer membrane (Millipore). This analysis showed the following sequence (using the simple one letter amino acid code):

GLFGSTGYTKTKYPIVLTHGMLGF 1 10 20 Cloning of BM-1 lipase gene Two 32-mer synthetic oligonucleotides 5'ACC GGC TAC ACC AAG ACC AAG TAC CCC ATC GT-
3 'and 5' ACC GGC TAC ACC AAG ACC AAG TAC CCG ATC GT−
3 ′ is amino acids 6-15 (TG) of the N-terminal sequence of the mature lipase protein described above in consideration of the codon preference of P. pseudomonas and the degeneracy of the genetic code.
YTKTKYPI). For use as a hybridization probe, this oligonucleotide is T4
End-labeled using polynucleotide kinase.

Chromosomal DNA is obtained by the Andreoli method (Molecular and General Genetics (Mol.Ge)
n.Genet.) 199 (1985) 372-380) and Pseudomonas pseudoalca.
ligenes) strain M-1 (CBS473.85), digested with several restriction endonucleases, and separated on a 0.8% agarose gel. Radioactively labeled as probe
Southern blot analysis of these gels with 32-mer oligonucleotides showed the following unique hybridizing DNA bands: 1.8 kb Bcl I, 2.0 kb Pvu II and 1.7 kb Sal I. Therefore, after separately digesting the M-1 chromosomal DNA with these three restriction endonucleases and fractionating by 0.8% agarose gel electrophoresis, the above hybridized fractions were combined with Schleicher and Schull. Biotrap BT10 commercially available from Sharp
Collected by electroelution on a 00 instrument.

The 1.8 kb Bcl I digested fraction was ligated to the vector pTZ18R / 19R (commercially available from Pharmacia, Warden, The Netherlands) which had been digested with BamHI and dephosphorylated. 2.
The 0 kb Pvu II digestion fraction was ligated to the vector pTZ18R / 19R, which had been digested with SmaI and dephosphorylated. The 1.7 kb Sal I digested fraction was digested with Sal I and then dephosphorylated vector pZT18R /
Ligation to 19R. These three ligation products were transferred to competent cells of E. coli JM101hsd S rec A (reported by Andreoli, supra), transformed, ampicillin, X-gel (5.
-Bromo-4-chloro-indolyl-β-D-galactopyranoside) and IPTG (isopropyl-β-D-thiogalactopyranoside) were plated on Luria medium agar plates.

Approximately 4000 white colonies were picked up on fresh agar plates and screened by colony hybridization to radiolabeled 32-mer oligonucleotides. By this method, 12 positive colonies were obtained. Plasmid minipreps were prepared from each of these positive colonies by the alkaline digestion method and digested with an appropriate restriction endonuclease according to the manufacturer's instructions. Six plasmids contained the expected hybridizing insert. Named pTMPv18A
One of the plasmids containing only the 2.0 kb Pvu II fragment was chosen for further analysis. A sample of Escherichia coli JM101hsd S rec A harboring plasmid pTMPv18A was registered in CBS on March 8, 1989, as CBS142.89.

A replica of the pTZ18R / 19R recombinant transformant of E. coli obtained as described above was taken and screened for lipolytic activity as described in Example 2. 5 × 10 ⁴ tested
None of the E. coli transformants showed lipolytic activity. Some possible reasons for this failure include: a) Gene expression initiation signal of the M-1 lipase gene is not recognized by the transcription / translation system of E. coli (for example, Molecular and General Genetics (Mol.
Gen. Genet.) 203 (1986) 421-429). b) This M
For the -1 lipase gene, there is a need to change the regulatory sequence or protein. c) Proper folding or secretion of M-1 lipase does not work well in E. coli.

Characterization and sequencing of the CM-1 lipase gene Plasmid pTMPv18A was digested with several restriction enzymes having a 6 bp recognition sequence. Analysis of the fragments resulting from these experiments allowed a preliminary restriction endonuclease cleavage map of the 2.0 kb Pvu II insert of pTMPv18A to be made. This map is shown in FIG.

The pTZ18 / 19R vector used provided a versatile “all-one system” that allows DNA cloning, dideoxy DNA sequencing, in vitro mutagenesis and in vitro transcription (Mead et al., Protein Engineering 1 ). (1986) 67
-74). Double-stranded plasmid is single-stranded by superinfection with helper phage M13K07 available from Pharmacia
Converted to DNA. 0 between the Xho I and EcoR V sites of pTMPv18A.
FIG. 12 shows the DNA sequence of the 94 kb DNA fragment. When translated in all possible reading frames, this DNA sequence, as determined by direct amino acid sequencing,
It was found to have a large reading frame including the NH ₂ terminal amino acid residue (residues 1-24) (see A in this example). Position-
The methionine at 24 is the initiation codon of the protein precursor because it precedes a series of amino acids typical of bacterial signal peptides (Von Heyn
e), Journal of Molecular Biology (J. Mol. Biol.) 192 (1986) 287-290). This signal peptide, consisting of 24 amino acids, is cleaved after the AEA (-3 to -1) signal peptidase recognition signal during the secretory process. It is noteworthy that the region around the cysteine residue in this signal sequence is largely missed by the lipid protein consensus signal peptide (Von Heyne, supra).

The amino acid sequence deduced from this DNA sequence is
-1 lipase is shown to consist of 289 amino acids ending with the TGA stop codon (see Figure 12). The expected molecular weight of this mature protein is 30,323, which is in good agreement with the molecular weight (MW31500) measured for M-1 lipase by SDS polyacrylamide gel electrophoresis (see FIG. 10).

Example 5 Pseudomonas aeruginosa
Molecular cloning of PAO lipase gene Pseudomonas aeruginos
The lipase a) is a lipid hydrolase (EC
3.1.1.3), which is secreted into the medium during late logarithmic growth (Stuer et al., Journal of
Bacteriology (J. Bacteriol) 168 (1986) 1070-10
74).

A. Cloning and properties Pseudomonas aeruginos
a) To clone the lipase gene of the PAO1 strain (ATCC15692), a global host cloning system was used that could screen gene banks directly by complementation to various mutant strains of Pseudomonaceae. PKT248 (Bagdasarian et al., Gene 16 (1981) 237) as a broad-range host vector
-247) was used. It is a broad host plasmid derived from the transposable R300B with streptomycin and chloramphenicol resistance.

Pseudomonas aeruginos
a) PAO1 DNA was partially digested with the restriction endonuclease SalI and ligated into the single SalI site of the pKT248 vector. The insert: vector ratio was 5: 1 to reduce the possibility of vector-vector ligation. The ligated PAO / pKT248 DNA was transformed into E. coli SK1108 competent cells as described in Example 1B (Donovan and Kushner, Gene 25 (1983) 39-48). E. coli SK1108 transformants were selected for streptomycin resistance (Sm ^R ) and counter-selected for chloramphenicol sensitivity (Cm ^S ).

6000 Sm ^R Cm ^S clones were obtained, which were screened for lipolytic activity as described in Example 2. None of these clones showed its activity, probably due to poor recognition of the Pseudomonas promoter in E. coli (Jeenes).
Etc.).

Therefore, the clone, Pseudomonas aeruginosa PAO2302 lipase deficient mutant 6-1 from E. coli (Wohlfarth)
And Winkler, Journal of General Microbiology (J.Gen. Microbiol.) 13
4 (1988) 433-440) was performed as follows. 6
The 000 clones were divided into 120 groups of 50 each. Make plasmid preparations from each group and use PA
O2302 (6-1) lip-mutant was used to transform competent cells. Pseudomonas
s) is described in Olsen et al. (J. Bacterio
l.) 150 (1982) 60-69). Selection was performed on calcium triolein (CT) agar plates (Wohlfarth and Winkler, supra) supplemented with streptomycin (50 μg / ml). 1 out of 5000 PAO transformants screened
0 showed lipase activity confirmed by white crystals at the tip of the colony. To further characterize, one of these positive PAO transformants, p
I chose SW1.

The plasmid contained therein was characterized by restriction analysis and agarose gel electrophoresis.
3.1 kb consisting of 7 kb and 0.76 kb Sal I subfragments
Was found to contain a Sal I insert. These inserts were subcloned into appropriate vectors for DNA sequencing and lipase gene expression.

Plasmid DNA (containing 1.3 kb and 0.97 kb inserts) of a subclone from pUC19, designated pSW103, was isolated and analyzed with restriction endonucleases. This plasmid pSW1
Fig. 13 shows the physical map of 03. A sample of E. coli JM101hsd S rec A harboring plasmid pSW103 was registered with the OBS on March 8, 1989 as No. 141.89.

B. Pseudomonas aeruginos
a) Sequencing and characterization of the PAO1 lipase gene The 2.3 kb SalI insert of pSW103 containing the PAO1 lipase gene was sequenced in two operations: bacteriophage M1
The three derivatives mp18 and mp19 (Yanisch-Pe
rron) et al., Gene 33 (1985) 103-119), using "Deininger""forced" or "shotgun" cloning sequencing (Analytical Biochemistry (Anal. Bio).
chem.) 129 (1983) 216-223) and Mizusaw
a) and other dideoxy chain termination technologies (Nucleic Acid Res.) 14
(1986) 1319-1324).

In the "forced" cloning sequencing operation, pSW103 2.
The SalI / PstI and SalI / EcoRI fragments from the 3 kb insert were purified and ligated into the appropriate M13mp18 vector. The whole DNA sequence was determined by splicing the obtained DNA sequence fragments. Most DNA sequences were determined for both strands. When translated in all possible reading frames, this DNA sequence contains only the 0.97 kb Sal I fragment of pSW103 (see FIG. 14).
It was found to encode mature PAO1 lipase.

The 1.3 kb SalI fragment of the pSW103 subclone encodes the 24 N-terminal amino acid residues of the PAO1 lipase protein as determined by direct amino acid sequencing, even when translated in all possible reading frames. I didn't.

The amino acid sequence deduced from the DNA sequence of the 0.97 kb Sal I fragment shows one interesting domain (shown in FIG. 14A).

Domain A is a eukaryotic lipase (Wion, etc.)
Science 235 (1987) 1638-1641), Bodmer, etc., Biochem. Biophys. Acta 909 (1987) 237-244) and prokaryotic lipase (Kugiyamiya) etc. , Biochem Biofeas Research Communication (Bioc
hem. Biophys. Res. Commun.) 141 (1986) 185-190) encodes the amino acid sequence GHSHSG which has already been determined to be both active centers.

Example 6 A. Expression of Cloned M-1 Lipase in Bacteria To improve the expression level of M-1 lipase in a heterologous host, the expression of pTMPv18A containing the M-1 lipase gene as described in 2.
The 0 kb Kpn I-Hind III fragment was ligated to the pBHA / C1 vector digested with Kpn I and Hind III. The nucleotide sequence of the pBHA1 vector is described in EP-A-0275598. The pBHC1 and pBHA1 vectors are the same except that the expression of the following antibiotic resistance genes is different: the chloramphenicol acetyltransferase gene
It is expressed in the E. coli WK6 strain harboring the pBHC1 vector.
The lactamase gene is expressed in the E. coli WK6 strain harboring the pBHA1 vector. R. Zell for E. coli WK6
And HJ Fritz (E
MBOJ. 6 (1987) 1809).

Transformation of the WK6 strain and the resulting ampicillin (for pBHA1) and chloramphenicol (pBH1)
In the case of C1) As a result of analysis of resistant colonies, each plasmid pB
HAM-1 and pBHCH-1 were detected (see FIG. 15).

The next step is the introduction of an Hde I restriction endonuclease site at the ATG start codon of the M-1 precursor protein. Stanssens et al. ("Protein engineering and site-directed mutagenesis", 24 times Harder)
Conference, 1985, site-directed mutagenesis of the pBHA / CM-1 plasmid was performed according to the method of AR First (Feersht) and G. Winter (eds.).

After mutagenesis, restriction enzyme analysis and Mizusa (Mizusa)
Potential variants with related mutations were checked by sequence analysis using the dideoxy method (see Example 5). After these analyses, a suitable plasmid, named pBHAM1N1, was found (see FIG. 16). M in E. coli
To regulate -1 lipase expression, a 1.7 kb HdeI-HindIII fragment of pBHAM1N1 containing the M-1 lipase structural gene was isolated and ligated into two expression vectors.

-PTZ18RN: pTZ18R derivative containing a single Nde I site at the ATG start codon of β-galactosidase (Mead
Et al., Protein Engineering 1 (198
6) 67-74).

-PMCTN: a pMC derivative containing a single NdeI site after the tac promoter and ribosome binding site (Stanssens et al., Supra).

Transfer the two ligation preparations to E. coli JM101hsd S rec
After transformation into competent cells of A, this transformant was screened for lipolytic activity using a tributyrin agar plate containing 0.5 mM IPTG (isopropyl-β-D-thiogalactoside). After incubation of these tributyrin plates at 30 ° C. for 48 hours, followed by several days of refrigerator storage, some colonies formed pale halos exhibiting lipolytic activity. The plasmid properties of these tributyrin positive clones were examined by restriction enzyme analysis. The two plasmids sought were found:-pTZN1M1: with the M-1 lipase gene after the lac control sequence (see FIG. 17);-pMCTM1: with the M-1 lipase gene after the tac control sequence (see FIG. 17). See Figure 18).

To identify the lipolytic activity produced by the E. coli transformants harboring plasmids pTZN1M-1 and pMCTM-1, respectively, these clones were best isolated at 30 ° C.
The cells were grown in 100 ml of 2TY medium (16 g / Bacto tryptone, 10 g / Bacto yeast extract, 5 g / NaCl, pH 7.0), and then induced with 0.5 mM IPTG at 30 ° C. for 3 hours. PTZ18RN as negative control
E. coli JM101hsd S rec A strain harboring E. coli was used. After separating the culture supernatant by centrifugation of the cells, periplasm and periplasm were prepared according to the method of Tetuaki et al. (Appl. Env. Microbiol. 50 (1985) 298-303). Fractionated into membrane / cytoplasmic components. Each fraction was analyzed on an SDS polyacrylamide gel consisting of 13% separation gel and 5% stacking gel according to the method of Laemmli (Nature 227 (1970) 680-685). Electrophoresis is 60m until the bromophenol (BPB) marker dye reaches the bottom of the gel
Driving in A. Sample preparation and protein blotting procedures were performed as described in EP-A-0253455. For Western blot analysis, a polyvalent rabbit antiserum against purified lipase from P. pseudoalcaligenes strain M-1 was used. From the results shown in FIG. 19, E. coli harboring pTZN1M1 and pMCTM1, respectively, synthesized an M-1 lipase-specific 31.5 kDa polypeptide,
It can be concluded that it can be secreted into the periplasmic space (see FIG. 19, lanes B and C). The lipolytic activity of this 31.5 kDa polypeptide was confirmed by the soft agar overlay technique based on the β-naphthyl acetate / fast blue BB salt method described in Example 1C.

EP A 0275598 and EP-A-0253455 disclose methods for efficiently transferring vesicles containing a gene of interest to Bacillus and obtaining a Bacillus strain that efficiently secretes the desired polypeptide product. ing.
This method was used for both Thai IV 17-1 and M-1 lipase genes.

For the M-1 lipase gene, the pBHAM1N1 plasmid (described above) was digested with NdeI and religated. This ligation mixture is used to transform Bacillus lichenylformis (Bacillus
licheniformis) Transformed into T9 protoplasts. Many neomycin resistant tributyrin positive colonies were analyzed to obtain the correct plasmid. This plasmid contains the Hpa II promoter of pUB110 (Ziprian (Zy
prian) and Matsubara (Matubara), DNA 5 (1986) 219
-225), and was named pBHM1N1 (see FIG. 20). T9 transformants harboring plasmid pBHM1N1 were tested for their ability to hydrolyze β-naphthyl esters after fermentation in industrial media according to the method described in Example 2. The result is
The lipolytic activity of the enzyme produced by this T9 clone indicates that the parent strain Pseudomonas pseudoalkagens (Pseu
domonas pseudoalcaligenes) strain M-1.

For expression in Pseudomonas hosts, the constitutive promoter of the p78 gene of the Pseudomonas-specific bacteriophage Pt3 was used (RGM Luiten, "Filamentous Bacteriophage" Pf3: Molecular Genetic Studies ", PhD Thesi
s), 1987, Nijmegen Catholic University, The Netherlands). A synthetic DNA fragment encoding this Pf3 promoter: Was synthesized.

This fragment was cut with EcoR I and Kpn I to obtain the vector pTZ18.
L was ligated to generate plasmid pTZPf31A.

To express the cloned M-1 lipase gene in Pseudomonas, pTMPv18A, pM
The M-1 expression cassette present in CTM1 and pTZPf3M1 was transferred to the broad host vector pKT231 (Bagdasario (Bagdasario).
n) et al., Gene 16 (1981) 237-247), pLAFR3
(Staskawisz et al., Journal of Bacteriology (J. Bacteriol.) 169 (1987) 5789
-5794) and pJRD215 (Davison et al., Gene 51 (1987) 275-280). The broad-range host plasmid harboring the M-1 expression cassette was replaced with Friedman (Fr.
iedman) et al. (Gene 18 (1982) 289-296), followed by the next pseudomonad (Pseudomo
nas) strain.

-Lipase-deficient mutant 6-1 of P. aeruginosa PAO2302 strain (Wohlfarth and Winkler).

-Lipase-deficient P. putida KT2442 strain (Zeyer et al., Applied Environmental Microbiol. (Appl. Env. Microbiol.) 50 (1985) 140
9-1413).

-P. Pseudoalcaligenes
M-1 strain (CBS473.85).

-P. pseudoalcaligenes I
NII-5 strain (CBS468.85).

An alternative procedure for transfer of broad-range host plasmids from E. coli to Pseudomonas was by electric field transformation ("electroporation") following the Gene Pulser (Biorad Laboratories) instructions for use.

The resulting Pseudomonas transformant lipase production test was reported by Odera et al. (J.Ferment.Technol. 64 (1986) 363-371). Performed after fermentation in olive oil medium.

Table 2 below shows the lipolytic activity productivity of the various strains.

When the lipase produced by the transformant of Pseudomonas was analyzed by SDS / gel electrophoresis, it was found that P. pseudoalkagens (ps)
eudoalecaligenes) showed the same mobility as the lipase produced by the M-1 strain.

The introduction of a large number of M-1 expression cassettes into P. pseudoalecaligenes results in 2-4 compared to lipase levels produced by the donor strain.
A two-fold improvement is seen. Furthermore, the original gene expression initiation signal or promoter of the M-1 lipase gene is PAO1
It can be concluded that it has activity in Pseudomonas pseudoalcaligenes in contrast to the strain KT2442 and the strain KT2442.

B. In vitro expression of cloned M-1 lipase gene In vitro expression of clones containing M-1 lipase was performed using a eukaryotic DNA-dependent translation kit (Amersham International). This system allows for the in vitro expression of genes on the bacterial plasmid, if relevant control signals are present. The following four bacterial plasmids were analyzed.

B.1. Plasmid pTMPv18A encoding M-lipase and β-lactamase gene products and conferring ampicillin (Ap) resistance (see FIG. 11). In addition, pTMPv18A has its own regulatory signals, promoter, Shine-Dalgarno sequence and leader sequence.

B.2. Plasmid pMCTM1 having the M-1 lipase gene and the chloramphenylchol resistance gene (Cm) (see FIG. 18). The lipase promoter is strong in this construct
Replaced with tac promoter.

B.3. Plasmid pMCTbliM1 also has an M-1 lipase gene and a chloramphenicol resistance gene. In this construct, the lipase signal sequence has been exchanged for the α-amylase signal sequence (see EP-A-0224294). The promoter is the same as the construct pMCTM1.

B.4. Plasmid pTZ18RN was used as a negative control (see FIG. 17).

DNA (0.5 μg) of the above plasmid was transcribed in vitro. The reaction was performed with 0.5 μl of a 10 × TB / 10 × NTP mix (equivalent mixture of 20 × TB and 20 × NTP; 20 × TB contained 800 mM Tris
HCl, pH 7.5, 120 mM MgCl ₂ and 40 mM spermidine; 20 × NTP
Mix contains 10 mM ATP, 10 mM CTP, 10 mM GTP and 10 mM UTP
TP), 0.5μ 0.1MDTT, 0.5μ RNasin (40u / μ
, Promega) and 0.5 μl of T7 RNA polymerase (15
u / μ, Promega) or 1μ E. coli RNA polymerase (1 u / μ, Boehringer). The reaction mixture was incubated at 39.5 ° C. for 1 hour.

Translation of the RNA transcript in vitro was performed according to the manufacturer's instructions. M-1 lipase is from Van Mour
ik) (J. Biol. Chem. 260 (1985) 11300-11306) and immunoprecipitated using a monoclonal antibody against M-1 lipase.

PTZ18RN was used as a negative control (FIG. 21, lanes A and E). Immunoprecipitation of pTMPv18A (lane B) was unprocessed at 34 kDa M-1.
Lipase was shown. Immunoprecipitation of pMCTM-1 (lane C) showed an unprocessed 34 kDa M-1 lipase and a 31.5 kDa mature M-1 lipase.
On the other hand, immunoprecipitation of pMCTbliM1 (lane D) showed a mature M-1 lipase of 31.5 kD. From this in vitro translation experiment, it can be concluded that the M-1 lipase gene can be expressed in the S-30 extract of E. coli. Data obtained from in vitro transcription / translation experiments of pTMPv18A confirmed that M-1 lipase did not show lipolytic activity on tributyrin plates because it was not processed (see Example 4B).

Example 7 Screening of Enzyme Molecules Using Characterized Lipase Genes as Probes To further illustrate the general applicability of the present invention, the present application was used to search for lipase genes with similar properties from another microorganism. A published probe was used. Here, it was shown that a lipase gene encoding a lipase having the same or equivalent washing applicability can be selected.

In Example 8, we also showed that the present hybridization technique can clone allogeneic lipase genes from other microorganisms.

To do this, the following strain, Pseudomonas pseudoalcaligenes M
-1 (CBS473.85), P. pseudomonas pseudoalgens IN II-5 (CBS468.85), P. alkaligens (DSM
50342), P. aruginosa (PAC1R (CBS136.
85) P. aeruginosa PAO (ATCC15692) (Wohlfarth and Winkle
r), 1988, Journal of General Microbiology (J. Gen. Microbiol.) 134 , 433-440), P. stutzeri Thai IV17-1 (CBS461.85); P. stutzeri ) PG-I-3 (CBS137.89), P. stutzeri PG-I-4 (CBS138.89), P. stutzeri (st
utzeri) PG-II-11. 1 (CBS139.89), P. stutzeri (stut
zeri) PG-II-11.2. (CBS140.89), P.fragi (fragi) D
B1051, P. gladori (CBS176.86), Acinetobacter calcoa
ceticus) Gr-V-39 (CBS460.85) and Staphyrococcus aureus (ATCC2766).
The chromosomal DNA of 1) was isolated, and 5 µg thereof was digested and analyzed by Southern blotting technique (Maniatez (Ma
niatis) et al., supra). The DNA is transferred to a nitrocellulose filter (Schleicher & Schuel).
er & Schuell), BA85: 0.45 mM). Filter this filter with 6xSSC, 5x Denhardt's solution, 0.5% SDS and 100μg /
Prehybridized in ml denatured bovine thymus DNA.

After 2 hours of pre-incubation, inserts of 32 P-labeled pTMPv18A or pET3, respectively, were added and hybridization was performed at 55 ° C. for 16 hours.

Insert the inserted DNA into pTMPv18A and EcoR I with Xho I and EcoR V.
Each fragment was isolated by digestion of pET3 with
After fractionation by 8% agarose gel electrophoresis, they were collected by a glass milk operation (Gene Clean). pTMP
The 1 kb XhoI / EcoR V fragment, which is an insert of v18A, and the 3.2 kb EcoRI fragment, which is an insert of pETT3, are nick-translated with α32PATP to have high radioactivity in vitro (2-5 × 10 ⁸ cpm). / μg) labeled (Feinberg and Vogels)
tein), Analytical Biochemistry (Anal Bio)
chem.) 132 (1983) 6-13). Probe fragments average 300
It was shown to have a length of ~ 800 bases.

The filters were then washed twice in 6 × SSC, 0.1% SDS at 55 ° C. for 30 minutes. After drying the filter at room temperature -70
C. Using an intensifying screen (Kronex, Lighting Plus GH220020) at 1-3 ° C. on Kodak X-omatAR film or Kronex 4NIF100 X-ray film (DuPont).
An autoradiograph was obtained by exposing for one day.

The following equation was derived by analyzing the effects of various factors on the stability of the hybrid: Tm = 81 + 16.6 (log10Ci) +0.4 (% G + C) -600 / n-15% Protocol ", 1987-19
88, Ausubel et al. (Where n = minimum chain length of the probe Ci = ionic strength (M) G + C = base composition) was used to determine the homology detected in our experiments. Assuming a probe length of 300 bases, a gene showing at least 67% homology in a fragment of 300 bases or more could be detected. Pseudomonas (Ps
eudomonas) was assumed to have a GC content of 65% (Normore (Nor
more), 1973, Laskin and Lechevaria (Le)
chevalier) (eds.) "Microbiology Handbook" Volume II, CRC
Press, Bocalayton, Fla) FIG. 22A shows the hybridization pattern of a SalI digest of chromosomal DNA when EcoRI of pET3 was hybridized.

Most Pseudomonas strains are found to contain genes homologous to the pET3 clone. No homologous genes were detected in P. flagi DB1051 (lane M), A. calcoaceticus Gr-V-39 (lane O) and S. aureus (lane P). P. Alcaligenes
(Lane E), P. pseudoalgens
enes) (lanes C and D), P. aruginosa (aerug)
inosa) (lane F and lane G), a moderate hybridization signal was observed, while weak hybridization signal was observed in P. gladioli (lane N). A very strong hybridization signal is seen in P. stutzeri (lanes HL), which is not surprising since pET3 is originally derived from the P. stutzeri Thai IV 17-1 strain.

FIG. 22B shows the hybridization pattern using the EcoR V / Xho I insert of pTMPv18A.

Strong hybridization signals were obtained from chromosomal DNA from P. pseudoalcaligenes (lanes C and D), P. alcaligenes (lane E) and P. stutzeri strains (lanes HL). You can see that it is obtained. P. aruginosa (aerug
Inosa) shows weaker hybridization (lanes F and G), P. fragii (lane M), P. gladioli (lane N), and A. calcoaceticus Gr. -Chromosomes of V-39 (lane O) and S. aureus (lane P)
No hybridization was seen in the DNA.

Example 8 Cloning of Homologous Lipase Genes from Other Microorganisms To demonstrate the applicability of the invention disclosed herein, use of the plasmid pTMPv18A to clone homologous genes from P. alcaligenes (DSM50342). This will be described (see FIG. 22B, lane E).

In FIG. 22B, lane E, P. alkaligens (alcal
igenes) is a clear 5.0k that hybridizes with the probe
b shows a Sal I fragment and a thin 0.5 kb Sal I fragment. This indicates that P. alcaligenes contains a gene with at least 67% homology within a 300 base pair fragment to the XhoI / EcoRV insert of plasmid pTMPv18A.

To determine whether the 5.0 kb Sal I fragment of P. alcaligenes contained the gene encoding lipase, the Sal I fragment was cloned. The Sal I gene library was constructed using the vector pUC19 and
Transformed to M109 (Yanish-Perran
-Perron) et al., Gene 33 (1985) 103-119).

Using the conditions described in Example 7, the replica filter of the gene library was hybridized with the α32P-labeled insert of pTMPv18A. Three positive clones containing the 5.0 kb Sal I fragment were isolated from the library; pCH1, p
CH2 and pCH3. Using the SalI site present in the chloramphenicol resistance gene, the 5.0 kb SalI fragment of pCH1 was ligated into the vector pKT248 (Bagdasarian, etc.)
Gene 16 (1981) 237-247). A streptomycin resistant chloramphenicol sensitive transformant of E. coli JM109 was selected. One of these transformants containing the expected 5.0 kb SalI insert, pCH101, was transformed into the Pseudomonas aeruginosa 2302 (6-1) lip-strain (see Example 5). .

Colonies were grown on NB-calcium triolein agar. After growth, the plates were stored at 10 ° C. A few days later, a calcium precipitate appeared around the transformed colonies, and the untransformed P. aeruginosa 2302 (6-1) lip ^- strain grown on the same agar plate without antibiotics. Does not appear around. We have cloned a 5.0 kb Sal I fragment that complements the lip ^- expressed form of the P. aeruginosa 2302 (6-1) lip ^- strain and thus encodes an extracellular lipase that can be produced in a suitable host. Conclude that

Based on DNA hybridization experiments, it was concluded that these lipases show homology to M-1 lipase and consequently also to other lipases that hybridize to the pTMPv18A insert used as a probe. I can do it. The description of the cloning of the lipase of P. alcaligenes represents a general application method for cloning lipases showing homology to M-1 lipase.

Example 9 Pseudomonas pseudoalkagens
Comparison of nucleotide sequences of lipase from pseudoalcaligenes M-1 and other lipases
s pseudoalcaligenes) M-1 lipase (Fig. 12) was converted to Pseudomona
s fragi) (IFO-3458) lipase (Kugimi
ya) et al., Biochem. Biophys. Res. Comm. 141 (198)
6) 185-190), Staphylococcus hicus (Staphyl
ococcus hyicus) lipase (Gotz et al., Nucleic Acids Res.) 13 (19
85) 1891-1903) and Pseudomonas Arginosa (Pseu
domonas aeruginosa) PAO1 lipase (FIG. 14). M-1 lipase and P. aruginosa (aerugi)
nosa) 81% high homology between PAO1 lipase and M-1 lipase and P. fragi (IFO-34).
There was 62% homology between 58). However, this P.
The sequence of the fragi lipase is significantly shorter than that of the other two Pseudomonas lipases. No homology was observed between the M-1 lipase and the lipase of Staphylococcus hyicus.

These results indicate that when pTMv18A was used as a probe, Pseudomonas fragi and Staphylococcus aureu
Example 7 not hybridizing to s) -derived chromosomal DNA
We support the data obtained in

The amino acid sequence derived from the nucleotide sequence of P. pseudoalgens M-1 lipase was also compared to other lipases. M-1 lipase P. aruginos (aeruginos)
a) The overall homology between PAO1 lipase was found to be 78% and the homology between M-1 lipase and P.fragi lipase was found to be 48%. No homology was found between the amino acid sequences of M-1 lipase and Staphylococcus hyicus lipase. However, in the important serine 87 region of mature M-1 lipase, high homology was observed among the four lipases. Kugimiya and others are in this area GH
It was proposed that the sequence -SHG is the active center of the lipase enzyme.

Example 10 Detergent Compatibility of Homologous Lipases in the SLM Test This example follows the SLM test modification to test the compatibility of these enzymes in modern laundry detergents and demonstrates the M-1 gene and high DNA in the washing process. Showing sequence homology,
P. aruginosa, P. stutzeri
And the performance of lipase enzymes produced by P. alcaligenes strains.

The detergent used is a powder detergent (ALL ^R base, EP-A-02
18272) and liquid detergents (Liquid TIDE ^R, also reported in EP-A-0218272 but without protease inactivation). The preparation of these lipase enzymes was carried out by culturing the bacteria according to the following procedure: culturing the bacteria in 100 ml of Brain Heart Infusion (BHI) medium at 30 ° C. for 24 hours on a rotary shaker. To prepare an inoculation medium. Then, it was inoculated into a lab fermenter (2) containing a medium 1 having the composition described below. Culture land pairs formed Ingredients Concentration (g / kg) Brain Heart Infusion 18.50 (BHI) (Difco) yeast extract (Difco) 16.00 Calcium · 2H ₂ O 0.80 Magnesium sulfate chloride · 7H ₂ O 3.20 manganese sulfate · H ₂ O 0.030 soybean oil 5.0 dipotassium phosphate 6.4 Fermentation was performed at 30 ° C. Sixteen hours after inoculation, soybean oil was started to be injected at a rate of 1 g / h and fermentation was continued. The aeration speed was 60 / h and the stirring speed was 700 rpm. Fermentation was performed for a total of 64 hours.

After fermentation, the bacteria were removed by centrifugation. The supernatant was rapidly mixed with 2.5 volumes of acetone while stirring at room temperature. The mixture was then stirred for 10 minutes and allowed to stand, which was filtered off with suction. The filtered solid was washed with 70% acetone, then washed with 100% acetone, and dried under reduced pressure.

The lipase enzyme preparation thus obtained was tested by the SLM test method. The operation of the SLM test is described in EP-A-0218272. This operation was modified as follows.

A solution containing 10 mg of olive oil (25%) in acetone was spotted on a polyester swatch (3 × 3 cm) and air dried at room temperature. 10ml SHW (standard hard water:
A washing solution composed of a detergent dissolved in 0.75 mM CaCl ₂ , 0.25 mM MgCl ₂ ) or SHW was placed in a stoppered Erlenmeyer flask (25 ml) and kept in a shaking water bath at 40 ° C. The detergents tested were powder detergents (ALL ^R base) and liquid detergents (TIDE liquid).
The concentration of the ALL base cleaning solution was 4 g / (pH 9.5), and the concentration of the liquid TIDE was 1.5 g / (pH 7.5). The washing process involved adding the enzyme preparation to an Erlenmeyer flask and immediately placing the dirty specimen and shaking at 40 ° C for more than 40 minutes. Final lipase concentration was 2 TLU / ml.

After washing, the specimen was rinsed with SHW and dried at 55 ° C. for 1 hour. The so dried specimen was again washed in a second 40 minute wash cycle with fresh detergent and enzyme solution.
After a second washing cycle, the specimens were treated with 0.01 N HCl, rinsed and dried overnight at room temperature. The dried specimen was washed with 5 ml of solvent (n-hexane / isopropyl alcohol / formic acid; 975: 2
Extraction was performed by rotation in a glass tube containing 5: 2.5 (v / v), 1 ml / min). The resulting residue was measured for triglyceride, diglyceride and free fatty acid by HPLC.

HPLC equipment and conditions Column: CP Microsphere Si (Chrom Pack), 100
× 4.6mm Injection system: Wisp (Millipore) 10μ Pump: Model 2150 (LKB) Detector: Refractive index monitor (Jobine bonbon) Integrator: SP4270 (Spectraphysis) Eluent: m-hexane / isopropyl alcohol /
Formic acid 975: 25: 2.5 (v / v), 1ml / min Temperature: room temperature Triolane retention time is 1.2 minutes, 1,3 diglyceride is
2.5 minutes, 3.6 minutes for 1,2-diglyceride and 1.6 for oleic acid
Minutes. The peak area or peak height was measured as an indicator of triglyceride and fatty acid recovery. The recovered amount of triglyceride extracted from the unwashed sample was defined as 100%. The refractive index ratio of triolein and oleic acid was found to be 1.0 from the peak height.

The results of these SLM tests are shown in the table below. These tables show the triglyceride recovery and total lipid recovery. Total lipid recovery is the sum of triglycerides, 1,2 and 1,3 diglycerides, and free fatty acids. The difference between total lipid recovery and triglyceride recovery is a measure of lipase activity and performance. Differences between total lipid recovery and controls (without lipase enzyme) indicate removal of oil stains from the fabric, indicating that these enzymes are stable and effective in realistic wash conditions mimicked by SLM tests .

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:01 1:38) (C12N 1/21 C12R 1:07 1:19 1:38) Microorganism accession number CBS 460.85 Accession number of microorganisms CBS 461.85 Accession number of microorganisms CBS 468.85 (72) Inventor Cox Maria Mathilde Josephina Netherlands Nuel-1012 Carte Amsterdam Rokin 38-3 (72) Inventor Farin Fallock Nether-2394 Hersey Hazelswald Reindijk van Beethovenlan 20 (72) Inventor Vuhlfarth Suzanne Germany Day 4600 Dortmund Eichlinghoffen Pell Zebeckelstraße 45 (56) References JP-A-63-32489 (JP, A) JP-T-63-500423 (JP, A) Journal of General Microbiology, 134, (1988), P433-440 Biochemical and Biophysical Research, 141 [1] (1986), pp. 185-190 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C12N 9/20 WPI / L (DIALOG) BIOSIS (DIALOG)

Claims (13)

(57) [Claims] In the 5'-3 'direction of transcription, a transcription regulatory region and a translation initiation region that function in a host cell, a DNA sequence encoding a lipolytic enzyme having the following amino acid sequence, and a function in a host cell: A translation and transcription termination region,
A transformed microbial host cell comprising an expression cassette in which the expression of the A sequence is under the control of the transcriptional and translational regulatory regions. 2. The transformed host cell according to claim 1, wherein the expression cassette further comprises a marker gene. 3. The transformed host cell according to claim 1, wherein the DNA sequence contains a leader sequence which binds to a gene encoding a lipolytic enzyme in an appropriate reading frame. 4. The transformed host cell according to claim 1, wherein said host cell is a prokaryotic cell. 5. The transformed host cell according to claim 4, wherein said prokaryotic cell is a Bacillus, Escherichia coli or Pseudomonas cell. 6. The transformed host cell according to claim 1, wherein said DNA sequence is derived from a microbial cell. 7. The transformed host cell according to claim 6, wherein said DNA sequence is derived from Pseudomonas species. 8. The transformed host cell according to claim 7, wherein said DNA sequence is derived from P. pseudoalkagens. 9. The transformed host cell according to claim 8, wherein said DNA sequence is derived from P. pseudoalgens M-1 (CBS473.85). 10. The TLU measurement condition is 8 to 10.
It has an optimum pH range of 5 and a temperature of about 60 ° C or less and a pH of 7 to
A method for preparing a lipolytic enzyme exhibiting lipase activity in an aqueous solution containing a detergent at a concentration of up to 10 g / under the washing conditions described in (11). A transcription control region and a translation initiation region that function in a cell, a DNA sequence encoding a lipolytic enzyme having the following amino acid sequence, and a translation and transcription termination region that function in a host cell; Growing a host cell containing an expression cassette under the control of a regulatory region; (b) isolating the lipolytic enzyme. 11. A method for producing a lipolytic enzyme, comprising the steps of: (a) culturing the transformed microorganism host cell according to any one of claims (1) to (9) in a nutrient medium to produce a rich broth; And b) isolating the lipolytic enzyme from the medium. 12. A transcription regulatory region which functions in a microbial host cell in the direction of transcription, has an optimum pH range of 8 to 10.5 at a constant pH under TLU measurement conditions, and has a temperature of about 60 ° C. or lower and a pH of 7 to 10. 11
DNA sequence encoding a lipolytic enzyme having the following amino acid sequence that exhibits lipase activity in an aqueous solution containing a detergent at a concentration of up to 10 g / under washing conditions, and a DNA containing a transcription termination regulatory region that functions in host cells Construct. 13. The DNA construct according to claim 12, wherein
A DNA construct further comprising a selectable marker gene and one of a secretory leader sequence.