DK175634B1 - Transformed host cell and method for producing 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

in DK 175634 B1

The invention relates to the preparation of recombinant DNA technology of lipolytic enzymes with characteristics which make them suitable for use as detergent additives. More specifically, the invention relates to a transformed host cell of Pseudomonas pseudalcells, and to a process for producing a lipolytic enzyme characterized by: (a) having a pH otium in the range of 8 to 10.5 measured in a pH state under conditions for TLU determination, and 10 (b) exhibit lipase activity in an aqueous solution containing a detergent at a concentration of up to 10 g / l solution under washing conditions at a temperature of ca. 60 C or lower and at a pH value between 7 and 11.

A particular problem associated with washing is the removal of greasy stains. Currently, greasy dirt is emulsified and removed using a combination of elevated temperature and high alkalinity. In recent times, however, there has been a strong tendency to use rather low washing temperatures, viz. 40 C or less, and these conditions are particularly unsuitable for removing greasy stains. There is therefore a need for detergent additives effective at these lower washing temperatures, stable in highly alkaline detergent solutions, and stable under storage conditions in both solid and liquid detergent mixtures. One group of enzymes that hydrolyze triglycerides are lipases (E.C. 3.1.1.3).

Lipases are widely distributed as they occur in many different prokaryotic organisms as well as in eukaryotic cells. Depending on the source of the enzyme, both substrate specificity and other characteristics that

I DK 175634 B1 I

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Amongst stability under various conditions, be

In very different. Lipases have been used in detergent

In gene mixtures, however, the lipases used herein showed I

In low washing efficiency under washing conditions and had

In addition, the stability required for detergen- I

I ter. IN

In lipolytic enzymes that can exhibit lipase activity

In white under modern washing conditions, ie. such that I

You are stable and efficient at bending detergent concentrations

In 10 tions, high pH values and low washing temperatures, pro- I

You are duced by certain strains belonging to the species Pseudo-I

In monas pseudoalcallqenes, Pseudomonas stutzerl and acine-

In tobacter calcoacetlcus (see EPO patent application I

In EP-A-0218272). However, these species are potentially I

In 15 disease-causing plants and animals, and there are

In only a few data for the necessary fermentation conditions

I for efficient production of lipases using I

In of these microorganisms. IN

It is therefore desirable to develop an effective I

In a 20g safe manner for the preparation of lipolytic enzymes I

In with the desired characteristics of detergent additives. IN

It is further desirable that the lipases be separated

You read by the host organism to extract the enzyme di-I

You extracted from the extracellular fluid from the fermentation I

In the mixture. IN

You know about lipases produced by Pseudomonas I

Species that the cultivation conditions strongly influence

In the final localization of these enzymes (Sugiura, I

In I: "Lipases", editors B. Borgstrom and H. L. Brockman I 30 (1984) pages 505-523, Elsevier, Amsterdam).

You often have trouble obtaining effective expression of I

In heterologous genes in microorganisms, including incorrect ones

In folding the proteins that are formed, protein degradation I

3 DK 175634 B1 and incorrect location of the products. Harris, in: "Genetic Engineering", Volume 4 (1983), Academic Press,

New York. In EL coli, use of the secretion cloning vector generally permits transport of heterologous gene products into the periplasm and only the products are occasionally found in the culture medium; Lunn et al., Current Topics in Microbiol, and Immunol. 125 (1986) 59-74. When using EL coli as a host cell, cloned microbial lipases are only poorly secreted in the culture medium. See Gotz et al., Nucleic Acids Res. 13 (1985) 5895-5906, Kugimiya et al., Biochem. Biophys.

Res. Commun. 141 (1986) 185-190 and Odera et al., J. Ferment. Technol. 64 (1986) 363-371.

Wohlfarth and Winkler, J. Gen. Microbiol. 134 15 (1988) 433-440, discloses physiological characteristics of newly isolated lipase-defective mutants from Pseudomonas aeruginosa strain PAO 2302 and the chromosome mapping and cloning of the corresponding gene.

Bacillus species, especially Bacillus 20 subtilis strains, have been used with varying degrees of success as host strains to express both foreign and endogenous genes and to secrete the encoded protein products. An overview of this can be seen e.g. in Sarvas, Current Topics in Microbiology and Immunology 125 (1986) 103-125, H. C. Wu 25 and P. C. Tai, publishers Springer Verlag; see also Himeno et al., F.E.M.S. Microbiol. Letters 35 (1986) 17-21.

U.S. Patent No. 3,950,277 and GB Patent No. 1,442,418 disclose lipase enzymes combined with an activator and calcium and / or magnesium ions, respectively, which are used to soak up soiled fabrics and to remove triglyceride stains and dirt from polyester or polyester / cotton fabric. Suitable microbial

I DK 175634 B1 I

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In lipases described herein, such derivatives include

I from Pseudomonas, Aspergillus, Pneumococcus, Staphylo- I

In coccus, Mycobacterium tuberculosis, Mycotorula llpoly- I

In tica and Sclerotlnia. IN

In 5 GB Patent No. 1,372,034, a part I

In tergent mixture comprising a bacterial lipase pro- I

In duced by Pseudomonas stutzerl strain ATCC 19154. Pa- I

In the tent script further describes that the preferred li I

In polytic enzymes should have a pH optimum between 6-10 and 1

I 10 should be active in this area, especially between 7-9. (This one

In putative Pseudomonas stutzerl strain have been reclassified

In sifted to Pseudomonas aeruginosa). IN

In EPO patent application EP-A-0130064 discloses a single I

In zymatic detergent additive comprising a lipase iso- I

In the clay from Fusarlum oxysporum with a larger lipolytic I

In washing efficiency than conventional lipases. Lipolytic I

Detergent additives are also disclosed, e.g. i

GB Patent No. 1,293,613 and Canadian Patent No. I

I 835,343. IN

In 20 EPO patent applications EP-A-0205208 and I

EP-A-0206396 discloses the use of Pseudomonas and I

In Chromobacter lipases in detergents. Regarding a thorough I

For an overview of lipases as detergent additives, see Andree I

In et al., J. Appl. Biochem. 2 (1980) 218-229. IN

I 25 The transformed Pseudomonas pseudoalcall genes I

In the host cell of the invention is peculiar to that of I

The characteristic part of claim 1 stated. Furthermore, it is you

In particular processes for preparing one lipolytic I

In enzyme peculiar to the characterizing part of claim 3. Put another way, the inventive method of producing a lipolytic enzyme I is characterized by comprising in a nutrient medium culturing in a transformed host cell of the invention to achieve a nutritious herb and isolating the lipolytic enzyme from this herb.

In the drawing:

Figure 1: Strategy for cloning the lipolytic enzyme gene; for symbols, see the text of Figure 2;

Figure 2: A restriction map of pAT1; having determined a number of restriction enzyme recognition sites in the plasmid pAT1; 10 [I: pUN121 vector DNA, mm: Thai IV 17-1 DNA insert; The symbols used are: - - Ori E. coli: origin of replication; Apr: the pUN121 gene encoding ampicillin resistance; - Tcr: the pUN121 gene encoding tetracycline resistance; - cl: the bacteriophage lambda gene encoding the cl repressor;

The position where partially Sau3A digested chromosomal DNA from Pseudomonas stutzeri Thai IV 17-1 (CBS

25 461.85) was ligated to pUN121, is designated by BclI / Sau3A. The position of the gene encoding lipolytic activity is indicated by a dashed line;

Figure 3: A restriction map of pAT3, the symbols being as in Figure 2; Figure 4: A restriction map of pET1, the symbols being as in Figure 2;

Figure 5: A restriction map of pET3, the symbols being as in Figure 2;

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Figure 6: A restriction map of pAM1, with I

In the symbols are as in Figure 2; IN

Figure 7: A restriction map of pM6-5, with I

In the symbols are as in Figure 2; IN

Figure 8: A restriction map of pP5-4, with * I

In the symbols are as in Figure 2; the position in which I partly

In EcoRI *, chromosomal DNA from Aclnetobacter cal digested

In coacetlcus GR V-39 (CBS 460.85) was ligated to pUN121 I

I is designated EcoRI / EcoRI *; IN

I 10 Figure 9: An SDS 10-15% gradient Phastgel I

I (Pharmacia); IN

In Figure 9A: after staining with Coomassie Brilliant I

I Blue; IN

In Figure 9B: after staining with 8-naphthyl acetate / I

I 15 Fast Blue BB; IN

In Lane 1: lysate from E. coli JM101 hsdS recA strain with I

Contained with pUN122 heated with SDS for 10 minutes to 1

At 95 ° C; IN

In Lane 2: lysate from E ^ coli JM101 hsdS recA strain with I

In 20 contents of pET3 heated with SDS for 10 minutes to 1

At 95 ° C; IN

In Lane 3: culture supernatant from P. stutzerl Thai IV 17-1 I

In strain heated with SDS for 10 min to 95 ° C; IN

In Lane 4: same sample as in Lane 2, but heated with SDS I

For 251 5 minutes to 95 ° C; IN

In Lane 5: same sample as in Lane 3, but heated with SDS I

For 5 minutes to 95 ° C; IN

In Lane 6: the same sample as in Lane 2, but incubated with SDS I

I for 10 minutes at room temperature; IN

In Lane 7: the same sample as in Lane 3, but incubated with SDS I

I for 10 minutes at room temperature; IN

In Lane 8: low molecular weight protein markers from Pharmacia; IN

In Figure 10: SDS 13% polyacrylamide gel by color I

In co-op with Coomassie Brilliant Blue; IN

7 DK 175634 B1

Lane 1: purified M-1 lipase;

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

Figure 11: restriction map of pTMPv18A.

[? - ----- j: pTZ18R vector DNA

: M-1 DNA insert the symbols used are: 10 - Ori E. coli: E. coll origin of origin derived from pBR322 vector; - fl ori: origin of replication from the filamentous bacteriophage fl; p <7: promoter of the bacteriophage T7 in the preparation of in vitro transcripts; - Apr: gene encoding ampicillin resistance; and - Lip: gene encoding the M-1 lipase;

Figure 12: The nucleotide sequence (i.e., the first 942 nucleotides) of the M-1 lipase gene and the deduced amino acid sequence of the M-1 lipase; the termination codon TGA is denoted by an asterisk and the A square represents the active center of the lipase protein, in addition the arrow denotes the assumed signal peptidase cleavage site and the amino-terminal sequence of the mature lipase protein is underlined;

Figure 13: a restriction map of pSW103,

In 1: pUC19 vector DNA

30 flHH: PA0 1 DNA insert, the symbols used are: - Ori E. coli: E. coli origin of replication derived from pBR322 vector; In DK 175634 B1

I 8 I

In ”plac: promoter for E. coll lac operon; IN

I - Apr: gene encoding ampicillin resistance; and in

I - Lip: gene encoding PAO 1 lipase; IN

Figure 14: A partial nucleotide sequence of PAO li-I

In the 5 pass gene (i.e. from the internal SalI site) and the off-I

In directed amino acid sequence of the PAO lipase; termination co

In the donation TAG is denoted by a star, and A square I

represents the active center of the lipase protein network; IN

Figure 15: Construction of plasmids pBHAM1 I

I 10 and pBHCM1, the symbols used being: I

I - Bacillus ori: Bacillus origin of replication; IN

I - Kmr: the pUBHO gene encoding neomycin resi- I

resistance; IN

I - Qn: Tn9 transposon network encoding chlorine-I

I amphenicol resistance; and in

In “pHpaII: Hpall promoter from plasmid pUBHO; IN

In other symbols, as in Figure 11; IN

In Figure 16, the construction of the plasmid pBHAMIN1, I

In that the symbols are as in Figure 15; IN

In Figure 17 the construction of the plasmid ρτζΝίΜΙ, I

In that the symbols used are:

- lac Za: the N-terminal part of the LacZ gene that I

encodes the α domain of β-galactosidase; and in

I - Plac: the promoter of E. coli lac operon; IN

The other symbols are as in Figure 11; IN

In Figure 18, the construction of the plasmid pMCTM1, I

In that the symbols used are:

I - Cmr: the gene encoding chloramphenicolresl- I

I stens; and in

In 30 ptac: hybrid trp-lac E. coli promoter; IN

the other symbols are as in Figures 11 and 15; IN

Figure 19: Immune blot detection of M-1 lipase-I

In protein produced by transformed coli cells, I

9 DK 175634 B1, wherein lanes A-D contain periplasmic fractions of E. coli cells containing the following constructs:

Lane A: pTZ18RN; Lane B: pTZNIM1;

Lane C: pMCTM1;

Lane D: purified M-1 lipase from Pseudomonas pseudoalcali gene strain M-1; the molecular weights of the marker proteins (Rainbow, Amersham) are expressed in kDa on the right;

Figure 20: Construction of the plasmid pBHM1N1, the symbols being as in Figure 15;

Figure 21: An autoradiography of 35S labeled proteins synthesized in vitro as

Lanes A-D: show immunoprecipitation of the in vitro translated samples by monoclonal antibodies against M-1 lipase;

Lanes E-H: in vitro transcription / translation products of the following plasmids:

Lanes A and E: pTZ18RN; Lanes B and F: ρΤΜΡνίβΑ;

Lanes C and G: pMCTM1; and lanes D and H: pMCTbliMl;

Figures 22A and 22B: detection of lipase-coding sequences in bacterial DNA, digesting 25 '5 ng of plasmid DNA, and 5 µg of chromosomal DNA with restriction enzymes as depicted on 0.8% agarose gel, exposed to nitrocellulose filters and hybridized with the notch-translated insert of pET3 and ρΤΜΡνίβΑ, respectively; Figure 22A: The autoradiography after hybridization with the pET3 EcoRI insert;

Figure 22B: autoradiography after hybridization with ρΤΜΡνίβΑ xhol-EcoRV insert;

I DK 175634 B1 I

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In lane A: HindIII / SStI digestion of the plasmid pTMPv1SA; IN

In Lane B: EcoR1 digestion of plasmid pET3; IN

In BRL DNA gel marker with molecular weights of 0.5, I

I 1.0, 1.6, 2.0, 3.0, 4.0, 5.0, 6.0, 7, 8, 9, 10, I

In 5 11 and 12 kb; IN

In Lane C: Saline digestion of P. pseudoalcallqenes M-l I

I (CBS 473.85); IN

In Lane D: Sall digestion of P. pseudoalcallqenes IN II-5 I

I (CBS 468.85); IN

In 1 ° Lane E: Saline digestion of p. Alkaliqenes DSM 50342; IN

In Lane F: Saline digestion of P. aeruginosa PAC IR (CBS I

I 136.89); IN

In Lane G: Saline digestion of P. aeruginosa PA02302 (6-1); IN

In Lane H: Sall digestion of P. stutzerl Thai IV 17-1 I

I 15 (CBS 461.85); IN

In Lane I: Sall digestion of P. stutzerl PG-I-3 (CBS I

I, 137.89); IN

In Lane J: Saline digestion of p. Stutzerl PG-I-4 (CBS I

I, 138.89); IN

In 20 Lane K: Sall digestion of P. stutzeri PG-II-11.1 (CBS I

I 139.89); IN

In Lane L: Sall digestion of P. stutzerl PG-II-11.2 (CBS I

I 140.89); IN

In Lane M: Sall digestion of P. fraql serm. DB1051 (= I

I 25 Ferm BP 1051); IN

In Lane N; Saline digestion of p. Gladioli (CBS 176.86); IN

In Lane 0: Sall digestion of A. calcoacetlcus Gr-V-39. IN

I (CBS 460.85); and in

In Lane P: Sall digestion of S. aureus (ATCC 27661). IN

I 30 I

In accordance with the invention, DNA construct 1 is provided

In tions and host cells comprising DNA encoding enzyme I

In more. IN

11 DK 175634 B1

The lapas of interest in connection with the invention have a pH optimum between about 8-10.5, exhibits an effective lipase activity in an aqueous solution containing a wash mixture with concentrations up to about 5%. 10 g per 1 under washing conditions and a temperature 0 0 of 60 C or lower, preferably 30-40 C, and a pH between about 7-11, preferably ca. 9-10.5. Plasmid constructs comprising a DNA sequence encoding a lipase gene with the desired characteristics are used to transform a host cell which may be either a eukaryotic or prokaryotic cell. Then, the transformed host cell is grown to express the gene.

The method for isolating the lipase gene belongs to the prior art including synthesis, genomic DNA isolation, cDNA preparation or combinations thereof. The various methods of manipulating the gene are well known and include restriction digestion, resection, ligation, in vitro mutagenesis, primer repair and the use of linkers and adapters and the like. See Maniatis et al., "Molecular Cloning", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.

In general, the method comprises preparing a genomic library from an organism expressing lipase with the desired characteristics. Examples of such lipases are those obtainable from Pseudomonas and Aclnetobacter and especially from strains belonging to the species Pseudomonas alcaligenes. Pseudomonas pseudoalca ligigenes, Pseudomonas aeruginosa, Pseudomonas stutzerl and Aclnetobacter calcoacetlcus. A number of these lipases and strains are described in more detail in EP-A-0218272.

The genome is isolated from the donor microorganism and

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Iterate it using an appropriate restriction enzyme

You as Sau3A. The obtained fragments are combined with an I

In a vector molecule previously cleaved by an I

In this, compatible restriction enzyme. An example of an I

In suitable vector, plasmid is pUN121 that can be cleaved by I

In restriction endonuclease Bell. In addition, you can use- I

In the amino acid sequence to construct a probe for I

In scanning a cDNA or genomic library I

I made from mRNA or DNA from the cells of interest I

In 10 as donor cells for a lipase gene. IN

In addition, it is easy to clone structurally related I

In genes found in other microorganisms using li- I

You bag DNA or a fragment thereof as hybridization I

In probe. In particular, we think of isolating genes from I

In 15 organisms expressing lipolytic activity during I

Using oligonucleotide probes based on nueleo- I

In the time sequence of lipase genes obtainable from organ I

In the niches described in EP-A-02118272. Alternatively, you can

You derive these oligonucleotides from the amino acid sequences I

20 from Upaser of interest. Such probes may be significant

In part shorter than the whole sequence, but should have a long I

In those of at least lye, preferably at least 14 l

In nucleotides. Longer oligonucleotides are also used

right up to the full length of the gene, but preferably not

In 25 me (3 an X length of more than 500, especially not more than 300 I)

In nucleotides. Both RNA and DNA probes can be used. IN

In use, the probes are typically labeled on an I

In a detectable manner (eg with 32P, 35S, 3H, biotin or I

avidin) and incubate them with single-stranded DNA and RNA I

In 30 from the organism in which one is looking for a gene. Man I

demonstrates hybridization by labeling after I

single-stranded and double-stranded I have been separated

I (hybridized) DNA (DNA / RNA) (typically using I

13 DK 175634 B1 nitrocellulose paper). Hybridization methods for use in oligonucleotides are known to those skilled in the art.

Although probes are usually employed with a detectable marker for easy identification, unlabeled oligonucleotides can also be used both as precursors for labeled probes and for use in methods that provide direct detection of double-stranded DNA (or DNA / RNA). Thus, the term "oligonucleotide probe" must refer to both labeled and unlabeled forms.

In a preferred embodiment of the invention, the sequence of Pseudomonas pseudoalcaligen's lipase from the strain CBS 473.85 (M-1) is cloned. Surprisingly, the cloned lipase from Pseudomonas aeruginosa PAO (ATCC 15692) after sequence splitting showed a high degree of sequence homology with the lipase gene sequence from M-1. Even more surprising was finding a similarly high degree of sequence homology between the lipase gene sequence of the M1 strain and chromosomal DNA from a number of Pseudomonas stutzerl isolates [namely PG-I-3 (CBS 137.89), PG-I-4 20 (CBS 138.89), PG-II-11.1 (CBS 139.89), PG-II-11.2 (CBS 140.89)] and Pseudomonas alcallgenes DSM 50342. A "high degree of hybridization" will in this connection be defined as a continuous sequence of DNA having a length of at least 300 bp in which at least 25% homology is found.

Lipase enzymes from P. aeruginosa and P. stutzeri were prepared and tested for their washability by the 'SLM test. Surprisingly, all the enzymes which had a high degree of homology to the M-1 lipase gene were found to also have superior stability, efficiency and performance under conditions reminiscent of a modern washing process. According to Ausubel et al., Current Protocols in Molecular Biology, 1987-1988, the Southern hybrid

I DK 175634 B1 I

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In dissection, prove homologous! up to this level. This one

In found homology could not be observed with P. gladioli, I

As described in EP-A-0205208 and EP-A-0206390 or Humicola I

In the lanqulnosa lipase, described in EP-A-0305216. IN

I 5 You can identify clones with the contents of it

I inserted DNA fragment using a direct el-I

You laugh positive selection method, such as what you are

In E. coli (Kuhn et al. Gene 44 (1986) I)

I 253-263) and for B. subtilis (Gryczan and Dubnau, Gene 20 I

I 10 (1982) 459-469). An example of such a positive selection

In tions vector for E. coli, pUN121 (Nilsson et al., Nu- I

In cleic Acids Res. 11 (1983) 8019-8030). IN

In addition, one can, for example. identify clones, I

In which they express the lipolytic enzymes, using I

In a suitable indicator plate assay, such as agar medium with I

Containing tributyrin or olive oil combined with I

rhodamine B (Kouker and Jaeger, Appl. Env. Microbiol. 53 I

In (1987) 211). In addition, replica cocolo I can be searched

In kidneys using a custom soft I technique

In 20 agar based on the procedure described for detection I

of esterase activity (Hilgerd and Spizizen, J. I

In Bacteriol. 114 (1978) 1184). In addition, one can identify

In ficier clones expressing lipolytic enzymes, by using genetic complementation in an appropriate lipase-senegative recipient strain, such as that described by I. Wohlfarth and Winkler, J. Gen. Microbiol. 134 (1988) I 433-440.

Once a complete gene has been identified either as cDNA or chromosome DNA, it can then be hypothesized in a variety of ways to obtain expression. You can use micro hosts that, for example. may include bacteria, yeasts and fungi such as E. coli, I. Kluvveromyces, Aspergillus, Bacillus and Pseudomonas. For the purposes of this invention, Pseudomonas pseudoalcaligenes are used. Thus, when the gene is to be expressed in a host that recognizes wild type transcriptional and translational regulatory regions 5 of the lipase, the whole gene with its wild type 5 'and 3' regulatory regions can be introduced into an appropriate expression vector. Various expression vectors exist that utilize prokaryotic cells replication systems. See, for example, Pouwels et al., "Cloning Vector, A Laboratory Manual," Elsevier, 1985.

Such replication systems have been developed to provide markers that allow one to select transformants as well as provide convenient intersection sites where the gene can be inserted.

15 When the gene is to be expressed in a host that does not recognize the naturally occurring wild-type transcriptional and translational regulatory regions, further manipulation is needed. In addition, a number of 31 transcriptional regulatory 20 regions are known and must be inserted downstream of the stop codons. The non-coding 5 'region upstream of the structural gene can be removed by endonuclease intersection, Bal31 resection or the like. Alternatively, when an appropriate restriction site is found near the 25 'terminus of the structural gene, the structural gene can be intersected and an adapter used to bind the structural gene to a promoter region, the adapter yielding the lost nucleotides of the structural gene. .

Various strategies can be used to provide an expression cassette having in the 5 '- * 3' transcriptional direction a transcriptional regulatory region and a translational initiation region which also

I DK 175634 B1 I

I 16 I

You can Include regulatory sequences that allow In-

In duction of the regulation; an open reading frame encoding I

I for the lipolytic enzyme, which should preferably include an I

In a sequence of excretion recognized by the desired I

In 5 sheath host cell; and translational and transcription I

In nell termination regions. The expression cassette can i

In addition, include at least one marker gene. Initial I

In the ring and taming regions must work in the host cell · - I

In the county and may be either homologous (derived from the original I

In 10 equal hosts) or heterologous (derived from the original I

Host) or heterologous (derived from a foreign source or I)

In clay from synthetic DNA sequences). The expression box- I

Thus, the substance may be wholly or partially derived from the substance

In turbulent sources and either wholly or partly derived from I

In 15 sources that are homologous to the host cell or are hot

In rologue to the host cell. The various DNA constructs

I sequences (DNA sequences, vectors, plasmids, expression I)

In cassettes) according to the invention are insulated and / or I

You are purified or synthesized and thus are not

In 20 "naturally occurring". IN

In selecting suitable regulated sequences, you should

In considering the following factors that influence expression, I

In consideration. Regarding the regulation of transcription I

In this, the amount and stability of messenger RNA is important

In 25 ge factors affecting expression of gene products- I

I ne. The amount of mRNA is determined by the copy number of the re- I

In levante, gene, the relative efficacy of its promoter I

I and the factors that regulate the promoter, such as

In stronger or repressors. The stability of mRNA is controlled I

In 30 of the susceptibility of this mRNA to ribonuclease enzyme I

In more. Generally, exonuclease digestion is inhibited by I

In the presence of structural processes at the ends of I

In mRNA; palindrome structures, altered nucleotides or I

17 DK 175634 B1 specific nucleotide sequences. It is believed that endonuclease digestion occurs at special recognition sites within mRNA, and stable mRNA is lacking at these sites.

There is also a certain likelihood that mRNA, which can greatly undergo translation, is protected from degradation by the presence of ribosomes on mRNA.

Regarding translation regulation, when mRNA is present, expression can be regulated by affecting the rate of initiation (binding of ribosomes to 10 · mRNA), the rate of extension (translocation of the ribosome across mRNA), the rate of post-ruptured ionic modifications, and the stability of the gene product. The rate of elongation is likely to be affected by codon usage, as using codons for a rare tRNA can reduce the rate of translation. It is believed that the initiation takes place in the region located just upstream of the beginning of the coding sequence.

This prokaryote will in most cases contain a consensus nucleotide sequence AGGA, designated the Shine-Dalgarno sequence. As this sequence is characteristic of the ribosome binding site, it is evident that sequences both upstream and downstream thereof may influence successful initiation.

There is also evidence that within the coding region there are nucleotide sequences that may affect the binding of ribosomes, possibly through the formation of certain structures whereby the ribosome can recognize the initiation site. The location of the AGGA sequence relative to the initiating ATG codon may also affect expression. Thus, it is an interaction of all these factors that determines a particular rate of expression. However, the genes for expression have developed a combination of all these factors to

I DK 175634 B1 I

I I

I 16 I

In reaching a certain rate of expression. If you

You construct an expression system to obtain an I

At the high level of the gene product, one must not merely consider you

In the particular regions intended to influence I

In the 5 expression, but also how these regions (and so- I

In which the sequences therein) affect each other. IN

I Illustrative transcriptional regulatory re- I

In gions or promoters Includes e.g. such se- I

In sequences derived from genes overexpressed in I

In 10 industrial production strains. IN

In the transcriptional regulatory region, you can

In addition, include regulatory sequences that allow- I

In that one can modulate the expression of the structural I

In real genes, e.g. in the presence or absence of I

In 15 nutrients or expression products in growth media I

In one, temperatures etc. For example. can in prokaryotic cells I

In the expression of the structural gene is regulated by I

Using the temperature, using a regulating I

In sequence comprising the bacteriophage λ PL promoter co-I

I 20 but with the bacteriophage λ 0L operator and a temperature I

In sensitive repressor. Regulation of the promoter I is achieved

I by interaction between the repressor and the operator. By I

Of particular interest are expression cassettes that can be exported

In primers a lipolytic enzyme using the regulator I

In the sequences of Bacillus amylase and protease genes. The ten

In the current structural gene, downstream of the ribo-I is introduced

In the binding site so that it is under regulatory control

I of the transcriptional regulatory region and the I

In translational initiation region. IN

In addition, a fused I can be prepared

In gene by providing a 5 'sequence to the structural I

In real genes that encode a leader sequence for secretion I

In relief and a processing signal. If the signal sequence I

! 19 DK 175634 B1 from the lipase gene itself acts in the selected host cell, this can be used. Illustrative heterologous secretory leader sequences include the secretory leader sequences from penicillinase, amylase, protease and yeast α factor. If in the correct reading frame a secretory leader sequence and the structural gene in question are fused, the final lipolytic enzyme can be excreted in the culture medium.

The expression cassette may be included in a replication system for episomal maintenance in a suitable host microorganism or may be provided without a replication system where in this case it can be integrated into the host genome. The way the host microorganism is transformed with the various DNA constructs is not critical to the invention. DNA may be introduced into the host of the prior art, such as by transformation using calcium phosphate, precipitated DNA, conjugation, electroporation, transfection by contact of the cells with a virus, microinjection of 20 DNA into the cells or the like. The host cells may be whole cells or protoplasts.

As a host organism, any microorganism suitable for the production and extraction of a lipolytic enzyme may be used; The host organism 25 is preferably also capable of secreting the enzyme produced, which is why the enzyme can be recovered from cell-free fermentation liquid. Furthermore, the host microorganism is preferably a non-pathogenic organism. Examples of host organisms that meet the above requirements include E. coli, Pseudomonas putida and Bacillus strains, particularly B. subtilis and B. licheniformls, Streptomyces strains and fungi and yeast strains such as Aspergillus and Kluyveromyces. In the invention, Pseudomonas pseudoalcanes are used.

DK 175634 B1 I

20 I

The host strains may be laboratory strains or

may include industrial strains of microorganisms. IN

The special thing about industrial strains is that they are counter-

resistant to genetic exchange such as phage infection

5 tion or transformation. Such strains are stable

and may or may not form spores. They are prototrophic and I

modified to give high yields of endogenous I

protein products such as the enzymes α-amylase and miscellaneous I

proteases. The yield of an endogenous protein product is obtained

10 in an industrial production process can go up to I

at least 5 g / l (0.5% w / v). industrial strains out- I

also separates DNAs, leading to a breakdown I

of DNA in the medium and provides protection against genetic I

exchange. IN

Once the structural gene is introduced into an I

appropriate host, the host cell can be cultured to obtain I

of expression of the structural gene. Production I

the level of lipolytic activity may be comparable- I

equal to or higher than the original strain I

20 mer, from which the genes are derived. One can cultivate

the high density host cells in a suitable medium for I

formation of a nutritious herb. In the event that the promo- I

If the tor is inducible, permissive conditions must be used

eg. changes in temperature, full utilization

25 or excess of a metabolic product or nutrient

ring agent or the like. IN

When excretion is provided, you can

isolating the expression product from the culture medium at I

prior art. One can increase the release of the pro- I

30 induced lipolytic enzyme by diluted I

surfactant solutions. When there is no- I

brought secretion, one can harvest the host cells and I

brighten them by prior art. Then isolate and purify

The desired product is known in the prior art, such as by chromatography, electrophoresis, solvent extraction, phase separation or the like.

The present compositions can be used in a variety of ways. The transformed host microorganisms can be used for enhanced lipase production with characteristics that make it useful in detergent mixtures. The cloned lipase genes may also be used in the search for lipase genes, including identification of a lipolytic gene as a lipase, rather than as an esterase.

They can also be used for enzyme construction of the prior art in conjunction with random or location-directed mutagenesis to obtain lipases with the desired altered characteristics.

The lipolytic enzyme compositions can be used in detergents together with a detergent and optionally other ingredients commonly used in detergents. These ingredients may include at least one of the following: surfactants, water softeners such as complex phosphates, alkali metal silicates and bicarbonates; fillers such as alkali metal sulfate; other enzymes such as proteases, amylases, bleaches, and various components such as fragrances, optical white, etc.

In such enzymatic detergents, lipase activity is preferably in the range of 1-20,000 TLU / g of agent, with the activity of proteolytic enzyme preferably in the range of 50-10,000 Delft units / g of detergent. A 30 TLU (real lipase unit) is defined as a titre-only fatty acid equivalent to the amount of 1 µmol NaOH / min released from olive oil (gum arabic emulsion at pH 8.0, 25 ° C. The delphine unit is defined in J. Amer. Oil Chem. Soc 60 (1983) 1672.

I DK 175634 B1 I

I 22 I

Such detergents can be prepared according to I

In the prior art, e.g. by mixing the components I

In or by preparing a premixture which then I

You are done by mixing the other ingredients. IN

I 5 In one possible manufacturing route, mix one or more I

In re lipase preparations with one or more of the other compounds

In ponents for preparing a concentrate having a formula I

In definite enzymatic activity and this concentrate, you can

You then mix with the other desired components. IN

The lipolytic enzymes of the invention provide I

I is preferably in the form of an enzymatic detergent I

In additives. This additive may also contain one or more

In other enzymes, e.g. eh protease and / or an amylia I

You see that can be used in modern detergents and one or I

In 15 other other components commonly used herein, I

In e.g. a non-ionic, salt, stabilizing agent I

In and / or coating agent. The enzymatic detergent I

In gene additive, in addition to a lipase, it may include a protease and I

Optionally an α-amylase. The proteolytic enzymes are I

Compatible with the lipolytic enzymes in this I

In preparation. The enzymatic detergent additives are usually mixed with one or more detergents and

In the prior art components for obtaining I

In detergents. The enzymatic detergent additive is used

I usually in the range of 102-107 TLU / g additive and I

In any protolytic activity present

You are in the range 5 x 104-106 Delft units / g. IN

In these enzymatic detergent additives, e.g. IN

Available as granules or prills prepared by I

In 30 prior art. See in this connection e.g. GB patents

In Nos. 1,324,116 and 1,362,365 as well as U.S. Patents Nos. I

In 3,519,570, 4,106,991 and 4,242,219. IN

In the enzymatic detergent additive may be present

In liquid form together with an enzyme stabilizer,

23 DK 175634 B1 e.g. propylene glycol. They may also be in the form of organic or inorganic slurries, emulsions or be in encapsulated form immobilized on a soluble or insoluble carrier or in an aqueous or anhydrous solution in the presence of one or more stabilizers. Such additives are preferably used in liquid detergent mixtures.

The following examples are merely illustrative.

Ordinary cloning technique as described in Maniatis et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory, 1982, CSH, New York, was used. All DNA modifying enzymes were obtained from commercial suppliers and used according to suppliers' instructions. Materials and apparatus for DNA purification and separation were used according to the supplier's instructions.

Example 1 Molecular cloning of trlacylglycerol acyl hydrolases A. Source of DNA and selection vector.

EP-A-0218272 discloses several bacterial strains producing lipases suitable for use in 25 detergents. Among these selected were Acinetobacter calcoaceticus Gr V-39 (CBS 460.85), Pseudomonas stutzeri Thai IV 17-1 (CBS 461.85), Pseudomonas pseudoalca ligigenes IN II-5 (CBS 468.85) and Pseudomonas pseudoalca ligene 47 Ml ) as sources of lipolytic genes.

Plasmid vector pUN121 (Nilsson et al., Nucleic Acids Research 11 (1983) 8019) bearing a ampicillin resistance gene, a tetracycline gene, was obtained.

I DK 175634 B1 I

I 24 I

In the clin resistance and the cl repressor gene from bacteriophage λ, I

I from Dr. M. Uhlén, Royal Institute of Technology, De- I

In Partment of Biochemistry, Teknikringen 10, S-10044 I

In Stockholm, Sweden. Transcription of the tetracycline network I

I 5 is prevented by the cl repressor. Insertion of foreign

In DNA at the unique restriction sites (Bell, Smal, Hindlll I

I and EcoRl) lead to activation of the tetracycline network. IN

I This allows direct (positive) selection of recombi- I

In nante transformants on Luria-herb agar plates with ind. I

In 10 teams of 8 µg / ml tetracycline and 50 µg / ml ampicillin. IN

B. Manufacture of gene labs (see Figure 1)

Plasmid and chromosomal DNA were isolated as I

Written by Andreoli, Mol. Gen. Genet. 199 (1985) I

I 15 372-380. Partially chromosomal DNA was digested, isolated I

I from Thai IV 17-1 and M-1, respectively, using I

In Sau3A. This DNA was then ligated using T4 DNA

In gases ligated to Bell digested pUN121 DNA as described in I

In Maniatis et al. (see above) and transformed into com

In 20 petent cells of E. coll strain JM101 hsdS recA I

In (Dagert and Ehrlich, Gene 6 (1979) 23-28). You provide- I

You brought E. coli JM101 recA from Phabagen Collection I

I (deposit number PC2495), Utrecht, Netherlands. You go out

In selected transformants resistant to tetracycline I

In 25 clin at a level of 8 µg / ml, on Luria-herb agar plates. IN

In C. Selection of Transformants I

In the gene library I obtained as described above

I was replicated and searched for lipolytic activity

I know by the following two methods. By the first I

In the process, the replica colonies of Li-I were searched

In polytic activity on peptone agar medium containing I

In the tributary. Lipolytic activity as an I was detected

25 DK 175634 B1 clear zone (glory) around a colony due to degradation of the obscure lipid emulsion. In the second method, replica colonies for esterase activity were searched using a soft agar overlay technique 5. The method was based on the method of Hilgerd and Spizlzen, J. Bacteriol. 114

(1987) 1184. Essentially, a mixture of 0.4% low melting agarose, 0.5 M potassium phosphate (pH

7.5), 0.5 mg / L β-naphthyl acetate dissolved in acetone and 0.5 10 mg / L Fast Blue BB (see Example 2) in addition to transforman. States. Within minutes, colonies with esterase or lipase activity were stained purple.

Of the 1,200 tetracycline-resistant transformants obtained from the Thai IV 17-l / pUN121 gene bank, 15 produced three clones of glory on tributyrin agar plates. Of the 12,000 recombinant transformants tested from the M-1 / pUN121 gene bank, only one clone showed weak lipolytic activity.

The tributyrin positive clones were grown overnight in 2TY medium (16 g / l Bacto tryptone, 10 g / l Bacto yeast extract, 5 g / l sodium chloride, pH 7.0) and analyzed for both plasmid content (see below ID) and the ability to convert various β-naphthyl substrates, a sign of lipolytic activity (see Example 2A).

25 D. Plasmid Insulation D.l Content of Thai IV 17-1 lipase gene

The plasmids isolated from the Thai IV 30 17-l / pUN121 transformants were designated pAT1 and pAT3. Their structure is shown in Figure 2 and Figure 3. A third clone designated pAT2, respectively, contained a plasmid identical to the pAT1 construct. Genet

I DK 175634 B1 I

I 26 I

In coding for the lipolytic activity, I I was located within a 2.7 kb EcoRl fragment of pAT1 (Figure 2, I I dotted line) and within a 3.2 kb EcoRl fragment I I of pAT3 (Figure 3, dotted line). The two EcoRl fragments were subcloned into appropriate vectors both for DNA sequencing and to obtain high yields of lipolytic activity. In cloning the 2.7 kb EcoRl fragment from pAT1 into the I I vector pUN121, the recombinant plasmid generated pET1 I I 10 (Figure 4). A sample of E. coli JM101 hsdS recA containing pET1 was deposited with CBS on February 5, 1987 with deposit number CBS 157.87. I I Cloning of the 3.2 kb EcoRl fragment from pAT3 into I

the vector pUN122 generated the recombinant plasmid pET3 I I 15 (Figure 5). A sample of E. coli JM101 recA containing content I I of plasmid pET3 was deposited with CBS on February 5, I, 1987 with accession number CBS 155.87. IN

D.2 Content of the M-1 lipase gene I I The recombinant plasmid was isolated from the I I M-1 / pUN121 transformant named pAM1 and characterized by

saw it (Figure 6). However, biochemical characterization of the I I lipolytic activity of pAM1 showed that this I plasmid did not encode a desired lipase (see I I 25 also Examples 2 and 3). Therefore, strategies had to be developed, which will be illustrated later. IN

The plasmid pAM1 in E. coli JM101 hsdS recA was deposited with CBS on February 5, 1987 with deposit I-number 154.87.

I DK 175634 B1 I 27 I D.3 Content of IN II-5 lipase gene I Partially cloned Sau3A digested Pseudomonas I pseudoalcalligenes IN II-5 DNA fragments in E.coll K12 I DH1 strain (ATCC 33849) using plasmid I 5 vector pUN121. After ligation and transformation into 1 competent DH1 cells prepared as described by Hana-Han, J. Mol. Biol. 166 (1983) 557-580, approx.

In 1500 transformants resistant to 50 µg / ml am-I picillin and 8 µg / ml tetracycline. Transformants were selected which could hydrolyze tributyrin and β-naphthyl-I acetate as described under 1C. From a positive colony I, plasmid DNA was isolated and characterized by determining several restriction enzyme recognition sites. The mapping of this plasmid named pM6-5 is shown in Figure 15.

The activity of E. coli DH1 (pM6-5) I against β-naphthyl esters was determined (Table 1). A sample of E. coli DH1 I containing plasmid pM6-5 was deposited with CBS on February 5, 1987, with deposit number 153.87.

I 20 I D.4 Content of Gr v-39 Lipase Gene I Partially mixed EcoRI * digested (conditions I according to Gardner et al., DNA 1 (1982) 109-114) Acineto-I bacter calcoaceticus Gr V-39 DNA with EcoRI linearized pUN121 DNA. Following new cyclization by T4 I polynucleotide ligase, the DNA mixture was introduced into I E. coli DH1 (ATCC 33849) by the transformation procedure described earlier in this example. All of the I obtained 1800 tetracycline resistant transformants were searched for lipolytic activity as described under 1C.

Three clones in this Gr v-39 / pUN121 gene library produced a lipolytic enzyme. Plasmid DNA was isolated from one of these clones named pP5-4 and characterized by restriction endonucleases. The formation of this plasmid pP5 ~ 4 can be seen in Figure 8. Then, the hydrolysis of β-naphthyl esters was determined using 5 E. coli DH1 (pP5-4) crude enzyme preparations (Table I1). The plasmid pP5-4 in E. coli DH1 was deposited with CBS on February 5, 1987 with deposit number 151.87.

In Example 2 I 10

Characterization of cloned lipolytic I enzyme preparations.

A. Determination of Lipolytic Activity 15 Tributyrin positive E. coli colonies were inoculated into 100 ml of 2TY medium containing ampicillin and tetracycline in a 500 ml conical flask. E. coli cultures were shaken for 40 hours at 30 ° C in a rotary shaker

at 250 rpm. The strains of Pseudomonas and Acinetobacter H

20 was grown at 30 C. After 40 hours, optical density was measured at 575 nm and centrifuged in a Sorvall RC5B centrifuge in a GSA rotor at 6,000 rpm for 10 minutes, then supernatant stored at 4 ° C until enzyme assay was performed. .

The cells were resuspended in 4 ml of lysis buffer (25% sucrose, 50 mM Tris-HCl pH 7.5). Lysozyme was added and, after 30 minutes incubation at 21 ° C, 20 µg / ml DNase was added and incubation was continued for 30 minutes at 37 ° C. Now 0.1 v / v 30 triton-X100 was added and sonicated cell suspensions on ice using a Labsonic 1510 sonifier set (5 shocks of 30 seconds duration, 100 watts, 1 minute intervals).

Cell debris was then removed by centrifugation in B1

For 15 minutes at 12,000 rpm in a Hettich Micro Rapid / K

In the centrifuge. The supernatant obtained was then analyzed for lipolytic activity. The assay is based on hydrolysis of β-naphthyl esters by lipolytic I · 5 enzymes. Released β-naphthyl reacts with the diazonium salt Fast Blue BB to give an azo dye that you absorb at 540 nm. The process is essentially the same according to McKellar, J. Dairy Res. 53 (1986) 117-127, I and was carried out as follows.

In the tube contained a final volume of 2.0 L ml: 1.8 ml 55 mM TES (N-tris (hydroxymethyl) methyl-2- L aminoethanesulfonic acid, Sigma); 0.02 ml 100 ml of β-I naphthyl ester dissolved in dimethyl sulfoxide (DMSO, Merck)

I or methylcellusolvacetate (Merck), 0.1 ml 120 mM NaTC I

In 15 (Na taurocholate, Sigma), and 0.1 ml of an enzyme preparation. IN

Controls without enzyme and β-naphthyl-I were also used

standards (Sigma) without enzyme and substrate. IN

Corning centrifuge tubes (15 ml) were incubated

O I

containing the reaction mixture at 37 ° C or at 1 ° C

20 specific temperatures for 30 minutes. Now added- I

tea to 0.02 ml of 100 mM FB solution (Fast Blue BB salt I

(Sigma), dissolved in DMSO) and continued incubation for 10 L

minutes, after which the reaction was terminated by addition

addition of 0.2 ml of 0.72 N TCA (trichloroacetic acid, I

25 Riedel-De Haen) and extracted the colored complex I

by vigorously mixing with 2.5 ml of 1-butanol (Merck). Man I

separated layers by centrifugation at 5,000 rpm for 5 L

minutes in a Heraeus Christ minifuge RF, and measured ab- I

the sorbance of the upper layer at 540 nm during application I

30 view of an LKB ultrasound II spectrophotometer. After subtracting control values, readings were converted to TLU (True Lipase Units) using Candida cyllndracea lipase (L1754, Sigma) as standard. A TAU

---------

I DK 175634 B1 I

I 30 I

You are defined as titratable fatty acids which equals I

In the amount of 1 µmol NaOH / min (see also EP-A-0218272). Re- I

The results are shown in Table 1 below

I Comparison of hydrolysis data for β-naphthyl-I

In the 5 esters of varying length of the acyl chain from C ^ C ^ g I

You suggest that: 1. The clones containing pAT3 and pET3 I

In the plasmids, real lipases produce; and 2nd clones- I

In ne containing pAT1, pET1, pAM1, pP5-4 and pM6-5 I

In the plasmids, enzymes with essentially I produce

In 10 esterase activity. Most of the lipolytic enzymes, I

I synthesized by E. coli carrying recombinant I

In plasmids, were found in the cell lysates. IN

DK 175634 B1 31

Table 1

Colormetric Determination of II Polytic Activity (1 TLU. 1-1) 1 Samples from Donor Milk Chemisms and E.coll 5 Transformants.

Host strain Plasmid β-naphthyis substrate 0-NB (4) 0-NC (8) 0-NL (12) 0-NO (18) E. coli K12 -------- sup ly- sup ly- sup ly- sup lysat sat sat sat JM101 hsdS recA pATl 22 80 4 66 1 20 1 10 JM101 hsdS recA pETl 2 66 4 88 1 25 1 8 JM101 hsdS recA pAT3 24 80 4 132 4 42 2 60 JM101 hsdS rSSA pET3 5 90 4 22 1 12 1 54 15 JM101 hsdS recA pAMl 1448141 1 JM101 hsdS rs £ A pUN121 11241211 (control) (vector) DH1 pP5-4 124 6 1 4 16 DH1 pM6-5 124 7 1 15 17 DH1 (control) pUN121 11241211 (vector) pseudomonas Ml ND. 5 8 23 40 14 34 11 20 20 IN II-5 N.D. 50 52 58 66 17 37 7 22 \ THAI IV 17-1 N.D. 23 28 10 60 9 40 31 25

Acinetobacter GR V-39 N.D. 6 10 17 21 15 20 5 12 25 N.D. = not determined.

30 I DK 175634 B1 I 32 B. Further characterization of cloned II polytic I preparations The cloned lipolytic enzyme I preparations were characterized using SDS gel electrophoresis 5 on a Phastgel system (Pharmacia) under a Phastgel I gradient 10-15% according to the manufacturer's instructions. Cell-free extracts from the E. coll clones of pET3 and DH1 strain were compared with the pUN121 vector with partially purified enzyme from the donor strain Thai IV 17-1 (Stuer et al., I 10 J. Bacteriol. 168 (1986) 1070). 1079).

Preparation I

In Man, four volumes of a suitable formula were mixed

In thinning the enzyme preparations with one sample buffer with I

In 15 contents of 10% SDS, 10% β-mercaptoethanol in 0.5 M Tris-I

In HCl, pH 6.8. This solution was divided into three equal I

In large parts. Some received no further treatment, but you

I was stored at room temperature until gel electrophoresis

In travel.

In 20 The second and third portions were heated to

IN

95 C for 5 and 10 minutes respectively, respectively

In ferries they were cooled in ice and stored at room temperature

trip until gel electrophoresis. IN

I Electrophoresis I

In the treated samples, in duplicate experiments were sub-I

I threw electrophoresis on the gelling system at 65 rpm. IN

I A gel was stained for protein with Coomassie Brillant I

In Blue according to Pharmacia Development technique file no I

In 30200. Figure 9A shows the polypeptide patterns after staining I

In with Coomassie Brillant Blue. Another gel was washed

I with 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100 for distillation.

In response to SDS and to reactivate enzyme activity. IN

I The presence of lipolytic activity in the washed I gel was made apparent by soft agar overlay technique I based on the β-naphthyl acetate / Fast Blue BB salt process as described in Example 1C. After incubation for 30 minutes for 5 minutes at 30 ° C, purple bands could be seen against a clear background. As shown in Figure 9B, the lipase from the E. coli pET3 clone and native P. stutzerl Thai IV 17-1 I lipase (molecular weight 40 kDa) had identical mobilities by I SDS gel electrophoresis. Both enzymes show a so-called I10 heat modifiable. A similar heat modifiable

I is described for protein from the outer membrane (OmpA

In gene product) from E.coll K12, Freudl et al., J. Biol.

In Chem. 261 (1986) 11355-11361.

Example 15 I Construction of a Pseudomonas pseudoalcallgenes gene gene I-Blotec in vectors with broad utility in hosts I 20 As an alternative strategy for the cloning of the li-I gene from the P. pseudoalcallgenes M1 strain, a binary cloning system used in a wide variety of hosts that allowed one to search the gene directly by complementing against various human strains of Pseudomonaceae. Two broadly applicable vectors in hosts pLAFR1 (Friedman et al., Gene 1_8 I (1982) 289-296) and pKT248 (Bagdasarian et al., Gene 1_6 (1981) 237-247) were used. Plasmid pLAFR1 is an RK2-derived cosmid with broad utility in hosts that transmit tetracycline resistance and can be mobilized but not transmitted themselves. Plasmid pKT248 is a mobilizable R300B-derived plasmid with broad utility in hosts that confer streptomycin resistance and chloramphenic acid. I DK 175634 B1 I 34

In colresistance. Mobilization of these vectors from E. I

coli for Pseudomonas was performed using plasmid I

In pRK2013 which contains RK2 transfer functions

(Ditta et al., Proc. Natl. Acad. Sci. U.S.A. 77 (1980))

I 5 7347-7351) by the triparenteral coupling procedure I

In, according to Friedman et al. ((Gene lj) (1982) 289-296). Man I

I used a lipase negative mutant 6-1 of P. aeruginosa in strain PAO 2302 (from S. Wohlfarth and UK Winkler, in Ruhr University, Bochum, DE) as Pseudomonas recipient I 10 (J. Gen. Microbiol. 134 (1988) 433-440).

In the preparation of in vitro λ phage packing extracts and the packing of pLAFR1 DNA was performed essentially as described by Ish-Horowicz and Burke I (Nucleic Acids Res. 9 (1981) 2989-2998). Briefly, total P. pseudoalkalcene M-1 DNA sections are cut through.

Either with EcoRI or SalI and ligated to either EcoRI

In cut pLAFR1 DNA or SalI cut pKT248 I DNA. The ratio of inserts to vector was I 5: 1 to reduce the possibility of vector-vector alignment. The ligated M-1 / pLAFR1 DNA was packed in vitro H into λ phage heads and injected into E. coli DH1 (Maniatis H et al., Supra, 1982).

Approximately 2,500 tetracycline resistant I transducers were obtained. pg P. pseudoalcaliqenes M-l. Assuming that P. pseudoalcaligenes have a genome size of 5,000 kb and that at least 50% of the H tetracycline resistant transducers have a 20 kb insert, 2,300 independent clones would be required for to obtain a 99% probability of finding a particular DNA sequence (Clark and Carbon, Cell 9 (1976) 91). Since the gene library contained more than 8,000 In various recombinant colonies, it was likely to contain the entire P. pseudoalcaligen genome.

35 DK 175634 B1

The ligated M-1 / pKT248 DNA was transformed into competent E.coll cells as described in Example 1.

Transformants of E.coll JM101 hsdS reeA were selected for streptomycin resistance (SmP) and counter-selected for chloramphenicol sensitivity (Cms). There were obtained 5,000 SmR Cms clones. The two M-1 gene libraries obtained in E. coli host were replica-plated and scanned for lipolytic activity as described in Example 1. None of the 13,000 recombinant transformants studied showed 10 lipase activity.

Therefore, a mobilization of the clone from E.coll to P. aeruginosa PAO 2302 (6-1) was performed as follows. Recombinant plasmids were transferred to Pseudomonas recipients by replication plating of donor strains 15 to seed of the recipient strain (PA02302 / 6-1) and the helper strain (E. coli MC1061 or DH1 containing the PRK2013 plasmid). After overnight growth of donor recipient and helper strain on a cardiac infusion agar plate, exconjugants were selected by replica plating on ml-20 agar medium containing 0.2% citrate, 10 µg / ml methionine and streptomycin or tetracycline.

Citrate is not metabolized by E. coli. Recovery of the lip phenotype of the lipase-defective mutant 6-1 with recombinant plasmids was tested by replication plating of P. aeruginosa exconjugants on nutrient-rich agar plates containing trioleoylglycerol and the fluorescent dye rhodamine B as described by Kouker and Jaeger. A V. Microbiol. 53 (1987) 211-213.

Four of the 13,000 probed exconjugants showed 30 lipase activity according to the development of orange fluorescence glories, visible at 360 nm, around the bacterial colonies after 40 hours of incubation at 37 C. One of these positive clones, pALM5, was further selected characterization.

I DK 175634 B1 I 36

Finally, enzyme samples were prepared and subjected to

did both biochemical analysis (see Example 2) and SLM

In the test (as described later in Example 10) to ensure that the lipase produced by PAO 2302 / 6-1 exchonant containing pALM5 had the desired characteristic exhibited by the lipase from P pseudoalcalianes in the M1 strain. The results of these experiments indicated that the lipolytic activity of the enzyme produced by the I pALMS clone had similar characteristics to the enzyme 10 obtained from the original M-1 strain.

In Example 4

Molecular cloning of the Pseudomonas pseudoalcaligenes M-1 I 15 II pathogen A. Protein purification and sequencing

The fermentation and preparation of a freeze-dried supernatant of the Pseudomonas pseudoalcaligenes M-1 strain is described in EP-A-0218272. The lipolytic enzyme from this supernatant was purified essentially according to Wingerder et al., Appl. Microbiol. Biotechnol.

27 (1987) 139-145. After purification, the protein preparation was more than 80% pure according to SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant

Blue (see Figure 10).

N-terminal sequence analysis was performed after SDS

gel electrophoresis and electroblotting on Immobilon transfer membrane (Millipore) according to Matsudaira (J. Biol.

Chem. 262 (1987) 10035-10038). This analysis yielded the following sequence (using conventional single-letter amino acid code):

I GLFGSTGYTKTKYPIVLTHGMLGF

I 1 10 20 37 DK 175634 B1 B. Cloning of the M-II IIase gene

Two synthetic 32-mer oligonucleotides were deduced: 5'ACC GGC TAC ACC AAG ACC AAG TAC CCC ATC GT-3 * 5 5'ACC GGC TAC ACC AAG ACC AAG TAC CCG ATC GT-3 'from amino acids 6-15 ( TGYTKTKYPI) in the N-terminal sequence of the mature lipase protein, as described above, after taking into account codon affinity for Pseudomonaceae and degeneration of the genetic code. For use as hybridization probes, the oligonucleotides were end-labeled using T4 polynucleotide kinase.

Chromosome DNA was isolated from the Pseudomonas pseudo-cell lines M-1 strain (CBS 473.85) according to Andreoli (Mol. Gen. Genet. 199 (1985) 372-380), digested with several restriction endonucleases and separated on 0.8% agarose gel. Southern blotting of these gels using radiolabeled 32-mer oligonucleotides as a 20 probe showed the following unique hybridizing DNA bands: 1.8 kb Bell, 2.0 kb PvuII and 1.7 kb SalI. Therefore, M-1 chromosomal DNA was digested separately with these three restriction endonucleases, partitioned by 0.8% agarose gel electrophoresis, and recovered the hybridizing fractions 25 as described above by electroelution in a bio-trap BT 1000 apparatus from Schleicher and Schull.

The 1.8 kb Bell digested fraction was ligated into Bam-HI-cut dephosphorylated vector pTZ18R / 19R (purchased from Pharmacia, Woerden, NL). Furthermore, the 2.0 kb PvuII digested fraction was ligated

I DK 175634 B1 I

I 38 I

I in SmaI-cut dephosphorylated vector pTZ18R / 19R I

I and one ligated the 1.7 kb Sal digest fraction in I

In SalI cut-through dephosphorylated vector pZTI8R / 19R. IN

In All three ligations were transformed into competent E. I

In 5 coll JM101 hsdS recA cells (as described by Andreoli, I

See above) and plated on Luriaurt agar plates with I

Containing ampicillin, X-gal (5-bromo-4-chloro-3-indolyl-I)

In β-D-galactopyronoside) and IPTG (isopropyl-B-D-thioga-I)

In lactoside). IN

In 10 People proclaimed approx. 4 x 103 white colonies on I

In fresh agar plates and searched by colony hybrid I

Seriously to the radiolabelled 32-bit probe, this one knows

In the process, 12 positive colonies were obtained. From I

In each of these positive colonies an I was prepared

In 15 plasmid mini-preparation by alkaline lysis and pre-I

You were dealing with the relevant restriction endonuclease, since I

You followed the supplier's instructions. Six plasmids I

You contained the expected hybridizing inserts. IN

I One of these plasmids containing only 2.0 kb PvuII I

In the 20 fragment, designated ρΤΜΡνίδΑ, was selected for detail

In fair analysis. A sample of E. coli JM101 hsdS recA with I

Containing plasmid pTMPv1SA was deposited with CBS on I

In March 8, 1989 with deposit number CBS 142.89. IN

In Man, they replicated the E. coli pTZ18R / 19R recombinant

In 25 binant transformants obtained as described above and I

You searched for lipolytic activity as described in I

In Example 2. None of the 5 x 104 E. coli I examined

In transformants, lipolytic activity showed. There may be- I

There are several reasons for this failure:

In a) the gene expression initiation signals from M-1 lipase-I

In the genes either E. coli transcript I was not recognized

In the tions translation system (see, e.g., Jeenes et al., I

In Mol. Gen. Genet. 203 (1986) 421-429), b) it is necessary

B1 you with a regulatory sequence or protein to activate this common lipase gene, c) the folding may be incorrect or the secretion of M-1 lipase in E. coli may be insufficient.

5 C. Characterization of sequence splitting of M-1 lipase gene

Plasmid ρΤΜΡνΙΘΑ was digested with a series of restriction enzymes with 6 bp recognition sequences. Ana-10 lysis of the fragment sizes from these experiments allowed one to make a preliminary restriction endonuclease cleavage map over the 2.0 kb PvuII insert of pTMPv18A. This map is shown in Figure 11.

The pTZ18R / 19R vectors used provide a flexible "all-in-one" system and allow DNA cloning, didesoxy xy DNA sequencing, in vitro mutagenesis and in vitro transcription (Mead et al., Protein Engineering I_ (1986) 67 -74). The double-stranded plasmids were converted to single-stranded plasmids by superinfection with a helper phage M13K07, from Pharmacia.

A DNA sequence analysis of a 0.94 kb DNA fragment between XhoI and EcoRV in ρΤΜΡνίβΑ is shown in Figure 12. This DNA sequence, when translated in all 25 possible reading frames, showed a large open reading frame that included the NH2-terminal amino acid residues from lipase protein as determined by direct amino acid sequence division (residues 1-24), see this example under A. Methionine at position -24 is the initiation codon for a preprotein, as this methionine precedes a stretch of amino acids typical of bacterial signal peptides. (see Von Heijne, J. Mol. Biol. 192 (1986) 287-290). This 24-amino acid signal peptide is cleaved off under I 175634 B1

I 40 I

In the distinction following A E A (-3 to -1) signal peptidase gene I

In the place of judgment. It should be noted that the region around I

In the cystine residue in this signal sequence is very similar to a Li-I

In pop protein consensus signal peptide (see Von Heyne, ibid.). IN

I The amino acid sequence predictable from DNA I

The sequence suggests that mature M-1 lipase is a protein I

I with 289 amino acids terminating with a TGA stop codon I

I (see Figure 12). The calculated molecular weight for this is I

that protein is 30,323, which is in strong agreement

In accordance with the molecular weight determined for M-1 lipase I

I (MW 31,500) using SDS-polyacrylamide gel electrophoresis

trip (see Figure 10). IN

In Example 5 I

I 15

In Molecular cloning of Pseudomonas aeruginosa PAO lipase-I

In the gene I

The lipase from Pseudomonas aeruginosa is a lipid I

In 20 hydrolyzing enzyme (EC 3.1.1.3), with a molecular weight I

I in approx. 29,000, which is excreted in the medium during the late I

In exponential growth phase (see Stuer et al., J. I

Bacteriol. 168 (1986) 1070-1074).

I ^ A. Cloning and characterization I

I To clone the lipase gene from pseudomonas aeru-

In ginosa PAO 1 strain (ATCC 15692) a clone I was used

In widespread use system in hosts, whereby one

You could directly crawl the gene bank by complementing I

In 30 against various mutant strains of Pseudomonaceae. The I

I used vector pKT248 with wide use in hosts I

In (Bagdasarian et al., Gene 16 (1981) 237-247), a mobilizable R300B-derived plasmid is widely used in vectors that transmit streptomycin resistance and chloramphenicol resistance.

Pseudomonas aeroqinosa PAO 1 DNA was partially digested with the restriction endonuclease SalI and ligated into the sole SalI site of the pKT248 vector. The insertion-vector ratio was 5: 1 to reduce the possibility of vector-vector ligation. The ligated PAO / pKT248 DNA was transformed into competent E. coli SK1108 cells (Donovan and Kushner, Gene 25 (1983) 39-48) as described in Example 1B. Transformants of E. coli SK1108 were selected for streptomycin resistance (SmR) and counter-selected for chloramphenicol sensitivity (Qns).

There were obtained 6,000 SmRCm clones and tested for lipolytic activity as described in Example 2. None of the clones showed such activity, probably due to the poor recognition of Pseudomonas promoters in E. coli (Jeenes et al., See above).

Therefore, a transfer of the clones from E.coll to the lipase negative mutant 6-1 of Pseudomonas aeruginosa PAO 2302 (Wohlfarth and Winkler, J. Gen. Microbiol. 134 (1988) 433-440) was performed as follows: clones in 120 parts, each consisting of 50 25 clones. From each part, plasmid preparations were obtained and used to transform competent cells of the PAO 2302 (6-1) lipid mutant. Competent Pseudomonas cells were prepared according to Olsen et al., J. Bacteriol. 150 (1982) 60-69, and made the selection on calcium triolein (CT) agar plates (Wohlfarth and Winkler, see above) supplemented with 50 µg / ml streptomycin.

Ten of the 5,000 PAO transformants showed lipase activity, which could be seen by white crystals

I DK 175634 B1 I

I 42 I

In the top of the colonies. One of these positives was chosen

In PAO transformants, pSW1, for further characterization

In the ring. IN

The plasmid therein was characterized by restriction I

In 5 tions analysis followed by electrophoresis on agarose gels, and I

It was found that it contained a Sal insert I

3.1 kb I composed of a 1.3 kb, a 0.97 kb and a 0.76 - 1

In kb Sall sub-fragment. These inserts were un- I

I cloned in appropriate vectors for DNA sequence division I

In 10 and to obtain expression of the lipase gene. IN

Plasmid DNA was isolated from a pUC19 derived I

In subclone, designated pSW103 (containing insertion I

In the 1.3 kb and 0.97 kb pieces), I characterized

In it by restriction endonucleases. An over- I

15 sieves of this plasmid pSW103 are shown in Figure 13. An I

In sample of E. coll JM101 hsdS recA containing plasmid I

In pSW103 was deposited with CBS on March 8, 1989 with de- I

In filing number 141.89. IN

B. Sequence Breakdown and Characterization of Pseudomonas I

In the aeruginosa PAO 1 lipase gene I

In the Man sequence, the 2.3 kb Sall was divided into I

In sentence piece from pSW103 containing PAO 1 lipase-I

In the gene by two methods: both a "forced" cloning I

In 25 sequence splitting and a shot-gun cloning sequence splitting-I

According to Deininger (Anal. Biochem. 129 (1983))

I 216-223) on the Ml3 bacteriophage derivatives mplB and mpl9 I

I (Yanisch-Perron et al. Gene 33 (1985) 103-119) and Di-I

In the desoxy chain termination method of Mizusawa et

For 30 years, Nucleic Acids Res. 14 (1986) 1319-1324. IN

By the "forced" cloning sequence splitting, I

Sall / Pstl and Sall / EcoRI fragments of 2.3 kb I were seen

In the insert from pSW103 and ligated them into a passport

...... "I

DK 175634 B1 43 send M13mpl8 vector. The entire sequence was determined, compiling the collection of obtained DNA sequence fragments. Most of the DNA sequences for both strands have been determined. This DNA sequence, when translated into all possible reading frames, showed that only the 0.7 kb Sall fragment from pSW103 (see Figure 14) encodes complete PAO 1 lipase.

The 1.3 kb SalI fragment from the pSW103 subclone, when translated in all 10 possible reading frames, did not encode for the 24 N-terminal amino acid residues in the PAO 1 lipase protein as determined by direct amino acid sequence division.

The amino acid sequence that could be predicted from the DNA sequence of the 0.97 kb Sall fragment has one interesting domain (denoted by A in Figure 14).

Domain A encodes an amino acid sequence GHSHG already defined as the active center in both eukaryotic lipases (Wion et al. Science 235 (1987) 1638-1641; Bodmer et al., Biochem. Biophys. Acta 20 9Q9 (1987) 237 -244) and prokaryotic lipases (Kugimiya et al., Biochem. Biophys. Res. Commun. 141 (1986) 185-190).

Example 6 A. Expression of cloned M-1 lipase in bacteria

To improve the expression level of the M-1 lipase in heterologous hosts, the 2.0 kb KpnI-HindIII fragment from pTMPv18A carrying the M-l 30 lipase gene was ligated into KpnI and HndIII digested pBHA / Cl vectors. The nucleotide sequence of the pBHA1 vector is disclosed in EP-A-0275598. The pBHCl vector is identical to the pBHA1 vector except for differences in expression of

I DK 175634 B1 I

I 44 I

In the following genes for antibiotic resistance: chlorampheni- I

In the colacetyl transferase gene (Cm) is expressed in E. coli I

In the strain WK6 containing the pBHCl vector, and β-lacta- I

In the mesh gene (Aβ), in E. coli strain WK6 is expressed with I

In 5 contents of the pBHA1 vector. E. coli WC strains are seen be- I

In written by R. Zell and H.J. Fritz, EMBO J. 6 (1987) I

In 1809. I

I After transformation of WK6 and analysis of either I

In the obtained ampicillin-resistant colonies (in the case of I

In 10 pBHCl) or the chloramphenicol resistant co-obtained

In lonies (in the case of pBHCl) the corresponding I was found

In plasmids pBHAM-1 and pBHCM-1, see Figure 15. I

The next step was the introduction of an Ndel restriction

At the tions endonuclease site of the ATG initiation cocoon in M-1 I

In the preprotein. A position-directed mutagenesis I was performed

In the pBHA / CM-1 plasmids as described by Stanssens et al

In al., In "Protein Engineering and Site-Directed I

In Mutagenesis ", 24th Harder Conference (1985), Ed. A. R. I

In Fersht and G. Winter. IN

After performing the mutagenesis, I

In the possible mutants for the relevant mutation at both I

In restriction enzyme analysis and sequence analysis under I

In terms of the didesoxy process of Mizusawa et al., I

See Example 5. Following these analyzes, it was found that I

In 25 straight plasmid designated pBHAM1N1 (see Figure 16). In order that you

In obtaining the regulated expression of M-1 lipase in E. coli I

The 1.7 kb Ndel-HindIII fragment I was isolated

I from pBHAM1N1 containing the M-1 structural lipase gene and I

You ligated it into two expression vectors; IN

I 30 - pTZ18RN, a pTZ18R derivative containing an I

At the unique NdeI site of the ATG initiation codon for β-galac-I

In tosidase (see Mead et al., Protein Engineering 1 (1986) I)

I 67-74); IN

45 DK 175634 B1 - pMCTN, a pMC derivative (see Stanssens et al., See above) containing a unique NdeI site behind a tac promoter and ribosome binding site.

After transformation of the two ligations into competent E.coll JM-101 hsdS recA cells, the obtained lipolytic activity transformants were examined on tributyrin agar plates containing 0.5 mM IPTG (iso-propyl-β-D-thiogalactoside). After incubating these tributyrin plates at 30 ° C for 48 hours, followed by storage of the plates in the cold for several days, certain 10 colonies formed a weak halo, which was a sign of lipolytic activity. Plasmid genes from these tributyrin positive clones were characterized by restriction enzyme analysis. Two plasmids were found: - pTZNIM1 containing the M-1 lipase gene behind the lac 15 control sequences (see Figure 17); and - pMCTM1 containing the M-1 lipase gene behind the tac control sequences (see Figure 18).

In order to identify the lipolytic activity produced by E.coll transformants containing plasmids pTZNIM-1 and pMCTM-1, respectively, these clones were grown overnight in 100 ml of 2 TY medium (16 g / l Bacto tryptone , 10 g / l Bacto yeast extract, 5 g / l NaCl, pH 7.0) at 30 C followed by a 3 hour induction with 0.5 mM IPTG at 30 ° C. E. coli 25 strain JM-101 hsdS recA containing pTZ18RN was used as a negative control. The cells were separated from the culture supernatant by centrifugation and then fractionated into periplasmic and membrane / cytoplasmic components according to Tetsuaki et al., Appl. Environmental Microbiol.

30 So (1985) 298-303. All the fractions were analyzed on SDS-polyacrylamide gels according to Laemmli, Nature 227 (1970) 680-685, which consisted of a 13% separator.

I DK 175634 B1 I

I 46 I

In tions gel and a 5% stacking gel. The gels were run at you

At 60 mA until the bromophenol blue (BPB) marker reached the bottom I

of the gel. The samples were prepared and a test was performed

In dough blot as described in EP-A-0253455. Western blot I

I 5 was analyzed using polyvalent rabbit I

In antisera against purified lipase from p. Pseudoalcalia I

In strain Μ-l. From the results in Figure 19, one can conclude, I

E. coli strains with Contents of pTZN1M1 and pMCTM1 I

Each can synthesize an M-1 lipase specifically I

31.5 kDa polypeptide and secreted this into the periplasm I

I (see Figure 19, lanes B and C). The lipo-I was confirmed

In lytic activity of this 31.5 kDa polypeptide at I

the soft agar overlay technique based on β-naph- I

In thyl acetate / Fast Blue BB salt method, described in Example I

In column 1C. IN

EP-A-0275598 and EP-A-0253455 disclose a prior art

In the method of efficient transfer of a vehicle which I

contains an interesting gene for Bacillus, which you

You bring about Bacillus strains that effectively secrete the I

In 20 desired polypeptide products. This procedure was followed

I used both for Thai IV 17-1 and the M-1 lipase genes. IN

In the case of the M-1 lipase gene, I was digested

In the pBHAMIN1 plasmid (see above) with Ndel and related I

In it. The ligation mixture was transformed into Bacillus I

In 25 licheniformis T9 protoplasts. A series of I was analyzed

In neomycin resistant tributyrin positive colonies and op- I

You reached the correct plasmid. This plasmid was called I

In pBHMINI (see Figure 20) with the M-1 preprotein behind Hpall I

In the promoter from pUBHO (see Zyprian and Matsubara, DNA jj I

In 30 (1986) 219-225). T9 transformants were tested with I

Containing the pBHMINI plasmid for their ability to hybridize

In drolysers β-naphthyl esters after fermentation in indu- I

radial herb by the method described in Example 2. I

47 DK 175634 B1

The results suggest that the lipolytic activity of the enzyme produced by the T9 clones has similar characteristics to the activity of the lipase obtained from the original Pseudomonas pseudoalcaligenes strain M-1.

5 Used for expression in Pseudomonas hosts

one is the basic promoter of the p78 gene of the specific bacteriophage Pf3 of Pseudomonas (see R.G.M. Luiten, "The Filamentous Bacteriophage Pf3: A Molecular Genetic Study", PhD these 1987, Nijmegen Catholic University 10, NL). A synthetic DNA was prepared

fragment coding for this Pf3 promoter:

This fragment was ligated into vector pTZ18R

cleaved with EcoRI and KpnI to obtain plasmid pTZPf31A.

The plasmids pTZPf31A and pMCTM1, both with BamH1 and HindIII, were cut and ligated and transformed into competent E. coli JM-101 hsdS recA cells. The correct plasmid in which the M-1 lipase gene is controlled by the Pf3 promoter could be obtained; this was designated pTZPf3M1.

To obtain expression of the cloned M1 25 lipase gene in pseudomonads, M1 expression cassettes found in plasmids ρΤΜΡνίδΑ, pMCTM1 and pTZPf3M1 were inserted into vectors pKT23l with wide use in hosts (Bagdasarion et al., 23 Gene 16 (1981) ), pLAFR3 (Staskawisz et al., J. Bacteriol. 169 30 (1987 (5789-5794) and pJRD215 (Davison et al., Gene 51 (1987) 275-280). They obtained plasmids with wide use in hosts and with content of the M1 expression cassettes was transferred by the triparental pairing procedure of Friedman et al., Gene 1J3 (1982) I 289-296) to the following Pseudomonas strains: I - the lipase negative mutant 6-1 of p. aeruginosa in strain PAO 2302 (Wohlfarth and Winkler, see above), I 5 - the lipase negative P. putlda strain KT2442 (Zeyer et al., Applied Environmental Microbiol. 50 (1985) I 1409-1413), I - P. pseudoalcallqenes strain M1 (CBS 473.85), I - P. pseudoalcaligenes strain IN II-5 (CBS 468.85).

I 10 As an alternative method of transferring the plasmids with wide use in E. coli hosts

I to Pseudomonas used the method of electr

In tric field-mediated transformation ("electroporation")

I according to manual from Gene Pulser (Bio-root Laboratories). IN

In 15, the obtained Pseudomonas transfor- I was tested

In manners for lipase production after fermentation in media

I based on olive oil as described by Odera et al., I

In J. Ferment. Technol. 64 (1986) 363-371.

Table 2 below shows the lipolytic pro- I

In the ductivity of the various strains. IN

DK 175634 B1 49

Table 2

The lipase product of certain transformed Pseudomonas strains containing the content of the M1 lipase gene I Pseudomonas Breat usable vector LiDase strain containing the M-1 expression productivity cassette from the% * PAO2302 (6-l) incubator (vector) 0 PA02302 (6- 1) ρΤΜΡνΙβΑ 2 PAO2302 (6-1) pMCTMl 40, _ PA02302 (6-1) pTZPf3Ml 60 15 ---------------------------------- vector) 0 KT2442 ρΤΜΡνΙβΑ 1 KT2442 pMCTMl 30 KT2442 pTZPf3M1 40 Kl u. Insert (vector) 100 Ml ρΤΜΡνΙβΑ 340 20 Ml pMCTMl 240 W Ml pTZPf3M1 260 IN II-5 u. ρΤΜΡνΙβΑ 420 IN II-5 pMCTM1 350 IN II-5 pTZPf3M1 270 25 30 * Lipolytic productivity was determined on culture supernatants by the procedure described in Example 2.

I DK 175634 B1 I 50

The lipases produced by these I Pseudomonas transformants were analyzed by SDS gel electrophoresis and I migrated identically to the lipase produced from the original P. pseudoalcaligenes strain I 5 M-1.

It can be seen that the improvement achieved by inserting multiple copies of the M-1 expression cassette into the strains of the P. pseudoalcaligen is 2-4 times compared to the level of lipase produced by the donor strain.

It can further be concluded that the original gene expression initiation signal or promoter of the M-1 lipase gene is active in Pseudomonas pseudoalcaligenes I strains as opposed to PAO 1 and KT2442 strains.

'15 B. In vitro expression of the cloned M-1 lipase gene H In vitro expression of clones containing M-1 lipase was performed using a prokaryotic DNA-directed translation set (Amersham International). This system allows in vitro expression of genes found on a bacterial plasmid, provided that the relevant control signals are present. The following four bacterial plasmids were analyzed: In B.l. Plasmid ρΤΜΡνίβΑ (see Figure 11), which encodes both I 25 M-1 lipase and β-lactamase gene products and provides ampicillin-I lineage resistance (Aβ). Furthermore, ρΤΜΡνίβΑ carries its own H regulatory signals, promoter, Shine-Dalgarno and leader sequence.

B.2. Plasmid pMCTM1 (see Figure 18) carrying the M-1 lipase gene and the chloramphenicol resistance gene (Om). In this construct, the lipase promoter is exchanged with a strong tac promoter.

51 DK 175634 B1 B.3. Plasmid pMCTbliMl also carries the M-1 lipase gene and the chloramphenicol resistance gene. In this construct, the lipase signal sequence was exchanged for the α-amylase signal sequence (see EP-A-0224294). The promoter was the same as 5 in the construct pMCTM1.

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

0.5 µg of DNA was transcribed from said plasmids in vitro. This reaction was performed by adding 0.5 μΐ 10 x TB / 10 x NTP mixture (a mixture of equal volumes 20 x TB and 20 x NTP mixture; 20 x TB contains 800 mM Tris HCl pH 7.5, 120 mM MgCl2 and 40 mM spermidine; 20 x NTP mix contains 10 mM ATP, 10 mM CTP, 10 mM GTP and 10 mM UTP), 0.5 µl 0.1M DTT, 0.5 µl RNasin (40 µl / µl, Promega) and 0.5 µl of T7 RNA polymerase (15 µl / µl, Promega) or 1 µl of E. coli RNA polymerase (1 µl / µl, Boehringer). The reaction mixture was incubated for 1 hour at 39.5 ° C.

The in vitro translation of the RNA transcripts was performed according to the manufacturer's instructions. Immunoprecipitated M-1 lipase as described by van Mourik (J.

Biol. Chem. 260 (1985) 11300-11306) using monoclonal antibodies against M-1 lipase.

As a negative control, pTZ1BRN was used (Figure 18, lanes A and E). Immunoprecipitation of pTMPv1SA (lane B) shows an unprocessed M-1 lipase of 34 kDa. Immunoprecipitation of pMCTM-1 (lane C) shows a non-processed M-1 lipase of 34 kDa and the final M-1 lipase of 31.5 kDa, whereas immunoprecipitation of pMCTbliMl (lane D) shows the final M-1 lipase of 31.5 kDa. In the experiments with in vitro translation, one can conclude-

I DK 175634 B1 I

I 52 I

In tea, the M-1 lipase gene can be expressed in S-30 extract-I

In ter of E.coll cells. Data obtained by in vitro I

In the transcription / translation of ρΤΜΡνίβΑ, the absence supports I

I of lipolytic activity on tributyrin plates (see Example I

In 5 column 4B), there is no machining of M-1 lipase. IN

In Example 7 I

In Molecular Enzyme Search with characterized lipase I

I ^ seqener sum without I

I For further demonstration of the general an- I

In accordance with the invention, the probes were used

I - written in this application to search for lipase genes

In 15 with similar characteristics and derived from others I

In microorganisms. One could hereby select lipase genes, I

In encoding lipases of the same or comparable I

In washability. IN

I As an example, the use of DNA I is described

In 20 inserts from plasmids pET3 and pTMPvlSA as I

In hybridization probes to show that homologous genes from I

Both are found in most of the microorganisms analyzed

In niches. There is no cross hybridization between the two

In lipase genes (lane A and lane B), suggesting that I

In each of these sequences encoding lipase I originate

In from various progenitor genes. IN

In Example 8, it is further shown that this hybrid I

In dicing technique, one can clone homologous li- I

pathogens from other microorganisms. IN

In order to achieve this, chromosome DNA I was isolated

I from the following strains; Pseudomonas pseudoalcallqenes M-l I

I (CBS 473.85), P. pseudoalcallqenes IN II-5 (CBS I

I 468.85), P. alcallqenes (DSM 50342), P. aeruginosa (PAC I

53 DK 175634 B1 IR) (CBS 136.89), P. aeruginosa PAO (ATCC 15692) (Wohlfarth and Winkler, 1988, J. Gen. Microbiol. 134, 433-440). P. stutzerl Thai IV 17-1 (CBS 461.85), P. stutzerl PG-I-3 (CBS 137.89), P. stutzerl PG-I-4 (CBS 5 138.89), P. stutzerl PG-II-11.1 (CBS 139.89), P. stutzerl PG-II-11.2 (CBS 140.89), P. fragi DB1051, P. gladioli (CBS 176.86), Acinetobacter calcoacetlcus Gr-V-39 (CBS 460.85) and Staphylococcus aureus (ATCC 27661), digested 5 µg and analyzed using 10 Southern Blot (Maniatis et al., See above). DNA was transferred to a nitrocellulose filter (Schleicher & Schuell, BA85; 0.45 mM). The filters were prehybridized in 6 x SSC, 5 x Denhardt, 0.5% SDS and 100 µg / ml denatured calf DNA.

After two hours of pre-incubation, 32P was added

labeled inserts from pTMPv18A or pET3, respectively, and performed the hybridization at 55 ° C over 16 hours.

DNA inserts were isolated by digesting pTMPv18A with xhol and EcoRV and digesting pET3 with EcoRI, followed by separation of the fragments by 0.8% agarose gel electrophoresis and recovery of the fragments by the glass-milk process (Gene Clean). The insert in ρΤΜΡνΙΘΑ, a 1 kb XhoI / EcoRV fragment, and the insert in pET3, a 3.2 kb EcoRI fragment, were labeled in vitro with high specific activity (2-5-10 cpm / pg) with a32P ATP at chin translation (Peinberg and Vogelstein, Anal. Biochem. 132, 1983, 6-13). The probe fragments were shown to have an average length of between 300-800 bases.

The filters were then washed twice for 30 minutes at 55 ° C in 6 x SSC, 0.1% SDS and dried at room temperature, then autoradiographed in

I DK 175634 B1 I

I 54 I

For 1-3 days, affecting KODAK X-omat AR film or I

Cronex 4 NIF 100 X-ray film (Dupont) with reinforcing I

In monitors at -70 ° C (Cronex lighting plus GH 220020). IN

The following equation derived from I was used

I 5 by analyzing the influence of various factors I

I on hybrid stability:

I Tm = 81 + 16.6 (log10Ci) + 0.4 (% G + C) -600 / n -1.5% I

poor adaptation (Current protocols in molecular bio- I

logy 1987-1988, published by Ausubel et al.), in which I

I n = the length of the shortest chain in the probe,

I Ci = ionic strength (Μ), I

I G + C = base composition, I

I to determine the homolog! That could be detected in I

In the experiments. Assuming a probe length of I

In 15,300 bases, a homologous gene showing I could be detected

at least 67% homology within a 300 I fragment

In bases or more. In determining percent homo- I

In accommodation, it was assumed that the GC content of Pseudomonas is 65% I

In (Normore, 1973, in Laskin and Lechevalier (ed). Handbook I

In 20 of Microbiology Vol II, CRC press, Inc. Boca Raton. IN

I Fla.) I

In Figure 22A, the hybridization pattern of Sal I shows

In digested chromosome DNA when hybridized with EcoRI I

In the insert from pET3. It can be seen that the majority of I

Twenty-five of the Pseudomonas strains contain homologous genes to I

In that pET3 clone. In P. fragi DB 1051 (lane Μ), I

A. calcoacetlcus Gr-V-39 (lane O) and S. aureus (lane P) I

homologous genes could not be detected. I was observed

moderate hybridization signals in P. alcallqenes (lane I

In E), P. pseudoalcallqenes (lane C and lane D), P. aeruqi- I

In nosa (lane F and lane G), whereas weak hybridis I was seen

In ring in P. gladioli (lane N). You saw a very strong one

In hybridization in P. stutzeri strains (lanes H-L), which I

55 DK 175634 B1 is not surprising since pET3 was originally derived from P. stutzeri ThailV 17-1.

Figure 22B shows the hybridization pattern with the EcoRV / xhol insert of pTMPv18A.

Strong hybridization signals of chromosome DNA can be seen from p. Pseudoalcalgenees (lanes C and D), P. alcaligenes (lane E) and P. stutzeri strains (lanes H-L). There was weaker hybridization in chromosome DNA from P. aeruginosa (lanes F and G) and no hybridization at all with chromosome DNA from P. fraqi DB1051 (lane Μ), P. gladioli (lane N), A. calcoacetlcus Gr-V- 39 (lane 0) or S. aureus (lane P).

Example 8 15

Cloning of homologous IL pathogens from other microorganisms

To demonstrate the utility of the invention, the use of plasmid ΡΤΜΡνΙΘΑ for cloning a homologous gene derived from P. alcaligenes (DSM 50342) is described (see Figure 22B, lane E).

In Figure 22B, Lane E, it can be seen that P. alcaligenes shows a distinct 5.0 kb SalI fragment and an unmistakable 0.5 kb SalI fragment that hybridizes to the probe.

This suggests that P. alcaligenes contains a gene with at least 67% homology within a fragment of 300 base pairs or more, to the XhoI / EcoRV insert of the pTMPv1BA plasmid.

To determine whether the 5.0 kb Sall fragment of P. alcaligenes represents a gene encoding a lipase, Sall fragments were cloned. A SalI gene library was constructed using vector pUC19 and

I DK 175634 B1 I

I 56 I

I transformed in E. coll JM109 (Yanish-Perron et al., I

In Gene 21 (1985) 103-119). Replica file I hybridized

In three from the gene library with the a32P-labeled insert I

Injection piece from ρΤΜΡνίβΑ using condition I

I 5 looks as described in Example 7. Three I could be isolated

In positive clones, all carrying a 5.0 Sal fragment from I

In the library: pCH1, pCH2 and pCH3. 5.0 I was again cloned

In kb Sall, the fragment from pCH1 in the vector pKT248 (Bagda- I

sarian et al., Gene 16 (1981) 237-247) using Sa-I

In 10 l of the site found in the chloramphenicol resistance gene. IN

Streptomycin-resistant chloramphenicol-I was selected

In Sensitive Transformants in E. coli JM109. One of these

In transformants, pCHIO1 containing the expected 5.0 I

In kb Sall insert, was transformed into Pseudo-I

In 15 monas aeruginosa 2302 (6-1) lip- (see Example 5). IN

In Man colonies were grown on NB calcium triolin agar. IN

I ° I

After growth, the plates were placed at 10 ° C and allowed

After several days, see a calcium precipitate around the I

In transformed colonies and not around non-transformed I

20 aerated P. aeruginosa 2302 (6-1) lip- grown I

In similar agar plates without antibiotics. Of which, I

In that, it cloned the 5.0 kb SalI fragment to complement I

Iterate the lip phenotype of P. aeruginosa 2302 (6-1) lip ”I

You and therefore encode an extracellular lipase that can

25 is produced in a suitable host. IN

Based on the DNA hybridization experiments I

You can conclude that this lipase shows homology to I

M-1 lipase and, consequently, to the other lipases, the I

You hybridize with the ρΤΜΡνίβΑ insert which I-

30 is used as a probe. The description of the cloning of P. alca- I

In the lipase of the body, a generally applicable method represents I

In a manner to clone lipase genes exhibiting homologous I

You give to M-1 lipase. IN

DK 175634 B1 57

Example 9

Comparison of the nucleotide sequence of the lipase obtained from Pseudomonas pseudoalcallgens M-1 with other II phases 5

The nucleotide sequence of Pseudomonas pseudoalcaligenes M1 lipase (Figure 12) was compared with the sequence of Pseudomonas fraql (IFO-3458) lipase (Kugimiya et al., Biochem. Biophys. Res. Commun. 141 10 (1986) 185-190), Staphylococcus hyicus lipase (Gotz et al. Nucleic Acids Res. 13, (1985) 1891-1903) and Pseudomonas aeruginosa PA01 lipase (Figure 14). A close homologue was found! of 81% between M-1 lipase and P. aeruginosa PAO1 lipase and a homology of 62% between M-1 lipases and 15 P. fragi (IFO-3458). However, the sequence of this P1q1 lipase is remarkably shorter than the sequence of the other two Pseudomonas lipases. No homology was found between M-1 lipase and Staphylococcus hyicus lipase.

These results support data from Example 7 in which no hybridization could be detected with (chromosome DNA) from Pseudomonas fraql and Staphylococcus aureus when using pTMPv18A as a probe.

The amino acid sequence derived from the nucleotide sequence of P. pseudoalcaligenes M-1 lipase was also compared with other lipases. A total homology between M-1 lipase and P. aeruginosa PAO1 lipase was found (78%) and a homology between M-1 lipases and P. fraql lipase of 48%. again, no homology could be found between the amino acid sequence of M-1 lipase and Staphylococcus hyicus lipase. However, there is a close homology between the four lipases in the region of the essential serine 87 in the final M-1 lipase. Kugimiya et al., (See above) believed.

I DK 175634 B1 I

I 58 I

In that the sequence in this region G-H-S-H ^ -G is the active I

At the center of lipase enzymes. IN

In Example 10 I

I I

In Detergent Compatibility for Homologous Ipases at SLM-I

In the test I

In this example, the performance of the lipase I-illustrated

In 10 zymes produced by P. aeruginosa, P. stutzeri and P. I

In the strains of the alkalis, showing a high degree of DNA sequence

In female homology to the M-1 gene, by a washing process at the I

In modified SLM tests that test compatibility I

In that of such enzymes in modern detergents. IN

The detergents used were a powder detergent

In admixture (ALL base, described in EP-A-0218272) and an I

In liquid detergent formulation (Liquid TIDE, also I-

Written in EP-A-0218272, but without the inactivation of I

In the protease). The lipase enzymes were prepared by:

I cultivate the bacteria by the following procedure:

You cultivated an inoculation culture by growing the bacteria I

I in 100 ml Brain Heart Infusion (BHI) medium in a rat I

I I

running shaker at 30 ° C for 24 hours. Then inoku- I

You were taught a 2 liter laboratory fermenter I

I 25 containing ι, Ό 1 of a medium of the following composition

In terms of composition. IN

59 DK 175634 B1

Composition of the medium

Component Concentration (q / kq) 5 Brain Heart Infusion (BHI) (Difco) 18.50 Yeast Extract (Difco) 16.00

Calcium chloride, 2H 2 O 0.80

Magnesium sulfate, 7H 2 O 3.20

Manganese sulphate, 1H20 0.030 10 Soybean oil 5.0

Dicalium Phosphate 6.40

The fermentation was run at 30 ° C. Sixteen hours after inoculation, soybean oil 15 was added at a rate of 1 g / hour and continued for the remainder of the fermentation. The aeration rate was 60 l / h and the stirring was 700 rpm. The fermentation was run for a total of 24 hours.

After the fermentation, the bacteria were centrifuged. The supernatant was rapidly mixed with 2.5 volumes of acetone at room temperature and stirred for 10 minutes, allowed to settle and filtered through a glass filter with suction. The filter cake was then washed with 70% ace-25 tone, then with 100% acetone and the filter cake dried under vacuum.

The lipase enzyme preparations obtained in this way were then tested with the SLM test procedure. The procedure for the SLM test is described in EP-A-0218272. This procedure was modified as follows:

A volume of 20 μΐ containing 10 mg of olive oil dissolved in acetone (25%) was placed on a 3 x 3 cm polyester sample as a stain

I DK 175634 B1 I

I 60 I

In and air dried at room temperature. They placed an I

In wash solution / consisted of 10 ml of SHW (water with standard I)

In hardness 0.75 mM CaCl2, 0.25 mM MgCl2) or detergent

Gently dissolved in SHW, in a 25 ml Erlenmeyer flask with an I

In 5 plugs and kept under shaking in a water bath at I

At 40 DEG C. the tested detergents were a powder detergent.

In admixture (ALL base) and a liquid formulation (TIDE I

In liquid). The concentration of ALL-base washing solution- I

The gene was 4 g / l (pH 9.5). The concentration of liquid I

In TIDE, 1.5 g / l (pH 7.5). The solutions were prepared

I with 0.1 M Tris / HCl. You started the wash by putting in

In the enzyme preparation and immediately thereafter it stained I

In sample to Erlenmeyer flask and shaking I

I I

for 40 minutes or longer at 40 ° C.

The .15 lipase was 2 TLU / ml. IN

After washing, the sample was cleaned with SHW and I

I I

dried it at 55 C for one hour. The dried specimens

You were washed again in another wash cycle as they were washed

In using new detergent and enzyme solution in 40 l

For 20 minutes. After the second wash cycle, sample I was treated

Stir with 0.01N HCl for 5 minutes, rinse and dry

You at room temperature overnight. They were extracted

In dried specimens under rotation in a glass tube with I

Containing 5 ml of solvent (n-hexane / isopropylal-I)

In carbonic / formic acid: 975: 25: 2.5 (v / v), 1 ml / min). Back in

In residues of triglyceride, diglyceride and formed

In free fatty acids was determined by HPLC. IN

61 GB 175634 B1 HPLC equipment and conditions:

Column: CP Microspher-Si (Chrompack), 100 x 4.6 nun 5 injection system: Wisp (Millipore) 10 μΐ Pump: Model 2150 (LKB) Detection: Refractive index check (Jobin Jvon)

Integrator: SP 4270 (Spectra Physis) Eluent: n-hexane / isopropyl alcohol / formic acid: 975: 25: 2.5 (v / v), 1 ml / min.

Temperature: room temperature.

The retention time for triolein was 1.2 minutes, 15 for 1,3-diglycerides was 2.5 minutes, for 1,2-di-glycerides it was 3.6 minutes and for oleic acid it was 1.6 minutes. The area below the peaks or the height of the peaks was used as a measure for the recovery of triglyceride and fatty acid. The recovery of triglyceride after extraction of an unwashed specimen was set to 100%. The ratio of the refractive index responses between triolein and oleic acid could be set to 1.0 based on peak heights.

The results of these SLM tests are shown in the following tables. These tables show the recovery of triglycerides and the recovery of total lipids. Recovery of total lipids covers triglycerides plus 1,2- and 1,3-diglycerides plus free fatty acids. The difference between the recovery of total lipids and the recovery of triglycerides is a measure of lipase activity and performance. The difference between total lipid recovery and control (without lipase enzyme) is a sign of the removal of the oil stain from the clothing and shows that these enzymes are

DK 175634 B1 I

62 I

stable and efficient under realistic washing conditions- I

looks as they are simulated in the SLM test. IN

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Claims (4)

A transformed host cell of the Pseudomonas pseudoalcalligen, characterized in that it comprises an expression cassette comprising in the transcriptional direction 5 '- + 3': 5 (1) a transcriptional regulatory region and a translation initiation region of the lipase coding gene on pTMPv18A ) that functions in the host cell; (2) a DNA sequence encoding a lipolytic enzyme obtained from a prokaryotic microorganism, wherein this DNA sequence includes the sequence of the lipase-coding gene on pTMPv18A (CBS 142.89), wherein the enzyme is characterized by: (a) having an optimum in the range of 8 to 15.5 measured in a pH state under conditions of TLU determination, and (b) exhibiting lipase activity in an aqueous solution containing a detergent at a concentration of up to 10 g / l solution under washing condition - 20 looks at a temperature of approx. 60 ° C or lower and at a pH value between 7 and 11; and (3) translational and transcriptotic termination regions that function in the host cell, wherein the expression of this DNA sequence is regulated by said transcriptional and translational regions, and wherein the expression cassette further comprises a marker gene. Transformed host cell according to claim 1, characterized in that the DNA sequence comprises a leader sequence in the correct reading frame added to the 5 'end of a gene encoding the lipolytic enzyme. I DK 175634 B1 I I 66 I A process for the preparation of a lipolytic enzyme characterized by: II (a) having a pH optimum in the range of 8 to II 10.5 measured in a pH state under conditions of TLU II 5 determination and II (b) exhibit lipase activity in an aqueous solution II containing a detergent at a concentration II of up to 10 g / l solution under washing conditions II at a temperature of ca. 60 ° C or lower I I 10 and at a pH value between 7 and 11; II wherein the method is characterized in that II comprises II in a nutrient medium culturing a transformed II Pseudomonas paseudoalcaligen host cell comprising a II expression cassette comprising in the transcription direction 5 · - * II 31: II a transcriptional regulatory region and a translational initiation region of the lipase-coding gene on II pTMPv18A (CBS 142.89), which functions in the host cell; a II 20 DNA sequence encoding the lipolytic enzyme obtained II from a prokaryotic microorganism, wherein this DNA sequence includes a sequence of at least 300 bp from the II lipase-encoding gene on pTMPv1SA (CBS 142.89) and translational and transcriptional termination regions that function in the host cell, wherein the expression of this DNA II sequence is regulated by said transcriptional and II translational regions, and wherein the expression cassette II further comprises a marker gene. IN A process for producing a lipolytic enzyme, characterized by comprising in a nutrient medium to grow a transformed micro-host cell 11 according to any one of claims 1 or 2 to 11 to obtain a nutrient-rich herb; and to isolate the lipolytic enzyme from this herb. IN