EP0495790A1

EP0495790A1 - Hybrid plasmids for the production of subtilisin carlsberg in bacillus

Info

Publication number: EP0495790A1
Application number: EP19900912849
Authority: EP
Inventors: Harald Berger
Original assignee: Henkel AG and Co KGaA
Current assignee: Henkel AG and Co KGaA
Priority date: 1989-08-25
Filing date: 1990-08-16
Publication date: 1992-07-29
Also published as: WO1991002802A1; DE3928046A1

Abstract

Lorsqu'on utilise des plasmides hybrides pour la production de protéase dans des bacilles, comprenant un plasmide de base caractéristique des bacilles et une séquence d'ADN réalisant le codage pour une protéase du type subtilisine Carlsberg, le pouvoir d'expression doit être accru. Ce résultat est obtenu grâce au fait qu'on utilise comme séquence de protéase le fragment d'ADN situé entre les interfaces AvaI et SstI les plus proches du gène structural de protéase et réalisant le codage pour la subtilisine Carlsberg.When hybrid plasmids are used for the production of protease in bacilli, comprising a basic plasmid characteristic of bacilli and a DNA sequence coding for a subtilisin Carlsberg type protease, the power of expression must be increased. This result is obtained by using the DNA fragment located between the AvaI and SstI interfaces closest to the protease structural gene and carrying out the coding for the subtilisin Carlsberg as protease sequence.

Description

"Hybrid plasmids for the production of subtilisin Carlsberg in Bacillus"

The invention relates to new hybrid plasmids which make it possible to express the alkaline protease subtilisin Carlsberg and related proteolytic enzymes in increased amounts in Bacillus.

Numerous proteases are known which are formed by microorganisms, in particular bacteria and fungi, and are removed from the cell by active transport, so that they collect in the culture medium. Numerous enzymes of this type are already being used industrially in processes that involve the degradation of proteins. Important applications are, for example, detergents and cleaning agents or also animal foods.

Because of the technical importance of the enzymes, which is slightly higher for the protease subtilisin Carlsberg than for the protease subtilisin BPN ¹ , there have been numerous attempts in the past to find strains which produce one or the other enzyme and which are suitable for the technical extraction of the products. For example, reference is made to the following German ones Laid-open or patent documents: DE 1940488, DE 20 18451, DE 2044 161, DE 21 01 803, DE 21 21 397 and DE 29 25427 as well as on GB 1 263 765, US 3 623 957, US 4264 738 and EP-A 6638. The applications and patents mentioned are classic mutation processes in which the repeated repetition of mutation and selection steps ultimately leads to production strains that are optimized with regard to yield or product quality. It is known to the person skilled in the art that such a mutation process takes place statistically, so that it is hardly to be expected that tailor-made strains are produced which have the objectively highest possible level of performance. In addition, breeding strains of this type can also tend to reverse mutations, ie they lose their favorable properties, at least in part and poach.

German patent application DE 35 27 913 describes hybrid plasmids for microorganisms of the genus Bacillus which contain a double-stranded DNA which has the following structure: promoter which is recognized by the RNA polymerase of microorganisms of the genus Bacillus, ribosome binding site , Start codon, structural gene which codes for subtilisin Carlsberg or its parts with proteolytic activity or variants with proteolytic activity including their leader sequences, stop codon. Such a construct, in which the inserted DNA sequence originates from Bacillus licheniformis and codes for subtilisin Carlsberg and the remaining plasmid part originates from pBC16, is called plasmid pC50.

German patent application P 38 21 491 describes that plasmids which produce an even higher protease expression can be obtained by dividing the piece of DNA which is in pC50 between two relatively adjacent Aval sites and a part of the promoter is removed by cutting with Aval and ligating.

Against the background of this prior art, it is the object of the invention to provide plasmids which bring about an even higher protease expression.

The invention therefore relates to hybrid plasmids for generation. of protease in Bacillus, consisting of a Bacillus-common basic plasmid and a DNA sequence which codes for a protease of the Subti¬ lisin Carlsberg type, characterized in that a DNA sequence is inserted as protease sequence which is for a protease of the Subtilisin Carlsberg type is coded and upstream is limited by the Aval interface closest to the structure, downstream by the closest neighboring Sstl interface.

In any case, the cut between the SstI interface and the Aval interface closest to this interface is preferred in an output plasmid such as pC50. A particularly preferred starting material is a plasmid pC50 according to DE 35 27 913. Here it applies that if the Aval interface which is closest in relation to the reading direction before the protease gene and at the base pair 1 cuts, the restriction endonuclease SstI cuts at base pair 1539 behind the protease gene or behind its transcriptional terminator.

To generate the plasmids according to the invention, the starting point is plasmids which contain the structural gene from Subtilisin Carlsberg or an enzyme similar to Subtilisin Carlsberg.

An enzyme similar to Subtilisin Carlsberg is understood here to mean those enzymes which differ from Subtilisin Carlsberg in up to 1% of the amino acids and which also have protease activity in a comparable manner. have their circumference, the structural genes having to have the interfaces mentioned.

The preferred starting material is a plasmid pC50, produced from a pBC16 portion and the structural gene of Subtilisin Carlsberg from Bacillus licheniformis DSM641 or from Bacillus licheniformis DSM 5440. The corresponding starting plasmid is called pC50.

In order to have several additional restriction enzyme cleavage sites available for recloning, the Aval / SstI fragment from pC50 (FIG. 1) comprising subtilisin Carlsberg protease genes was cloned into the multiple cloning site of the E. coli vector pUC19 (1).

For this pC50 was cleaved with Aval and SstI and pUC19 (Figure 2) with Xmal and SstI. In order to prevent the cloning of the vector part from pC50 into pUC19, pC50 is additionally cleaved with PvuII, which leaves the Aval / SstI fragment carrying the protease gene part intact, but cuts the pBC16 vector part twice. After the incubation, the enzymes were extracted twice with half the volume of phenol, based on the DNA solution, then mixed in a quantitative ratio of 1: 1, the mixture was extracted 5 times with the same volume of ether and then with twice the volume of ethanol 20 Incubated for minutes at -20 ° C and the DNA pelleted by centrifugation. After drying, the DNA was dissolved in a suitable buffer. The DNA concentration was adjusted to 20 μg / ml and T4 DNA ligase was added in a concentration of 1 U / μg of the DNA used. The ligase reaction was carried out at 6 ° C. for about 18 h.

1 μg of the DNA fragments linked by means of T4 DNA ligase were then used to transform competent cells (2) from Escherichia coli JM103 (3). Selection was made on LB medium (Luria-Broth) with 1% agar with 100 μg / ml ampicillin. In Schnei1 lysates, the correct clone was selected on the basis of the correct cleavage pattern and designated as pH9 (FIG. 3) by cleavage with the restriction enzyme PstI, which cleaves in the insert and in the pUC19 vector part in the multiple cloning region.

The DNA was analyzed by the SDS-NaCl method by Guerry et al. (4), with subsequent cesium chloride density gradient centrifugation.

The bacillus vectors pUBUO (5; FIG. 4) and pBC16 (6; FIG. 5) were used as vectors for the cloning of the Aval / Sstl protease gene fragment. These two plasmids are largely homologous and differ essentially only in the resistance determinant (kanamycin for pUBUO and tetracycline for pBC16). For the purpose of cloning, both pH9 and pUBUO and pBC16 were cleaved with BamHI and EcoRI, the latter being only incompletely EcoRI cleavage, since two EcoRI cleavage sites are present in pBC16, of which the cleavage site is in position 1 how the corresponding EcoRI site in pUBUO should be used for the insertion of the EcoRI / BamHI-P300 protease fragment. BamHI and EcoRI sites in pH9 originate from the multiple cloning site of pUC19. Even in the case of pH9, the EcoRI cleavage must be carried out incompletely, since an additional EcoRI cleavage site exists in the protease gene.

For the ligation, pH9 to pUBUO or pBC16 in a quantitative ratio of 2: 1 was used. The total DNA concentration was 200 μg / ml.

In both cases, the transformation was carried out in Bacillus subtilis SB202 (7).

The transformants were placed on full medium agar plates with 15 μg / ml tetracycline for ligation with pBC16 as vector, and 10 μg / ml Kanamycin selected for ligation with pUBUO as vector. For the selection of protease-producing clones, an immunological method was used which is based on a method by Broo e and Gilbert (8, 9). This method was already used in the original cloning of the subtilisin Carlsberg protease gene in the published patent application DE 3527 913 AI.

Immunologically positive clones were identified by means of restriction analysis of rapid lysates and the pBC16 derivative was designated as pC51 (FIG. 6) and the pUBHO derivative as pH70 (FIG. 7).

B e i s p i e l e

Example 1: Expression of subtilisin Carlsberg protease in Bacillus subtilis

Bacillus subtilis SB202 pC51 and Bacillus subtilis SB202 pH70 were compared at 37 ° C to Bacillus subtilis SB202 in shake flasks in a complex medium suitable for protease production (9.1 g / 1 KH ₂ P0, 11.8 g / 1 K ₂ HP0 ₄ 9/1 gSθ4x7H2θ, 0.5 g / 1 MnSθ4x2H 0, 0.2 g / 1 CaCl2 H2θ, 3 g / 1 soy flour, 18 g / 1 casein acc. To Hammarsten,

180 g / 1 amylase-treated potato starch). Samples were taken at various times to determine the protease activity. The protease activity was measured using a Cenco auto analyzer (company Skalar). The enzyme-containing solution is incubated with the substrate dimethyl casein. Proteolysis produces primary amino groups which react with 2,4,6-trinitrobenzenesulfonic acid to form a stable yellow complex. The color measurement takes place in a continuous flow at 420 nm.

Strain relative protease activity

Bacillus subtilis SB202 1

Bacillus subtilis SB202pC51 ca.2

Bacillus subtilis SB202pH70 (DSM 5479) 60-80

Example 2: Transformation of Bacillus licheniformis DSM 5440

From the strains Bacillus subtilis SB202pC51 and Bacillus subtilis SB202pH70 (DSM 5479), plasmid DNA was processed (10) and protoplasts from Bacillus licheniformis DSM5440 were hereby trans- formed. The transformants were selected on DM3 agar (11) with 15 μg / ml tetracycline for the selection of pC51 and on mannitol agar (12) with 10 μg / ml neoycin for the selection of pH 70.

Colonies that were grown were inoculated onto calcium caseinate agar (Merck) with 15 μg / ml tetracycline (pC51) or 10 μg / ml kanamycin (pH70). Transformants which, after growth, had a lysis yard larger than E312 on these plates were checked for their agreement with the plasmid DNAs used for the transformation after restriction enzyme analysis of rapid lysates.

Example 3: Expression of pC51 / pH70 in Bacillus lichenifoππis DSM 5440

Bacillus licheπiformus DSM5440, which contains the plasmids pC50, pC51 or pH70, was shaken in a flask in a complex medium suitable for protease production (2.4 g / 1 H2PO, 1 g / 1 MgSθ4x7H2θ, 0.5 g / 1 MnSθ4x2H2θ, 0, 2 g / 1 CaCl2x2H ₂ 0.3 g / 1 soy flour, 12 g / 1 casein acc. To Hammarsten, 120 g / 1 amylase-treated potato starch) at 39 ° C and 34 ° C. Samples were taken at various times to determine protease activity. The protease measurements were carried out using N-CBZ-valine-p-nitrophenyl ester as the substrate, the substrate being cleaved into N-CBZ amino acid and p-nitrophenol ^* . The formation of p-nitrophenol was measured as a measure of the activity of the protease at 340 nm in the photometer (13). Strain Relative Protease Activity (%)

39 ° C 34 ° C

DSM 5440 with pC50 80 - 100% 120 - 130%

DSM 5440 with pC51 130% 160% accession number DSM 5478

DSM 5440 with pH70 100 - 120% 135%

* The data refer to DSM 5440 at 39 ° C.

At 34 ° C there is obviously a better protease yield with the recombinant plasmids pC50, pC51 and pH70; which is presumably due to the better oxygen supply to the strains with slower growth and greater stability of the plasmids.

Methods

Unless otherwise stated in the text, the manual by Maniatis, T., Fritsch, E.F. &

Sambrook, J., Molecular Cloning, A Laboratory Manual (1982):

* Phenolization of DNA

* DNA ethanol precipitation

* Ligation of DNA literature

1. Yanisch-Perron, C, Vieira, J. & Messing, J., Gene 33: 103 (1985)

Commercially available through e.g. Biolabs New England

2. Mandel, M. & Higa, A., J. Mol. Biol. 53: 154 (1970)

3. Messing, J., Crea, J.R. & Seeburg, P.H., Nucleic Acids Res. 9: 309 (1981)

4. Guerry, P., LeBlanc, D.J. & Falkow, S., J. Bacteriol. 116: 1064 (1973)

5. Bacillus Genetic Stock Center No. 1E6

6. Bacillus Genetic Stock Center No. 1E9 / DSM 402

7. Copeland, J.C. Marmur, J. Bacteriol. Rev. 32: 302-312 (1968)

8. Broome, S. & Gilbert, W., Proc. Natl. Acad. Sc. U.S.A. 75: 2746 (1978)

9. Buckel, P. & Zehelein, E. _f Gene 16: 149 (1981)

10. Godson, G.N. & Vapnek, D., Biochim. Biophys. Acta 299: 516 (1973)

11. Chang, S. & Cohen, S.N., Molec. Gene. Genet. 168: 111 (1979)

12. Bourne, N., Dancer, B.N., J. Gen. Microbio. 132: 251 (1986)

13 Dupaix, A., Bechet, J.-J. & Roncons, C, Biochem. Biophys. Res. Commun. 41: 464 (1979)

Claims

Patent claims

1. Hybrid plasmids for the production of protease in Bacillus, consisting of a Bacillus-common basic plasmid and a DNA sequence which codes for a protease of the subtilisin Carlsberg type, characterized in that a DNA sequence is inserted as the protease sequence encoded for a protease of the Subtilisin Carls¬ berg type and upstream is limited by the Aval interface closest to the structural gene, downstream by the closest neighboring SstI interface.

2. Hybrid plamide according to claim 1, characterized in that the basic plasmid is the Bacillus plasmid pBC 16 (plasmid pC51).

3. Hybrid plasmid according to claim 1, characterized in that the basic plasmid is the Bacillus plasmid pUBUO (plasmid pH70).

4. Transformed _. Strains of the genus Bacillus subtilis and / or licheniformis contain plasmids according to claims 1 to 3.

5. Transformed strains Bacillus subtilis SB202 pH 70 (DSM 5479) and Bacillus licheniformis pC 51 (DSM 5478).