WO1994006905A1

WO1994006905A1 - Mutated subtilisine-like serin proteases

Info

Publication number: WO1994006905A1
Application number: PCT/EP1993/002492
Authority: WO
Inventors: Andrea SÄTTLER; Detlev Riesner; Susanne Kanka; Karl-Heinz Maurer
Original assignee: Cognis Gesellschaft Für Bio- Und Umwelt-Technologie Gmbh
Priority date: 1992-09-23
Filing date: 1993-09-15
Publication date: 1994-03-31
Also published as: EP0662126A1; JPH08501447A; DE4231726A1

Abstract

In a subtilisine-like resin protease produced by the mutation of the structural gene of a wild strain and the expression of the mutated structural gene into a production strain, the temperature stability of the enzyme is to be raised. This is done by introducing a glutaminic acid radical into the protease in position 194 in the BPN' system and, if desired, introducing proline into position 188.

Description

"Mutated Subtilisin-Type Serine Proteases"

The invention relates to mutations in the structural gene of subtilisin proteins that lead to new enzymes with a changed amino acid sequence.

Subtilisin proteases are widespread in nature and are widely used for numerous technical applications. For example as detergent enzymes. For many of these applications, it is desirable to improve the stability properties of the enzymes in relation to the applications required in each case. A summary of such considerations is given by J. A. Wells and D. A. Estell in TIBS (Trends in Biochemical Sciences), 13, page 291 ff (1988). Another review article that quotes virtually all known sequences and structures of subtilisin-like serine proteases was by R. R. Siezen et al. in Protein Engineering, Vol. 4 (7), pages 719 to 737 (1991). In the area of detergents, the stability against the irreversible inactivation at elevated temperatures plays a role in the proteases. As described in the J.A. Wells et al. However, it is not possible to make reliable theoretical statements about the steps by which the temperature stability can be improved. Wells et al describe the introduction of disulfide bridges, but emphasize that this did not solve the problem. To improve the temperature stability, the person skilled in the art is therefore primarily dependent on the experiment.

International application WO87 / 5050 claims cloned subtilisin mutant genes which are intended to code for subtilisin proteases which are more active at higher temperatures than the corresponding wild types. The international application mentioned makes numerous suggestions for mutations. For example, in position 218, asparagine is said to be replaced by another amino acid. It is also suggested in Item 188 to replace serine with proline. However, the international application gives no indication as to whether this serine can actually be linked to the success of increased temperature stability by proline exchange or one of the many other measures mentioned there. A mutation S188P is therefore no longer claimed in European patent EP 260299 resulting from the international application mentioned.

International application WO89 / 6279 also proposes to improve the properties of subtilisin proteases by exchanging individual amino acids. Changes to approximately 60 amino acids of the molecule containing a total of approximately 275 amino acids are proposed there. One of the suggestions is; to carry out any exchange in position 194. In addition to the applications mentioned, the following applications also make suggestions for changing individual amino acids in subtilisin proteases: EP 130 756, EP 246678, EP 247 667 and EP 251 446.

Against the background of this prior art, it was the object of the invention to find a way to change the amino acid sequence of subtilisin proteases in such a way that the temperature stability is increased.

The invention therefore relates to a subtilisin-like serine protease, produced by mutating the structural gene of a wild strain and expressing the mutated structural gene in a production strain, characterized in that after the BPN 'count in position 194 of the protease, a glutamic acid residue and, if desired a proline residue is present in position 188.

The numbering of the positions to be exchanged in the proteases mutated according to the invention relates in each case to the protease BPN'-. In order to come to this numbering from any other serine protease of the subtilisin type, the person skilled in the art will place the amino acid sequence of this protease under the numbered sequence of the protease BPN 'in such a way that maximum agreement is achieved. In individual cases, this may require omissions or insertions of one or more amino acids. The type of numbering is described in the already cited W089 / 6279. To produce the mutated proteases according to the invention, the person skilled in the art can start from a large number of subtilisin-like serine proteases. For example, from the enzymes mentioned in the overview articles mentioned above or also from mutated enzymes. Common subtilisin-like serine proteases are, for example, Subtilisin BPN ¹ -, Subtilisin Carlsberg or also the Subtilisins from Bacillus lentus. It is clear to the person skilled in the art that in order to generate clones which produce new, mutated enzymes, he does not start from the enzyme itself, but from the structural gene coding therefor and subjects it to mutagenesis and then uses it again in a production strain.

According to one embodiment of the invention, it is particularly preferred to start from those proteases which carry the amino acid .alanine in position 194 (all positions according to BPN 'count), which is then exchanged according to the invention for glutamic acid. According to a further embodiment of the invention, those proteases are assumed which carry the amino acid alanine in 194 and the amino acid serine in 188, an exchange A194E and S188P being carried out. Suitable starting materials for a mutation of this type are, for example, proteases of the Subtilisin Carlsberg type and its variants. Among these variants, a subtilisin Carlsberg variant with a mutation N158S and S161N (BPN 'count) is particularly preferred. The structural gene of such a protease is described in European patent application EP 214435.

According to a further preferred embodiment of the invention, a protease from Bacillus lentus is used. In addition to proteases which are described in WO89 / 6279, a protease which is described in WO91 / 02792, FIG. 29, is also particularly suitable. In the count as it is carried out in WO91 / 02792, amino acid serine in position 182 corresponds to serine 188 after BPN 'counting. The difference here is due to the fact that the protein described therein contains some deletions from BPN '.

According to a particularly preferred embodiment of the invention, protease mutants are produced which, after subtilisin BPN 'count, contain the following amino acid sequence: 188P-189F-190S-191S-192V-193G-194E-195E-196L-197E-198V-199M. It is particularly preferred in the sense of the invention that this sequence is inserted into a protease, as described in WO91 / 02792, between positions 181 and 194.

The new protease mutants according to the invention show improved stability. The half-lives of the autoproteolytic degradation are extended over a wide temperature range, which increases the heat and storage stability at the same time. The prolongation of the half-life is also achieved under non-physiological conditions, for example in the presence of complexing agents. The proteases according to the invention can be produced with the aid of numerous methods. The methods of random in vitro mutagenesis (RIM) and directed mutagenesis are preferred. The modified structural genes produced in this way can be expressed and produced in a conventional manner in known expression systems. For example, please refer to W091 / 02792. For expression in a production strain and for the aforementioned applications W089 / 06279, EP 260299, EP 130756, EP 246678, EP 247647, EP 251 446.

The mutants according to the invention not only show increased stability of the protease when used in detergents and cleaning agents, but they can also be produced on an industrial scale during purification without additional cooling of the process medium.

Examples Example 1 1. Random in vitro mutagenesis

The starting point was the structural gene according to European patent application EP 214 435 and the expression vector pC51 mentioned there. This sequence was first provided with the restriction site Nar1 at position 597 of the gene sequence of pC51 by T after G exchange by directed mutation, which did not result in an amino acid exchange, the new plasmid being called pASl. Position 597 corresponds to the amino acid alanine in position 200 after BPN 'count. Restriction of pASl with Narl and Pstl results in a 142 bp gene fragment which contains the region of the subtilisin sequence which codes for the weak calcium binding site of the molecule. This double-stranded gene fragment was assembled in vitro from synthetic oligonucleotides and referred to as a gene cassette.

T C

Nucleotide sequence: GCT CCA TTC TCC AGC GTC GGA GAA GAG CTT GAA GTC ATG Amino acid sequence: A P F S S V G E E L E V M Position: 187 188 189 190 191 192 193 194 195 196 197 198 199

The gene sequence of the more stable S188P, A194E protease in the region of the mutagenic oligonucleotide III is shown. The nucleotide sequence is given according to the conventions in the 5'-3 'direction of the gene. The original wild-type nucleotides are indicated above the nucleotide sequence for each exchange found. Doped (= contaminated) phosporamidite solutions were used for the oligonucleotide synthesis. The more stable protease is due to a molecule of oligonucleotide III in the upper strand (FIG. 1). Figure 1 shows the representation of the synthetic cassette with division into the oligonucleotides. The mutagenic oligonucleotide III is marked. The amino acid sequence is given in the one-letter code, selected positions are numbered. The original wild-type nucleotide was indicated above or below the exchange at mutated positions outside the oligonucleotide III.

During the synthesis of oligonucleotide molecules III, all four phosphoramidite solutions were combined with the three remaining phosphoramidites contaminated. The concentration of a doping phosphoride in the solution was 2% of the concentration of the wild-type nucleotide. This doping was suitable for introducing an average of 1-2 nucleotide exchanges per synthesized molecule at any position via the oligonucleotide III sequence. All other oligonucleotides (I, II, IV, V, VI) were largely synthesized with wild-type sequences, directed nucleotide exchanges were inserted at a few positions, by which the amino acid sequence was not changed, but suitable restriction sites were inserted. After replication of the cassette, these interfaces served as reporter interfaces in the following cloning steps. Figure 1 shows the nucleotides of the wild type sequence above or below the sequence actually synthesized. The upper and lower strands of the cassette fragment were assembled from oligonucleotides with wild-type sequence and the mutated oligonucleotides and the strands were hybridized. ΔOpmol / oligonucleotide was used for the hybridization. The resulting synthetic fragments were trimmed with Pstl and Narl in order to convert any multimers into the monomers. The synthetic cassette fragments were purified from the free oligonucleotides by gel elution. 0.05 μg of the eluted cassette fragment was cloned into 0.5 μg Pstl, Narl, Smal-cut, dephosphorylated and purified pUC19 vector. The entire ligation product was transformed into Escherichia coli XL-1 and the transformants were amplified in four 250 ml cultures. The plasmids which were 50% replication products of the upper, mutagenic and 50% replication products of the lower wild-type strand were prepared from the amplified Escherichia coli cultures. 40 μg of the plasmid preparation were cut with Xhol, Pstl and Narl and the mutagenic, amplified cassette fragments were separated by gel electrophoresis. Wild-type fragments were cut up by this treatment and are no longer contained in the eluate. 0.08 μg of the eluted mutagenic cassette fragments were cloned into 1.7 μg of the expression vector pASl and transformed into Bacillus subtilis strain DB104 in aliquots of 0.25 μg / batch. This strain is deficient in the genomic and alkaline proteases. It only expresses the plasmid-encoded enzyme variants (Doi et al. (1986), Trends in Biotechnologie 4, pp. 232 - 235). All in all 25,000 clones per batch were generated in this way. The resulting mutant bank was called RIM2.

The stabilized protease variants were increased by temperature gradient gel electrophoresis to thermostability, i. e. increased autoproteolysis stability and increased structural stability, examined, for the screening of the mutant library. The method of W089 / 06279, page 24 can also be used.

The Pstl-Narl fragments of stabilized protease variant genes were cloned into the Escherichia coli vector pUC19 for the purpose of easier sequencing. Fourteen clones are selected from the mutant library, which express proteases with increased temperature stability.

Typically, 2 μg of the plasmid to be sequenced were prepared from the Bacillus clone of the RIM2 bank and cut with Pstl and Narl. The cassette fragment to be cloned is cut out. 0.006 μg of the cassette fragment was ligated into 0.055 μg of the Escherichia coli vector pUC19 and the ligate was transformed into Escherichia coli XL-1. The transformants were amplified and the plasmids of a clone prepared from a 4 ml culture. These plasmids were used for a conventional sequencing reaction in which the entire Pstl-Narl cassette was sequenced. The A194E and the S188P exchange were found. Example 1 shows the nucleotide sequence and the amino acid sequence of the A194E, S188P clone. The A194E exchange is responsible for the increased thermostability.

Example 2

Various methods of directed mutagenesis are available for the introduction of the stabilizing glutamate residue at the corresponding positions of related subtilisin genes and for the introduction of the sequence according to claim 3. A preferred method because of its high efficiency is the cassette mutagenesis described by Wells et al, Gene 34, 315-323, (1985) analogously to Example 1. The gene fragment to be mutated, the ends with restriction sites suitable for cloning, is composed in vitro of synthetic oligonucleotides. In the case of directed mutagenesis, the desired nucleotide exchanges are introduced during the oligonucleotide synthesis. With each exchange in oligonucleotides, which form the upper strand, the corresponding complementary exchanges are also introduced in the lower strand, so that no mismatches occur after hybridization to the double-stranded fragment.

The synthetic cassette is cloned into the target vector cut with the same restriction enzymes via the terminal restriction sites. The cut target vector is separated from the cut wild-type gene fragment by gel elution. For directed mutagenesis for the introduction of the sequence 188P-189F-190S-191S-192V-193G-194E-195E-196L-197E-198V-199M into the gene of the protease according to international application W091 / 02792, the Pstl described in Example 1 can be used. Narl cassette can be used, which covers the entire area of the weak calcium binding site. The following exchanges must be made: Q191S, Y192V, G195E, D197E, I198V, V199M. At the same time, the stabilizing exchange A194E and the S188P exchange are introduced. Since it is directed mutagenesis, the synthetic Pstl-Narl fragment with the introduced mutations can be cloned directly (ie without amplification in Escherichia coli, as necessary for Rando mutagenesis) into a Bacillus vector and into a suitable one Bacillus expression strain can be transformed.

Detection of the temperature stability of protease seminars

1. method

Vertical temperature gradient gel electrophoresis (TGGE) was used. This method is characterized by a temperature gradient which is applied to the gel perpendicular to the separation direction of the sample by means of a thermostatic plate. The 0.8 mm thick 10% polyacrylamide gel (20 cm x 20 cm) was polymerized on gel-yellow film and placed on the thermostatic plate made of Teflon-coated aluminum. The plate was cooled with the help of coolable water thermostats (Julabo) on one side and heated on the other side, so that a linear temperature gradient could develop over the entire width. The sample was placed in a 13 cm long, 0.3 cm wide application bag, which was perpendicular to the course of the temperature gradient. After 90 minutes at an initially uniform temperature of 25 ° C., the temperature gradient was applied (corner temperature selection depending on the application, but not higher than 80 ° C. The corner temperatures for those TGGEs which show the stabilization by the A194E exchange were 50 ° C. and 70 ° C. The separation with an applied temperature gradient was carried out at 350 V and a limited current of 110 mA for 30 minutes. Acetic acid, pH 5.0 was used and 0.1 M CaCl2 in the gel and electrode buffer were also used.

For the direct comparison between two samples (eg wild type and variant) in a vertical TGGE, two application pockets one below the other (distance approx. 7 cm) were used. The protoco formation was demonstrated after electrophoresis by activity and silver staining. The silver staining was carried out according to Blum et al (1987), the activity staining was carried out by adding the gel overnight in 0.2 μg / ml alpha-naphthyl acetate, 0.5 μg / inl Fast-Red, 0.1 M potassium -Phosphate buffer, pH 7.5 was shaken. The esterolytic activity of the native protease conformation is visible as a red color deposit. Due to the autoproteolytic reaction that occurs during the thermal unfolding of the molecules, active samples do not show any denatured conformation. Only the native and active conformation is detectable in the gel up to the autoproteolysis temperature. The structural transition from the native to the denatured conformation can only be demonstrated after inhibition of the proteases by incubation in 1 M phenyl-methyl-sulfonyl-fluoride. The course of the temperature gradient was measured by applying a Pt100 measuring resistor to the thermostatic plate.

The subtilisin Carlsberg variant according to EP 214 435 with an S188P mutation of the enzyme and with an S188P and A194E mutation of the enzyme were compared with one another by the method mentioned. The two mutants can be produced by directed or by random in vitro mutagenesis. The structural genes were cloned into expression vectors by generally known methods and expressed in a protease-free, generally accessible strain. Cultures of the cloned protease-free strain were used and, after a fermentation period of 24 hours, 20 ml of culture supernatant were obtained and concentrated. For the temperature gradient gel electrophoresis, a total of 8 ml of culture supernatant per protease type in Centricon 10 filtration tubes from Amicon was concentrated to 300 μl, mixed with 2 ml of a 0.1 M CaCl2 solution and again with Centricon 10 tubes to 200 μl concentrated. This last step is a dialysis step, which is essential for reducing the salt content of the sample, since only then can it be subjected to geelectrophoresis. The dialysis step was carried out immediately before electrophoresis, since the dialyzed sample is only stable for a few hours. The 300 ul of the dialyzed sample were diluted with 300 ul electrode buffer (see above) and analyzed in the TGGE.

The temperature gradient gel electrophoresis gave the following result:

The autoproteolytic degradation of the detected native conformation started for the subtilisin Carlsberg wild type (variant N158S, S161N) and a protease that only contained the S188P exchange at the same temperature (60.5 ° C), while this degradation temperature for the S188P, A194E mutant was increased by 1.5 ° C. Note: The determination of the absolute degradation temperature only applies to the solution conditions of the TGGE already stated (i.e. pH 5 and 0.1 mM calcium chloride). Table 1 summarizes the degradation temperatures:

Protease type degradation temperature

Subtilisin Carlsberg 60.5 ° C

S188P variant of the wild type 60.5 ° C S188P; A194E variant of the wild type 62.0 ° C

Claims

1. Subtilisin-like serine protease, produced by mutating the structural gene of a wild strain and expressing the mutated structural gene in a production strain, characterized in that after the BPN 'count in position 194 of the protease, a glutamic acid residue and, if desired, in position 188 Proline residue is present.

2. Serine protease according to claim 1, characterized in that it contains the mutation A194E, if desired in combination with the mutation S188P.

3. Serine protease according to claims 1 or 2, characterized in that it contains a structural element 188P-189F-190S-191S-192V-193G-194E-195E-196L-197E-198V-199M.

4. Serine protease according to claims 1 to 3, characterized in that it is identical in the remaining residues with Subtilisin Carlsberg or a variant N158S and S161N from Subtilisin Carlsberg.

5. Serine protease according to one of claims 1 to 3, characterized in that it corresponds in the remaining residues with an alkaline Bacillus lentus protease.