FR2676456A1 - Thermostable variants of Bacillus licheniformis alpha -amylase, process for preparing them and their use - Google Patents

Abstract

The invention relates to a thermostable variant of Bacillus licheniformis alpha -amylase, characterised in that, compared with the amino acid sequence of the natural form, its sequence contains, in the vicinity of histidine (His) in position 133, at least one substitution by an amino acid more hydrophobic than His, and/or, in the vicinity of alanine (Ala) in position 209, at least one substitution by an amino acid more hydrophobic than Ala. It also relates to a gene encoding such variants, an expression vector and the host cell for this vector, as well as a process for preparing these variants. It relates finally to the use of these variants in industry.

Description

 The subject of the invention is new thermostable alpha-amylases, their preparation process and their use in the textile, paper and brewery industries.

 The generic term "amylase" denotes any enzymatic activity capable of hydrolyzing all or part of the alpha 1-4 or alpha 1-6 bonds of the starch.

 In industrial practice, a so-called liquefying enzyme alpha-amylase is used to hydrolyze the starch after it has been converted into starch by heating to a temperature above 95 ° C. This type of enzymatic activity hydrolyzes the alpha bonds -amylase 1,4 glycosides, characteristics of amylose and amylopectin The use of a thermostable alpha-amylase is of immediate interest in that it avoids having to cool the starch before the On the other hand, the liquefaction operation can be carried out only after the starching or gelling of the native starch, after it has been stripped of its soluble constituents by soaking. performs at temperatures of the order of 100 to 115 ° C. It is therefore desirable to have an amylase which has enhanced thermoresistance characteristics, in particular to be able to perform simultaneously or at similar temperatures the two operations of setting and liquefaction.

 In addition, an increase in the reaction temperature makes it possible to increase the enzymatic kinetics and to reduce the viscosity of the mixture.

The Danish company Novo markets, under the name
Termamyl, a heat-stable alpha-amylase produced by fermentation of a strain of Bacillus licheniformis.

This enzyme is secreted in the culture medium. In the absence of any protector (Ca ++ ion and starch), it retains 90% of its activity after 10 minutes at 80 ° C. In the presence of 0.1 M Ca ions, it is 100% stable at 90 ° C. C for 15 minutes (Rosendal P., Nielsen BH,
Lange NK (1972) Stability of bacterial alpha-amylase in the starch liquefaction process. Die Starie 31, 368-372).

 On the other hand, the French patent publications FR-A-2,533,583 and FR-A-2,537,602 describe the cloning of the Bacillus licheniformis gene encoding heat-stable alpha-amylase and its expression by Escherichia coli and Bacillus subtilis. , the gene being localized on a multicopy plasmid. Synthesized by these two microorganisms, aphaamylase retains all its unchanged properties: molecular weight, thermostability, specific activity as a function of pH, immunological properties. The gene was sequenced and its sequence, as well as the corresponding amino acid sequence, are shown in Figure 1.

 The inventors have now developed variants of alpha-amylase, the sequence of which is shown in FIG. 1, for which the interesting properties of Bacillus licheniformis alpha-amylase are maintained and which, in addition, have the natural form, increased thermostability characteristics.

 The invention therefore firstly relates to a thermostable variant of Bacillus licheniformis alpha-amylase, characterized in that, with respect to the amino acid sequence shown in FIG. histidine (His) at position 133 at least one substitution by an amino acid more hydrophobic than His, and / or in the vicinity of Alanine (Ala) at position 209 at least one substitution with a more hydrophobic amino acid than Ala. The term "in the vicinity of" designates both the His or Ala positions themselves and some amino acids located on either side of these positions.

 The sequence shown in FIG. 1 corresponds to the mature sequence of alpha-amylase, that is to say after post-translational cleavage of the signal peptide.

With regard to substitution in the vicinity of position 133, the variant

<tb><SEP> 133 <SEP> 133
<tb> His <SEP>-> Tire <SEP>
<tb> is especially preferred. It is also necessary to mention the variants for which His is replaced by an amino acid selected from Phe, Leu, Tyr, Glu, Lys, Gln.

 The invention is based on the discovery that the region located in particular around the amino acid 133 is an important region, if not essential for the thermostability of the protein.

 Another important region for the stability of alpha-amylase is located near the 209 position of the mature protein. In particular, one or more amino acids naturally present in position 209 or in its neighborhood may be substituted by an amino acid chosen in particular from Val, Leu, Ile, to obtain a thermostable alpha-amylase variant.

For this position, a mutation particularly preferred
209 is that for which Ala is substituted by Val209.

 The alpha-amylase variants according to the invention may contain a mutation in only one of the two regions mentioned, or in both regions at the same time.

 Such doubly mutated variants have increased heat stability relative to each of the variant amylases and a half life at 90 ° C increased by a factor of 9 to 10 over the natural form.

 The variants according to the invention can be prepared by genetic engineering techniques, from a structural gene modified with respect to the natural gene. To this end, standard mutagenesis techniques are used and an oligonucleotide capable of hybridizing to the cDNA in the vicinity of position 133 is prepared.

 The Applicant has also developed a random mutagenesis strategy, from a thermosensitive variant called p841ts (having a double mutation at positions 164 and 165).

The subject of the invention is therefore also a gene encoding a thermostable variant of the alpha-amylase according to the invention. In a preferred embodiment, the subject of the invention is a gene which comprises the DNA sequence as shown in FIG. 1, except that the codon encoding His is replaced by a codon encoding Tyr and / or the codon. coding for Ala209 is replaced by a codon coding for Val209
The sequence of Figure 1 is not repeated in the body of the description to not weigh it down, but it is an integral part.

 The subject of the invention is also a vector for the expression of a thermostable variant of apha-amylase according to the invention in a host cell, that is to say a vector which contains a gene coding for alphaamylase such as described above as well as the elements ensuring its expression in said host cell.

 Different host cells may be envisaged within the scope of the invention and the elements ensuring the expression are chosen in relation to the host cell. There may be mentioned more particularly bacteria, preferably bacteria such as Bacillus licheniformis, Bacillus subtilis or E. coli.

 According to an advantageous characteristic of the invention, for the expression in Bacillus subtilis, the elements ensuring the expression of the gene coding for the variant of alpha-amylase contain the promoter-SacR regulatory region located upstream of the levan sucrase gene. of Bacillus subtilis. Such a construction is described in the patent publication FR-A-2,582,316. Thanks to this type of construction, the alpha-amylase is secreted in the external medium under conditions in which it is not naturally present. that is, during the exponential phase of growth.

 More generally, the regulatory region of the gene coding for Bacillus licheniformis alpha-amylase can be used to express the gene encoding the variant according to the invention in any host system.

 The subject of the invention is also the host cells transformed with an expression vector according to the invention as well as a process for preparing the variants in accordance with the invention, according to which host cells are cultured as defined above under conditions appropriate culture and the resulting variant is recovered.

 Finally, the subject of the invention is the use of the variants according to the invention in various textile, paper and brewery industries, in particular for the starch etching and liquefaction.

Other features and advantages of the invention will appear on reading the following detailed description, in relation to FIGS. 1 to 6 which show
FIG. 1: the DNA sequence of a structural gene of the thermostable apha-amylase of Bacillus licheniformis as well as the corresponding amino acid sequence.

 - Figure 2: the structure of the plasmid pINA90l.

 FIG. 3: in the form of a curve, the comparative evolution of the thermostability of the His133 alpha-amylase and of the Tyr133 variant as a function of the incubation time at 90 ° C. in the presence of 0.1 mM of Cas12.

 - Figure 4: the same curve as Figure 3 with 1 mM of poop ,.

 - Figure 5: the same curve as Figure 3 with 10 mM of poop ,.

 FIG. 6: a diagram representing the thermostability of different variants at position 133 at variable temperatures.

FIG. 7: Thermal inactivation diagram of B. licheniformis alpha-amylase relative to p841ts and p841ts variants
Val209, at 90 ° C, pH6, 1 mM CaCl2.

Figure 8: Thermal inactivation diagram of B. licheniformis alpha-amylase and alpha-amylase variants at 90 ° C and pH6, 1 mM CaCl2.
EXAMPLE 1 Preparation of the Tyr Variant of Bacillus licheniformis heat-stable alpha-amylase

1) gene preparation
An oligonucleotide of 25 nucleotides with the following sequence is synthesized: 5 'TCA GGA CTA GAA CTA ATT AAA GCC CT 3', capable of hybridizing to the DNA of the structural gene whose sequence is shown in FIG. 1, and of introduce there after using conventional techniques of site-directed mutagenesis, the TAC codon corresponding to the amino acid Tyr instead of the CAC codon corresponding to the amino acid His in position 133.

 In order to ensure that no modification other than the desired mutation has occurred, a fragment of the structural gene generated by the restriction enzymes Sst II and Cla I flanking the codon corresponding to amino acid 133 is isolated. determines its nucleotide sequence and is then introduced into a natural gene treated with the same enzymes Sst II and Cla I, in order to reconstitute a whole gene.

 Thanks to the gene thus reconstituted, it is certain that only the modification performed on position 133 is present and confers on the new enzyme its properties.

2) Preparation of the expression vector
The plasmid PINTA901, the structure of which is shown in FIG. 2, is prepared. It is a low copy number vector (10 to 40) having several unique restriction sites in the alpha-amylase gene for ease of use. subcloning the mutated fragments. This plasmid pINA901 allows the expression of alpha-amylase genes at a moderate level, which is nontoxic for the cells but high enough for the characterization of the variants. It carries the ColEl origin of replication (ORI) of a low copy number plasmid derived from pJRD158 (Davison et al., 1984). Unlike pBR322, the ampicillin resistance gene (Ap) does not contain a PstI site. The deletion of a fragment
Xhol-SalI also eliminated the SalI site from pJRD158. A 2.9 kb EcoRI-HindIII fragment carrying the gene encoding wild-type alpha-amylase (Amy) is introduced. Thus constructed, this vector has 8 unique restriction sites that share the amylase gene in 120 to 600 kb segments and thus facilitate the subcloning of mutated fragments. The gene coding for alpha-amylase is then replaced by the previously prepared gene.

3) Expression of the gene in a strain of E.Coli
An E. coli strain is transformed with the previously prepared plasmid. The cells are cultured in LB liquid medium (Luria Broth) with 100 mg / l of ampicillin, then they are taken up in 1/2 volume of 25% sucrose and stirred for 15 minutes at 20 ° C., and then again. minutes at 20 ° C in the presence of 25% sucrose, 10 mM EDTA.

After centrifugation, the cells are taken up in ice water and centrifuged after 20 minutes at 0 C.

 4) Determination of the characteristics of the variant obtained.

 Thermostability is defined as residual amylolytic activity after heating.

 Amylolytic activity is measured by the dextrinizing action of the enzyme. The iodine method is used by measuring the optical density at 620 nm of a 1% starch solution (pH = 5.8) in the presence of an iodine-iodide solution. The units are expressed in OD relative to a control without incubation.

 An aliquot of culture supernatant is subjected to the action of the temperature for a given time and the residual enzymatic activity is measured.

 Residual amylolytic activity is expressed as a percentage of activity relative to the activity of an untreated aliquot.

 The determination of the half-life of the protein obtained is carried out at 90 ° C., in a 50 mM sodium acetate buffer, pH 6. It depends on the concentration of Ca ++ in the medium.The Calf ion does not intervene not in the catalytic reaction but plays a structural role for the maintenance of a biologically active structure.

The results obtained for different concentrations of Ca ++ ions are illustrated in FIGS.
for a concentration of 0.1 mM Cas12, the half-lives are 133 respectively of 8 and 5 minutes for the variant Tyr and the natural form.

 for a concentration of 1 mM CaCl 2 the half-lives are respectively 200 and 55 minutes.

 for a concentration of 10 mM CaCl2, the Tyr133 variant retains 95% of its activity after 5 hours at 90 ° C., compared to 50% for the natural form.

Example 2: Preparation of other variants in position 133

The procedure is as in Example 1, except that the mutagenesis is carried out so as to replace the codon coding for His by a codon coding respectively for Ala (A), Glu (E), Pro (P), Lys (K). ), Leu (L),
Ser (S), Gln (Q), Gly (G), Phe (F).

 Figure 6 shows the thermostability results (residual activity after heating) obtained for these different variants. It also makes it possible to observe a relationship between the degree of hydrophobicity of the substituted amino acid and the thermostability of the variant of the alpha-amylase obtained. It appears that the variants having at position 133 a substitution with a more hydrophobic amino acid than His actually exhibit increased thermostability compared to the natural form.

 The variants Pro, Ser, Gly, Ala are therefore indicated only as comparative examples.

EXAMPLE 3 Obtaining a mutated gene of Bacillus licheniformis alpha-amylase expressing a protein that is more thermostable than the wild-type protein.

 The very nature of the enzyme makes it difficult to obtain affected variants in thermostability, in fact thermostable amylolytic activity does not confer a selective advantage on the bacteria that express it, which makes the use of simple genetic screens difficult, or the use of biological screens, as has been described for obtaining thermostable variants of Kanamicin Nucleotidyltransferase (J. Biol Chem., 1985, 260, 15298-15303), by the use of thermophilic bacterial strains .

 A strategy of random mutagenesis from a thermosensitive form of the protein has therefore been developed, in order to isolate intragenic mutations capable of restoring all or part of the thermostability of the wild form.

 Such a thermosensitive variant has been obtained by site-directed mutagenesis, it is the double substitution Asp 164tAla and Trp 1653goy. The thermal denaturation kinetics at 90 ° C of this thermosensitive variant, designated p841ts, is shown in Figure 7. At 90 ° C in the presence of 50 mM acetate buffer containing 1 mM CaCl 2, more than 90% of the amylolytic activity is lost after 8 minutes of incubation while the wild protein canned almost 100%.

 It was necessary to choose a biological system that is not very sensitive to heat and easy to handle for mutagenesis, mutant sequencing and amylase production. The M13 single-strand phage system fulfills all these conditions: after heating for 45 minutes at 800 ° C., almost all the phage particles remain infectious; the alpha-amylase gene is expressed in both orientations from its own promoter; the amylolytic activity is easily identifiable on starch medium by the production of halos around the beaches, after staining with iodine; the handling facilities for both the nucleotide sequence and the mutagenesis are obvious.

The entire structural gene, coding for the thermosensitive variant of the alpha-amylase p841ts, as well as the promoter region, is cloned in the form of a 2.8 Kb EcoRI-HindIII fragment in the phage
M13mp18. The recombinant phage obtained is called M13 mpl8 p841ts. The transcription of the cloned gene is under the control of its own promoter.

 The phage particles are mutagenized with hydroxylamine (250 mM, pH6 acetate buffer) and used to infect E. coli TG1. The cells are plated on Luria broth medium, 1 mM EDTA and incubated overnight at 37 ° C. Aliquots corresponding to 1-10% of surviving phage particles are used for the experiment. 1mM EDTA is added to the medium in order to chelate the Calcium Amylase Stabilizer ions.The plates are then placed at high temperature (80 ° C for 45 min) and covered with a second layer of LB agar medium, 1 mM EDTA, containing 1% soluble starch. After 45 minutes of incubation at 50 ° C., the plates are stained with iodine vapors. Phages producing heat-resistant amylases are detected by the appearance of a halo, in which case the thermosensitive amylase does not produce of halo.

 Among 104 phages tested, it was possible to isolate a phage producing a thermostability amylase significantly greater than the thermosensitive form.

 Gene sequencing revealed a suppressive mutation located outside the original mutation site. The Ala209-> Val substitution was found in 3 independent variants, so it is likely that only a small number of positions is likely to produce increased thermostability variants.

 Introduced into the wild-type protein, the Ala209-> Val substitution increases by a factor of two the half life of the protein at 90 ° C., which constitutes an appreciable gain for this already extremely heat-sensitive protein.

 This same strategy applied to the cloned gene in the other orientation in phage M13mp19, as well as the use of another mutagen should allow the isolation of other suppressive mutations, as well as the use of another variant. thermosensitive.

Example 4
By in vitro recombination of the genes encoding variant proteins Val209 and Tyr133, the doubled protein variant was obtained.
Val 133 substituted -Tyr. The effects of these two substitutions on the thermostability of the protein add up, the half-life of the amylase
Val209 133 -Tyr, tested at 90 ° C. in the presence of 50 mM acetate buffer and
ImM CaCl2 is greater than 10 hours, while those of the wild-type protein, and of each of Val209 and Tyr variants are respectively 1 h 30, 3 h and 4 h (FIG. 8).

Claims (14)

 1. A thermostable variant of Bacillus licheniformis apha-amylase, characterized in that, with respect to the amino acid sequence shown in FIG. 1, its sequence comprises in the vicinity of histidine (His) in position 133 at least substitution with an amino acid more hydrophobic than His, and / or in the vicinity of Alanine (Ala) at position 209 at least one substitution with a more hydrophobic amino acid than Ala.  2. Variant according to claim 1, characterized in that the substitution in the vicinity of position 133 is performed by amino acids selected from Phe, Leu, Tyr, Glu, Lys, Gln.  3. Variant of Bacillus licheniformis alpha-amylase

<Tb> <tb> <SEP> Tyr <tb> His133 <SEP> <SEP> 133 <SEP>  4. Variant according to one of claims 1 to 3, characterized in that the substitution in the vicinity of the position 209 is carried out by amino acids selected from Val, Leu, Ile.  5. Variant of the alpha-amylase according to claim 4, characterized in that the Alanine in position 209 is substituted by a Valine in position 209  6. Gene encoding a variant according to one of claims 1 to 5.  7. Gene encoding a variant according to one of claims 1 to 5, containing the DNA sequence as shown in Figure 1, except that the codon coding for His is replaced by a codon encoding Tyr133 and / or the codon coding for Ala209 is replaced by a codon 209 coding for Val  8. Expression vector of a variant according to one of claims 1 to 3, in a host cell, characterized in that it contains a gene according to claim 6 or 7, as well as the elements ensuring its expression in said host cell.  An expression vector according to claim 8, characterized in that the expression elements comprise the regulatory region of the gene encoding Bacillus licheniformis alpha-amylase.  10. Expression vector according to claim 8, characterized in that the expression elements comprise the promoter regulatory region SacR located upstream of the gene encoding Bacillus subtilis levan sucrase.  11. Host cell transformed with an expression vector according to one of claims 8 to 10.  12. Host cell according to claim 11, characterized in that it is selected from Bacillus licheniformis, Bacillus subtilis and E.coli.  13. Method for preparing varlants according to one of claims 1 to 5, characterized in that the host cells are cultured according to one of claims 11 or 12, under appropriate culture conditions and recovering the variant obtained.  14. Use of the variants according to one of claims 1 to 5, in the textile industry, paper, brewery, especially for starching and liquefaction of starch.