FR2533583A1 - Novel recombinant DNA which is useful in the preparation of alpha -amylase - Google Patents

Abstract

The invention relates to recombinant DNAs containing a gene coding for a heat-stable alpha -amylase, which DNAs contain at least: - the DNA fragment of a Bacillus licheniformis strain, coding for a heat-stable alpha -amylase, and - a DNA fragment of a plasmid, of a phage lambda or of a phage lambda derivative. The invention also relates to a method for producing alpha -amylase, employing bacterial strains transformed with the above DNAs.

Description

 The present invention relates to the preparation of α-amylase.

 Starch, the glucose polymer, consists of two fractions, amylose, a linear polymer containing 250 to 300 units of glucose linked by a-1,4-glucosidic bonds, and amylopectin, a structural polymer. branched whose branches are formed by α, 6-glucosidic bonds but most of the glucose units are united by α-1,4-glucosidic bonds.

 The generic term amylase is used to denote any enzymatic activity capable of hydrolyzing all or part of the starch at the α-1,4 and a-1,6 bonds.

 The α-amylases fragment the starch into dextrins and the amylases release maltose.

 In industrial practice, hydrolytic enzymes of the amylase type are used to degrade the starch in order to obtain increasingly soluble products, the ultimate term for which is glucose.

 The starch solubilization operation, often called liquefaction, can be carried out by amylolytic enzymes of the α-amylase type.

 The α-amylases are characterized by the fact that they exert on the amylase and the amylopectin an essentially endohydrolytic action which results in a statistical cut of the α-1,4 bonds.

 Many industries use α-amylase, either for the purpose of directly using the hydrolysis products of the starch thus obtained, or in order to obtain from the starch a product having a liquid consistency which can be easily treated in later stages.

 Among the industries using α-amylase, mention should be made in particular of the food industries which use starch hydrolysates, directly or after subsequent treatment, as thickening agent for various food preparations, as well as the textile industries which use this type of enzyme at different stages of the preparation of fibers and textiles.

 Currently, α-amylase is prepared by bacterial fermentation of strains exhibiting α-amylolytic activity, in particular strains of Bacillus licheniformis.

 It has already been proposed in the prior art transformed bacterial strains making it possible to increase the production of α-amylase.

Thus, European patent application No. 0 034 470 describes a process using a bacteriophage X as a cloning vector for an amylase of Bacillus coagulens in Escherichia coli, but the properties of the enzyme obtained are not those of an a- amylase, namely
- that it is inactive on cyclodextrins, which excludes any endomolecular activity,
- that it produces mainly glucose and maltose and few higher oligosaccharides or maltotrioses, which is rather characteristic of an amyloglucosidase or an amylase.

European patent application No. 0 057 976 describes a process for preparing a thermostable a-amylase by incorporating the gene coding for a thermostable a-amylase from Bacillus stearothermophilus into a chimeric plasmid, which is used to transform a
E. coli or B. subtilis.

 However, the α-amylase obtained has a very limited pH range of use, which constitutes a serious drawback for use at the industrial stage. Indeed, as has been indicated previously, the α-amylase is an enzyme used in many industrial fields and the pH as well as the temperatures at which it must be used are very varied, which requires that this enzyme has, to be marketable, a pH range where it is active - which is very wide as well as a very high inhibition temperature.

 Currently, the industrial product marketed under the Termamyl brand is a product obtained by fermentation of a B. licheniformis.

The α-amylase of B. licheniformis has a broad spectrum of activity pH, from pH 4.5 to pH 8 at the temperature of 500C; on the other hand the a-amylase of
B. stearothermophilus has a relatively narrow spectrum of activity, pH 5.4 to 6.1 at a temperature of 650C.

 In addition, from the tests reported in this European patent, it appears that the α-amylase prepared, in particular from E. coli, can only be recovered by lysing the strains using in particular a phage T4 .

This obviously constitutes a significant drawback at the industrial level, both with regard to the cost of the operation and for its possible repercussions on the product thus obtained,
Finally, the strains of B. subtilis transformed by the plasmid described in the European patent in question are only stable in the presence of chloramphdnicol, this naturally limits considerably the possibilities of application of these strains on an industrial level.

The subject of the present invention is the preparation and use of α-amylolytic strains capable of producing an amylase having characteristics very close to or identical to the industrial product currently marketed under the name of
Termamyl.

 The present invention also relates to a process for the preparation of α-amylase by fermentation of an α-amylolytic bacterium, process in which the α-amylase located in the vicinity of the cell periphery can be recovered in the medium by applying bacterial cells a simple osmotic shock.

To do this, the present invention provides a
Recombinant DNA comprising a gene coding for a thermostable α-amylase, characterized in that it comprises at least
- the DNA fragment of a strain of
B. licheniformis encoding a thermostable α-amylase, and
- a DNA fragment of a plasmid, of a phage X or of a derivative of phage A.

 In a preferred embodiment of this type of recombinant DNA, the following will be used. of bacterial DNA fragment coding for the thermostable α-amylase, all or part of a fragment of about 3.3 kilobases limited by two HindIII restriction sites, and preferably a bacterial DNA fragment originating from the strain of B. licheniformis ATCC 6598 deposited in the American Type Culture Collection.

 When the recombinant DNA in question is a plasmid, it is possible to use the DNA of any plasmid capable of replicating and ensuring an amplification of the gene encoded in the bacterium into which the recombinant DNA is to be introduced.

 Thus, the plasmids derived from the plasmid pBR322, such as pHC79, are particularly interesting in the context of the present invention.

When the recombinant DNA is a phage, it is possible to use DNA fragments of phage X L47
The present invention also relates to the bacterial strains transformed by recombinant DNA of the type described above and in particular the strains of
Transformed E. coli.

 The present invention finally relates to a process for the preparation of a-amylase, characterized in that a transformed bacterial strain as described above is cultivated on an appropriate medium, and the a-amylase is recovered after fermentation, preferably by lysis using osmotic shock.

 Finally, the invention relates to thermostable α-amylases obtained by the implementation of this process.

 Recombinants according to the present invention were prepared from the DNA of B. licheniformis, which is an organism producing a thermostable α-amylase, by purification with DNA on a cesium chloride gradient.

 The DNA thus obtained and the vector DNA from phage X L47 derived from bacteriophage A are then treated with a restriction enzyme.

 The DNAs thus obtained are mixed and ligated under the action of a ligase.

The ligation mixture is then packaged invitro in order to obtain infectious phage particles which are then used to infect a strain of
E. coli.

 Is then isolated, by conventional techniques, the bacteriophages X amy which can be used to infect various strains of E. coli by making them producers of α-amylases.

 In the same way, but starting from the DNA of bacteriophages X amy and from a plasmid for example derived from pBR322, one will obtain plasmids which can be used to transform bacterial strains to make them producers of amylases.

 Of course, the process described above can be modified in order to obtain different recombinants but which remain within the scope of the present invention.

 The methods of lytic infection or transformation of the bacterial strains are known and will not be described in detail.

 The following examples are intended to illustrate various aspects of the present invention.

EXAMPLE 1
Preparation of a recombinant DNA of phage X L47 comprising the DNA coding for the alpha-amylase derived from a strain of
B. licheniformis Donor Crooranism microorganism
The donor microorganism is a strain of
B. licheniformis deposited with ATCC under number 6598. This strain produces a thermostable a-amylase whose characteristics are very similar to those described by FJ Morgan and FG Priest: Characterization of a thermostable amylase from B. licheniformis NCCB 6346
Journal of Applied Bacteriology (1981) vol. 50, pp107-114 and by N. Saito: A thermophilic extra cellular amylase from B. licheniformis; Archives of Biochemistry and
Biophysics (1973) 155, pp. 290-298.

- Its molecular weight is 22,500 daltons
- Its optimum temperature is 7-6 C at pH 9 - its optimum pH is quite wide, from 6 to 25 C.

 The DNA of B. licheniformis is purified by CsCl gradient equilibrium centrifugation. To the lysate is added a solution of ClCs saturated in the proportions 1/3 and 2/3 respectively. The mixture is centrifuged for 36 hours at 45KRPM in a Beckmann 70Ti rotor.

 The tube is then pierced through the bottom, the presence of DNA is revealed by the viscous appearance of the flowing liquid.

 After 24 hours dialysis against TE (10 mM tris 1 mM EDTA pH 7.5), the DNA is subjected to the action of phenol balanced against TE pH 8 and dialyzed again for 24 hours.

 By 0.3% agarose gel electrophoresis with phage'A DNA size standard (49 kb), the quality of the DNA whose size is greater than 120 kb is checked.

 Restriction and ligation 20 1 l t2 ssg) - of phage A L47 DNA are subjected to the action of the enzyme HindIII, the deleted part can be replaced by a DNA fragment of 7.1 to 21.6 kg base pairs. 100 μl (8 ssg) of B. licheniformis DNA are subjected to the action of the same enzyme HindIII for 2 hours at 370C. The enzymatic reaction is stopped by heating for 15 minutes at 650C.

 The two DNAs are then mixed and precipitated with absolute alcohol in the presence of 0.3 M NaCl. The precipitate is taken up in 20 l of ligation buffer and subjected to the action of T4 DNAligase in the presence of ATP and of dithiothreitol. overnight at 100C.

 An aliquot of each DNA and the ligation mixture are removed and analyzed on gel.

Encapsulation of the ligation mixture
2 μl (1 g) of the ligation mixture are packaged according to the protocol of B. Hohn and J. Collins. Cosmids:
A type of plasmid gene cloning vector that is packageable in vitro in bacteriophage lambda leads; Proc. Natl. Acad.

Sci. USA, vol. 75, n 9, pp 4242-4246 (1978).

-EXAMPLE It
Preparation of an E. coli strain producing α-amylase and α-amylase obtained
Infection of indicator bacteria
The indicator bacteria infected by the packaging system prepared previously is a strain of
E. coli with a lysogenic phage P2.

 This bacterium is cultivated in the presence of 0.4% maltose until the end of the exponential phase. After centrifugation, the cells are taken up in a volume of 10 mM MgSO 4.

Results 4
About 2.5 x 10 phage particles were obtained, a few hundred were tested, two lysis ranges were found to be positive X amy 1 and X amy 2.

 The phages of the two positive lysis ranges are subcultured and amplified by reinfection of a gifted sensitive strain. coli. In order to study the characteristics of amylase a liquid infection is carried out.

The receptor bacterium is cultured in a liquid medium in the presence of 0.4% maltose up to an optical density of 0.4. The phage suspension is added in order to carry out an infection with a multiplicity of 1
Bacterial lysis occurs 4 to 5 hours after infection.

Amylolytic activity dosage
Two methods can be used to perform this assay.

 a) A method for the determination of the reducing sugars which appeared during the hydrolysis of starch, with dinitrosalicylic acid (DNS). The results are expressed in Maltose Equivalent.

 b) A method which consists in measuring the optical density at 620 nm of a starch solution in the presence of iodine.

Properties of the amylase thus produced by
E infection, coli
An aliquot of supernatant is subjected to the action of temperature for a given time and the residual enzymatic activity is measured.

 300 Rl of supernatant are subjected for 10 minutes to increasing temperatures. The residual activity is then assayed.

 To these 300 seconds, 1 ml of 1% soluble starch solution is added (NaH2PO4 Na2HPO4 buffer, pH 5.6) and the mixture is incubated for 10 minutes at 50 ° C. The OD is then measured at 620 nm of the mixture in the presence of 2 ml of lugol.

 The results are expressed as a percentage of the activity of an untreated aliquot (fig. 1).

Results ++
In the absence of any protective agent (Ca ion and starch), the enzyme retains 90% of its activity after 10 minutes at 80 C.

 In the presence of 0.1 M Ca ions, the enzyme is 100% stable at 900C for 15 minutes (result obtained by assay at the DNS).

 These properties are identical to those of the α-amylase of B. licheniformis described by Saito.

Characteristics of a starch hydrolyzate
A native starch liquefaction test was carried out - Starch concentration: 30% - pH 6.2 (addition 1 O / oo Ca (OH) 2) - Temperature: 95 C - Enzyme level: equivalent active at 2 ml
Termamyl 60 for 1 kg of starch
DE obtained - DE obtained: 15.2. (dextrose equivalent).

Carbohydrate distribution
- FROM 15.2
glucose 0.4
maltose 2.7
G3 5.5
G4 2.9
G5 5.4
G6 8.0
G7 6.5
G8 3.6 Gg 2.4
G10 1.8
G11 - G 18.5
G20 42.3
Results expressed as a percentage of the dry product.

(The abbreviation G n indicates glucose oligomers with n glucose units).

Immunological comparison
After precipitation with 65% ammonium sulphate and concentration 100 times of a bacterial lysis supernatant, the amylase produced by E. coli is compared with the α-amylase from B. licheniformis purified by the immunoprecipitation method of Ouchterlony
5 ml of purified agar in 1/15 M phosphate buffer pH 7 + 0.01% sodium azide are poured into a Petri dish 5 cm in diameter, wells of 5 mm spaced 1.5 cm are filled 50 fl of antiserum and 100 l of the two amylases.

 The wells are arranged in such a way that the antiserum can react with the two amylases at the same time.

The box is left overnight in the oven at 37 -C.

 No immunological difference is detectable by this method. All the antigenic determinants carried by the purified α-amylase of B. licheniformis are present on the α-amylase produced by E. coli as shown by the absence of spur at the meeting of the two precipitates.

EXAMPLE III
Under cloning in a bacterial plasmid replicating in Escherichia coli and identification of a restriction fragment containing the v-amylase gene
The bacterial plasmid pBR322 (marketed by Bethesda Research Laboratories) is capable of conferring on the bacteria which contain it resistance to the antibiotics ampicillin and tetracycline. This plasmid contains a number of unique restriction sites allowing cloning by insertion and inactivation of one of the resistance genes. This is the case for the enzyme HindIII which is located in the tetracycline resistance gene.

 The DNAs of bacteriophage X amy + obtained in the previous examples and of plasmid pBR322 are subjected to the action of the enzyme HindIII for 1 hour at 370C; the enzyme is then destroyed by heating for 15 minutes at 650C. 0.5 ssg of bacteriophage X amy DNA and 1 ssg of plasmid pBR322 treated with the enzyme HindIII are mixed and precipitated with absolute ethanol in the presence of 0.3 M NaCl.

The precipitate is taken up in 15 μl of ligation buffer and subjected to the action of T4 DNA ligase in the presence of ATP and of dithiothreitol overnight at 100 ° C.
The ligation mixture is then used to transform a competent E. coli strain HB101.

The transformed bacteria are selected on a complete medium containing 100 mg per liter of ampicillin, then the clones containing a hybrid plasmid ApRTcS are identified on a medium containing tetracycline 25 mg / l.
The hybrid clones are tested for linker capacity to produce amylase in the presence of 1% soluble starch, after treatment with lysozyme.

 Amylolytic activity is detected by iodine staining of a bacterial lysate in the presence of soluble starch after 30 minutes of incubation at 45 C.

The plasmid of the Amy + Tus clones is
is purified after amplification in the presence of chloramphenicol, on a CsCl gradient in the presence of BET
After dialysis and concentration they are analyzed by different restriction enzymes. All contain a 3.3 kb Hindili fragment.

 The restriction map of the plasmid obtained is shown diagrammatically in FIG. 2.

 On this Hindili fragment, we find from one of the Hindili ends, as shown schematically in the figure - a SalI site at approximately 100 base pairs - a BglII site at approximately 450 base pairs - an EcoRI site i 750 pairs of bases - a Psti site at 1.7 kilopairs of bases - a PvuII site located between the EcoRI site and Psti.

 From the other HindIII end we find - a SalI site at approximately 700 base pairs - a KpnI site at approximately 1.3 kilopair of bases - an SstI site at approximately 1.9 kilopair of bases.

However, there are no Xho, Soma,
PvuI, HpaI and Sstii.

 All of these enzymes specifically cut DNA at six nucleotide sites.

When we try to reduce the fragment
HindIII from these two ends, it can be seen that the EcoRI-EcoRI fragment can be deleted without altering the expression of the amylolytic activity.

 On the other hand, the deletion of the Pst-Pst and Sal-Sal fragments inhibits amylolytic activity.

 The region containing the amylase structural gene therefore extends over at least 1000 base pairs between the Sal and Psi sites and on either side of these sites over non-delimited areas.

 The plasmids obtained by this technique are capable of conferring resistance to ampicillin to a competent strain of E. coli and of conferring on it an amylolytic activity.

 This amylolytic activity can be identified directly on Petri dishes containing starch at 1 t t, which suggests that the amylolytic activity is distributed at the periphery of the cells, no doubt in the periplasmic space.

 Under these conditions, it is possible to release the α-amylase by a simple osmotic shock of the bacterial cells, which on the industrial level constitutes a considerable advantage.

Claims (10)

 1) recombinant DNA comprising a gene coding for a thermostable amylase, characterized in that it comprises at least  the DNA fragment of a strain of Bacillus licheniformis coding for a thermostable amylase, and  - a DNA fragment of a plasmid, a phage A or a derivative of phage A.  2) DNA according to claim 1, characterized in that the bacterial DNA fragment coding. for the α-amylase comprises all or part of a fragment of approximately 3.3 kb bounded by two HindIII restriction sites.  3) DNA according to one of claims 1 and 2, characterized in that B. licheniformis is the strain ATCC 6598.  4) DNA according to one of claims 1 to 3, characterized in that the DNA of a phage A comes from phage X L47.  5) DNA according to one of claims 1 to 4, characterized in that the plasmid DNA comes from the bacterial plasmid pBR322 or from a plasmid derivative of pBR322.  6) A bactdriene strain transformed with a recombinant DNA according to one of claims 1 to 5.  7) A strain according to claim 6, characterized in that it is a strain of E. coli.  8) A process for producing an α-amylase, characterized in that a bacterial strain according to one of claims 6 and 7 is cultivated on an appropriate medium and the α-amylase is recovered.  9) Method according to claim 8, characterized in that after growth of the bacterial cells, these are lysed by an osmotic shock in order to release the α-amylase in the medium.  10) thermostable amylase obtained by implementing the method according to one of claims 8 and 9.