FR2537602A2 - New improved recombinant DNA which is useful in the preparation of alpha -amylase via Bacillus subtilis - Google Patents

New improved recombinant DNA which is useful in the preparation of alpha -amylase via Bacillus subtilis Download PDF

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FR2537602A2
FR2537602A2 FR8220904A FR8220904A FR2537602A2 FR 2537602 A2 FR2537602 A2 FR 2537602A2 FR 8220904 A FR8220904 A FR 8220904A FR 8220904 A FR8220904 A FR 8220904A FR 2537602 A2 FR2537602 A2 FR 2537602A2
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amylase
dna
plasmid
bacillus subtilis
fragment
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FR2537602B2 (en
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Philippe Joyet
Michel Guerineau
Henri Heslot
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Centre National de la Recherche Scientifique CNRS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus

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Abstract

The invention relates to a recombinant DNA containing a thermostable alpha -amylase-encoding gene, characterised in that it comprises at least: - the thermostable alpha -amylase-encoding DNA fragment of a Bacillus licheniformis strain, and - a DNA fragment from a plasmid or from a lambda phage derivative which can replicate inside B. subtilis. This DNA enables the expression of alpha -amylase in Bacillus subtilis.

Description

La présente invention concerne un perfectonnement aux ADN recombinants décrits dans le brevet principal nO 82 16069 au nom du demandeur et en particulier concerne des souches de Bacillus subtilis transformées par lesdits ADN recombinants et surproducteurs d'une a-amylase thermostable. The present invention relates to a complementation to the recombinant DNAs described in the main patent No. 82 16069 in the name of the applicant and in particular relates to strains of Bacillus subtilis transformed by said recombinant DNAs and overproducing a thermostable α-amylase.

Le brevet principal décrit des ADN recombinants comportant un gène codant pour a-amylase thermostable, ainsi que les souches obtenues par transformation à l'aide de ces ADN recombinants. Des expériences ont montré qu'il était possible de cette façon d'obtenir des souches de E.coli surproductrices d'une a-amylase thermostable. The main patent describes recombinant DNAs comprising a gene encoding heat-stable α-amylase, as well as strains obtained by transformation using these recombinant DNAs. Experiments have shown that it is possible in this way to obtain E. coli strains overproducing a thermostable α-amylase.

I1 est particulièrement intéressant de pouvoir utiliser au stade industriel Bacillus subtilis au lieu de E.coli, en effet Bacillus 'subtilis est un organisme non pathogène ne produisant pas d'endotoxine et ne présentant donc aucune limitation quant à son utilisation à des fins alimentaires ou médicales. It is of particular interest that Bacillus subtilis can be used in the industrial process instead of E. coli, since Bacillus subtilis is a non-pathogenic organism which does not produce endotoxin and therefore has no limitation as to its use for food or feed purposes. medical.

En outre Bacillus subtilis est un microorganisme déjà largement utilisé au stade industriel et dont les conditions de fermentation sont largement étudiées dans la technique antérieure. In addition Bacillus subtilis is a microorganism already widely used at the industrial stage and whose fermentation conditions are widely studied in the prior art.

Enfin, Bacillus subtilis comme la plupart des bactéries gram positives est connu pour être un organisme excrétant de nombreuses protéines (amylases et protéases) à la différence des bactéries gram négatives telles que E.coli. Finally, Bacillus subtilis like most gram-positive bacteria is known to be an excretory organism of many proteins (amylases and proteases) unlike gram-negative bacteria such as E. coli.

L'invention concerne donc un perfectionnement aux
ADN recombinants décrits au brevet principal qui peuvent exprimer un gène codé dans Bacillus subtilis. The invention therefore relates to an improvement to
Recombinant DNAs described in the main patent which can express a gene encoded in Bacillus subtilis.

Ces ADN recombinants comportant un gène codant une a-amylase thermostable sont caractérisés en ce qu'ils comportent- au moins
- le fragment d'ADN d'une souche de Bacillus licheniformis codant pour une a-amylase thermostable, et
- un fragment d'ADN d'un plasmide, ou d'un dérivé de phage X se répliquant dans Bacillus subtilis.These recombinant DNAs comprising a gene encoding a heat-stable α-amylase are characterized in that they comprise at least
the DNA fragment of a strain of Bacillus licheniformis encoding a heat-stable α-amylase, and
a DNA fragment of a plasmid, or a phage X derivative, replicating in Bacillus subtilis.

Le fragment drADN codant pour une a-amylase thermostable est de préférence constitué par tout ou partie du fragment d'environ 3,3 kb limité par deux sites de restriction HindIII qui a déjà été décrit dans le brevet principal, il ressort en effet des différentes expériences qutil s'agit là d'un fragment qui à lui seul contient l'information génétique nécessaire à la synthèse de l'amylase en cause. The fragment dDNA encoding a thermostable α-amylase is preferably constituted by all or part of the fragment of about 3.3 kb bounded by two HindIII restriction sites which has already been described in the main patent, it is clear from the different experiments that this is a fragment which alone contains the genetic information necessary for the synthesis of the amylase in question.

Parmi les plasmides pouvant se répliquer dans B subtilis il faut citer en particulier le plasmide
PUB 110 et de façon générale les plasmides se répliquant dans 13. subtilis conférant la résistance à la kanamycine.Among the plasmids that can be replicated in B subtilis, it is necessary to mention in particular the plasmid
PUB 110 and generally the plasmids replicating in 13. subtilis conferring resistance to kanamycin.

I1 est possible également d'utiliser des plasmides hybrides se répliquant à la fois dans E.coli et
Bacillus subtilis, c'est le cas du plasmide qui sera décrit dans les exemples et est constitué par fusion de PUB 110 issu destaphylococcusaureus conférant la résistance à la kanamycine et se répliquant dans
Bacillus subtilis et pBR 322 qui confère la résistance à la tétracycline et à l'ampicilline,et qui est capable de se répliquer dans E.coli.It is also possible to use hybrid plasmids replicating in both E. coli and
Bacillus subtilis, this is the case of the plasmid which will be described in the examples and is formed by melting PUB 110 derived from kanamycin resistance-conferring locaphylococcusaureus and replicating in
Bacillus subtilis and pBR 322 which confers resistance to tetracycline and ampicillin, and which is able to replicate in E. coli.

L'invention concerne également les souches de B. The invention also relates to B strains.

subtilis transformées par les ADN recombinants, les procédés de fermentation mettent en oeuvre ces souches transformees et l'a-amylase thermostable obtenu dans ces fermentations. subtilis transformed by the recombinant DNAs, the fermentation processes use these transformed strains and the heat-stable α-amylase obtained in these fermentations.

Les exemples ci-après sont destinés à illustrer d'autres avantages et caractéristiques de la présente invention. The following examples are intended to illustrate other advantages and features of the present invention.

Exemple 1
Matériel et méthode
a) vecteur de clonage utilisé et souche réceptrice
Le plasmide utilisé est un plasmide hybride obtenu à partir des plasmides pUB 110 et des plasmides pBR 322 (tous deux commercialisés notamment par la Société
Bethesda Research Laboratories, Zinc.). Example 1
Material and method
a) cloning vector used and recipient strain
The plasmid used is a hybrid plasmid obtained from plasmids pUB 110 and plasmids pBR 322 (both marketed in particular by the Company).
Bethesda Research Laboratories, Zinc.).

La souche réceptrice est une souche de B.subtilis
BS 151.The recipient strain is a B. subtilis strain
BS 151.

Les différentes enzymes sont utilisées dans les conditions prescrites par le fabricant. The different enzymes are used under the conditions prescribed by the manufacturer.

b) transformation de Bacillus subtilis
La souche de Bacillus subtilis a été transformée par le procédé décrit par D. Erhlich (B. Niaudet et coll. : In vitro Genetic labelling of Bacillus subtilis cryptic plasmid PHV400. Plasmid 2 : 48-58 (1979)). b) transformation of Bacillus subtilis
The Bacillus subtilis strain was transformed by the method described by D. Erhlich (B. Niaudet et al .: In vitro Genetic labeling of Bacillus subtilis cryptic plasmid PHV400, Plasmid 2: 48-58 (1979)).

Les milieux de sélection sont des milieux LB contenant 5 pg/ml de kanamycine et 1 % d'amidon soluble. Selection media are LB media containing 5 μg / ml kanamycin and 1% soluble starch.

Enfin l'activité amylolytique des clones transformés a été testée par exposition aux vapeurs d'iode. Finally, the amylolytic activity of the transformed clones was tested by exposure to iodine vapor.

c) Construction des Plasmides hybrides DUB 110, pBR 322. c) Construction of the hybrid plasmids DUB 110, pBR 322.

Les plasmides pUB 110 et pBR 322 sont traités par l'enzyme de restriction EcoRI qui dans chacun de ces plasmides constitue un site de restriction unique. Plasmids pUB 110 and pBR 322 are treated with the restriction enzyme EcoRI which in each of these plasmids constitutes a unique restriction site.

Les produits de restriction sont soumis à l'action de la ligase T4 afin de fusionner les deux plasmides par leur site unique.  The restriction products are subjected to the action of T4 ligase in order to fuse the two plasmids by their unique site.

Le mélange de ligation est ensuite utilisé pour transformer une souche de E.coli et l'on sélectionne les transformants présentant une résistance à la tétracycline, à l'ampicilline et à la kanamycine,' qui ont donc intégré un plasmide hybride de pUB 110 et de pBR 322. The ligation mixture is then used to transform an E. coli strain and the transformants having resistance to tetracycline, ampicillin and kanamycin, which thus integrated a hybrid plasmid of pUB 110, are selected. pBR 322.

d) Insertion du fragment Hindili de 3ff3 kb dans le plasmide hybride. d) Insertion of the HindIII fragment of 3ff3 kb in the hybrid plasmid.

Le plasmide hybride pUB 110 et pBR 322 préparé précédemment ainsi que le plasmide pBR 322 Amy+ qui a été décrit dans la demande de brevet principal sont restreints par l'enzyme Hindili mélanges puis soumis à l'action de la ligase T4. The hybrid plasmid pUB 110 and pBR 322 prepared above as well as plasmid pBR 322 Amy + which was described in the main patent application are restricted by the enzyme HindIII mixtures and then subjected to the action of T4 ligase.

Comme précédemment, les plasmides recombinants ayant intégré le fragment de 3,3 kb conférant le caractère Amy+ - sont sélectionnés dans des transformants d'E.coli en recherchant le phénotype Kanr Apr Tc5 TcS amy+.  As before, the recombinant plasmids having integrated the characterizing Amy + - 3.3 kb fragment are selected in E. coli transformants by searching for the Kanr Apr Tc5 TcS amy + phenotype.

Les transformants présentant le phénotype précédent sont alors traités comme cela a été décrit au brevet principal afin d'en extraire les plasmides pour les purifier sur un gradient de chlorure de césium. Transformants exhibiting the above phenotype are then treated as described in the main patent in order to extract the plasmids to purify them on a cesium chloride gradient.

e) Transformation de Bacillus subtilis
Les plasmides hybrides purifiés précédemment sont utilisés pour transformer Bacillus subtilis BS 151.e) Transformation of Bacillus subtilis
Hybrid plasmids purified previously are used to transform Bacillus subtilis BS 151.

L'activité amylolytique des clones transformés est testée par exposition aux vapeurs d'iode. The amylolytic activity of the transformed clones is tested by exposure to iodine vapor.

On constate que dans tous les cas où les clones présentent le phénotype Kanr, ils s'entourent également d'un large halo due décoloration correspondant à une activité amylolytique.  It is found that in all cases where the clones have the Kanr phenotype, they also surround themselves with a large halo due to discoloration corresponding to amylolytic activity.

f) Dosage de l'activité amylolytique
L'activité amylolytique d'un clone kanamycine résistant est dosée dans le surnageant d'une culture d'une nuit en milieu LB contenant 5 pg/ml de kanamycine.f) Determination of amylolytic activity
The amylolytic activity of a resistant kanamycin clone is assayed in the supernatant of a overnight culture in LB medium containing 5 μg / ml kanamycin.

La culture de la souche réceptrice ne contenant pas de plasmide est menée dans les mêmes conditions et en absence de kanamycine. The culture of the recipient strain containing no plasmid is conducted under the same conditions and in the absence of kanamycin.

L'activité amylolytique est dosée par la méthode au D.N.S. qui a déjà été décrite dans le brevet principal. The amylolytic activity is assayed by the D.N.S. method. which has already been described in the main patent.

Dans ces conditions, on observe les résultats suivants :
TABLEAU I

Figure img00050001
Under these conditions, the following results are observed:
TABLE I
Figure img00050001

<tb> <SEP> Souche <SEP> Amylase <SEP> activité <SEP> X <SEP> activité
<tb> <SEP> EO <SEP> MAL/ML/10' <SEP> 370C <SEP> relative
<tb> BS <SEP> 151 <SEP> DO-: <SEP> 0,032
<tb> <SEP> 0.09 <SEP> i
<tb> BS <SEP> 151 <SEP> / <SEP> 3,3 <SEP> kb <SEP> DO <SEP> : <SEP> 0,946
<tb> <SEP> 2.8 <SEP> 29.5
<tb>
On constate que dans ces conditions la culture d'un clone transformé par le plasmide hybride amy produit près de 30 fois plus d'activité que la souche réceptrice.<tb><SEP> Strain <SEP> Amylase <SEP> Activity <SEP> X <SEP> Activity
<tb><SEP> EO <SEP> MAL / ML / 10 '<SEP> 370C <SEP> Relative
<tb> BS <SEP> 151 <SEP> DO-: <SEP> 0.032
<tb><SEP> 0.09 <SEP> i
<tb> BS <SEP> 151 <SEP> / <SEP> 3.3 <SEP> kb <SEP> DO <SEP>: <SEP> 0.946
<tb><SEP> 2.8 <SEP> 29.5
<Tb>
It is found that under these conditions, the culture of a clone transformed with the amy hybrid plasmid produces nearly 30 times more activity than the recipient strain.

L'acquisition de la résistance à la kanamycine s'accompagne donc d'une surproduction d'activité amylolytique qui ne peut être que la conséquence de l'introduction du fragment HindIII et de son expression par l'intermédiaire du plasmide hybride.  The acquisition of kanamycin resistance is therefore accompanied by an overproduction of amylolytic activity which can only be the consequence of the introduction of the HindIII fragment and its expression via the hybrid plasmid.

Etude de la thermorésistance
Des aliquots de surnageant d'une culture d'une nuit d'un clone kanamycine résistant est soumis préalablement durant 10 minutes à des températures croissantes avant de doser l'activité résiduelle.Study of heat resistance
Aliquots of supernatant from overnight culture of a resistant kanamycin clone were preliminarily subjected for 10 minutes to increasing temperatures prior to assaying for residual activity.

La figure 1 représente le pourcentage d'activité résiduelle amylolytique en fonction de la température à laquelle a été soumis le surnageant. Figure 1 shows the percentage of amylolytic residual activity as a function of the temperature at which the supernatant was subjected.

On constate que la thermostabilité de l'activité amylolytique contenue dans le surnageant est comparable à celui de l'a-amylase de Bacillus licheniformis. Ceci confirme que les transformants de Bacillus subtilis excrètent bien une a-amylase thermostable en tout point identique à celle de Bacillus licheniformis. It is found that the thermostability of the amylolytic activity contained in the supernatant is comparable to that of Bacillus licheniformis α-amylase. This confirms that the transformants of Bacillus subtilis excrete a heat-stable α-amylase in every way identical to that of Bacillus licheniformis.

Ceci constitue sur le plan industriel un avantage considérable puisqu'il n'est plus nécessaire d'effectuer la lyse des-bactéries pour en récupérer l'a-amylase.  This is an industrial advantage a considerable advantage since it is no longer necessary to perform the lysis of bacteria to recover the α-amylase.

Claims (7)

REVENDICATIONS 1/ ADN recombinant comportant un gène codant pour une a-amylase thermostable caractérisé en ce qu'il comporte au moins 1 / recombinant DNA comprising a gene encoding a heat-stable α-amylase characterized in that it comprises at least - le fragment d'ADN d'une souche de Bacillus licheniformis codant pour une a-amylase thermostable1 et the DNA fragment of a Bacillus licheniformis strain encoding a thermostable α-amylase1 and - un fragment d'ADN dgun plasmide, ou d'un derivé de phage X se répliquant dans B subtilis. a DNA fragment of a plasmid, or of a phage X phage replicating in B subtilis. 2/ ADN selon la. revendication 1, caractérisé en ce que le fragment d'ADN bactérien codant pour 1'a-amylase comporte tout ou partie d'un fragment d'environ 3,3 kb limité par deux sites de restriction HindîlI.  2 / DNA according to the. Claim 1, characterized in that the bacterial DNA fragment encoding α-amylase comprises all or part of a fragment of about 3.3 kb bounded by two HindIII restriction sites. 3/ ADN selon l'une des revendications 1 et 2, caractériséen ce que le fragment d'ADN d'un plasmide est le fragment du plasmide pUB 110. 3 / DNA according to one of claims 1 and 2, characterized in that the DNA fragment of a plasmid is the fragment of plasmid pUB 110. 4/ ADN selon la revendication 3, caractérisé en e que 1'ADN d'un plasmide est un ADN hybride de pUB 110 et de pBR 322. 4 / DNA according to claim 3, characterized in that the DNA of a plasmid is a hybrid DNA of pUB 110 and pBR 322. 5/ Souche de Bacillus subtilis transforme par un ADN selon l'une des revendications 1 à 4. 5 / Bacillus subtilis strain transformed with a DNA according to one of claims 1 to 4. 6/ Procédé de production d'a-amylase caractérisé en ce que l'on cultive sur un milieu approprié une souche bactérienne selon la revendication 5 et on récupère l'a-amylase dans le milieu de croissance. 6 / A process for producing α-amylase characterized in that a bacterial strain according to claim 5 is cultivated on a suitable medium and the α-amylase is recovered in the growth medium. 7/ a-amylase thermos table obtenu par la mise en oeuvre du procédé selon la revendication 6.  7 / a-amylase thermos table obtained by carrying out the process according to claim 6.
FR8220904A 1982-09-24 1982-12-13 NOVEL IMPROVED RECOMBINANT DNA USEFUL IN THE PREPARATION OF A-AMYLASE BY BACILLUS SUBTIL Expired FR2537602B2 (en)

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FR8216099A FR2533583A1 (en) 1982-09-24 1982-09-24 Novel recombinant DNA which is useful in the preparation of alpha -amylase
FR8220904A FR2537602B2 (en) 1982-09-24 1982-12-13 NOVEL IMPROVED RECOMBINANT DNA USEFUL IN THE PREPARATION OF A-AMYLASE BY BACILLUS SUBTIL

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0124076A2 (en) * 1983-04-25 1984-11-07 Sanraku-Ocean Co., Ltd. Method for preparation of recombinant plasmids, microorganisms transformed by said plasmids and thermo-stable alpha-amylase
FR2582316A1 (en) * 1985-05-22 1986-11-28 Centre Nat Rech Scient A-AMYLASE EXPRESSION VECTORS IN BACILLUS SUBTILIS, STEM OBTAINED AND PROCESS FOR PREPARING A-AMYLASE
EP0252774A1 (en) * 1986-05-09 1988-01-13 Transgene S.A. Saccharomyces strains producing alpha-amylase

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0032238A2 (en) * 1980-01-07 1981-07-22 The University Of Rochester Recombinant bacteriophage for heterologous cloning of bacillus microorganisms and method for its production
EP0034470A2 (en) * 1980-02-15 1981-08-26 Cpc International Inc. Genetically engineered microorganisms for massive production of amylolytic enzymes and process for preparing same
EP0036259A2 (en) * 1980-03-10 1981-09-23 Cetus Corporation Method,vectors and organisms for transported cloned heterologous gene products in bacillus subtilis and E.coli
GB2091268A (en) * 1980-12-31 1982-07-28 Palva Ilkka Recombinant-DNA-molecules and methods for protein production

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0032238A2 (en) * 1980-01-07 1981-07-22 The University Of Rochester Recombinant bacteriophage for heterologous cloning of bacillus microorganisms and method for its production
EP0034470A2 (en) * 1980-02-15 1981-08-26 Cpc International Inc. Genetically engineered microorganisms for massive production of amylolytic enzymes and process for preparing same
EP0036259A2 (en) * 1980-03-10 1981-09-23 Cetus Corporation Method,vectors and organisms for transported cloned heterologous gene products in bacillus subtilis and E.coli
GB2091268A (en) * 1980-12-31 1982-07-28 Palva Ilkka Recombinant-DNA-molecules and methods for protein production

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0124076A2 (en) * 1983-04-25 1984-11-07 Sanraku-Ocean Co., Ltd. Method for preparation of recombinant plasmids, microorganisms transformed by said plasmids and thermo-stable alpha-amylase
EP0124076A3 (en) * 1983-04-25 1986-06-04 Sanraku-Ocean Co., Ltd. Method for preparation of recombinant plasmids, microorganisms transformed by said plasmids and thermo-stable alpha-amylase
FR2582316A1 (en) * 1985-05-22 1986-11-28 Centre Nat Rech Scient A-AMYLASE EXPRESSION VECTORS IN BACILLUS SUBTILIS, STEM OBTAINED AND PROCESS FOR PREPARING A-AMYLASE
EP0205371A1 (en) * 1985-05-22 1986-12-17 Centre National De La Recherche Scientifique (Cnrs) Expression vectors for alpha-amylase in bacillus subtilis, strains obtained and process for the preparation of alpha-amylase
EP0252774A1 (en) * 1986-05-09 1988-01-13 Transgene S.A. Saccharomyces strains producing alpha-amylase

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