BE1007313A3 - Pullulanase, micro-organisms producing same, methods for preparing saidpullulanase and uses thereof - Google Patents

Abstract

The invention relates to a thermostable pullulanase, having the property tohydrolyse alpha-1,6 type glucoside bonds in amylopectin and showing an enzymeactivity in acidic media and at a temperature of approximately 60 degrees C.The invention also relates to micro-organisms producing said pullulanase andmethods for preparing said pullulanase. The invention also relates to theuses thereof and formulations comprising said pullulanase. The invention alsorelates to a DNA molecule and a vector containing said DNA molecule.<IMAGE>

Description

       

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   Pullulanase, microorganisms producing it, processes for preparing this pullulanase and uses thereof
The invention relates to a new pullulanase. The invention also relates to a new strain of microorganisms producing this pullulanase and the methods for preparing this pullulanase. The invention also relates to the uses and compositions comprising it. The invention also relates to a DNA molecule containing the gene for this pullulanase and an expression vector containing this DNA molecule, useful for expressing pullulanase in strains of Bacillus.



   Starch, the essential constituents of which are amylose and amylopectin, can be converted into simple sugars by an enzymatic process carried out in two stages: a stage of liquefaction of starch and a stage of saccharification of liquefied starch . In order to achieve a high conversion rate of starch, it has already been proposed to add during the saccharification of the liquefied starch an enzyme hydrolyzing the glucosidic bonds a-1, 6, such as, for example, a pullulanase.



   In European patent 0 063 909, a so-called debranching enzyme is described, that is to say capable of hydrolyzing the glucosidic bonds 1, 6 in amylopectin, which has a pullulanase activity and has an optimum of activity at a pH of 4-5 at 60 C. This enzyme is derived from a strain of Bacillus acidopullulyticus.



   Furthermore, in US Pat. No. 5,055,403, a pullulanase has been proposed which exhibits enzymatic activity in

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 acid medium and derived from a strain of Bacillus naganoensis. This enzyme has maximum activity at a pH of around 5
 EMI2.1
 measured at 60 C and a maximum of activity at a temperature of approximately 62.5 C measured at a pH of 4.5.



   Although active at acidic pH and at a temperature of around 60 ° C. and therefore usable during the saccharification of liquefied starch, the pullulanases of the prior art have the drawback of being very unstable under such conditions temperature and pH, their half-life at a temperature of 60 C and a pH of about 4.5 in the absence of substrate not exceeding a few tens of minutes.



   Consequently, there is currently a need for a pullulanase which can be used during the saccharification of liquefied starch, which is very stable over a wide range of temperature and pH, and in particular at a temperature of around 60 ° C. and at a pH d 'about 4.5.



   The present invention aims to provide a new pullulanase, active at acidic pH, having a thermostability at acidic pH very much greater than that of the pullulanases of the prior art and a half-life of several hours under these aforementioned conditions.



   The present invention also aims to identify, isolate and provide a strain, and in particular a strain of Bacillus, which naturally produces said pullulanase.



   The present invention also aims to isolate and provide a nucleotide sequence coding for said pullulanase.



   The present invention also aims to prepare and provide an expression vector containing the nucleotide sequence coding for said pullulanase.



   The present invention also aims to prepare and provide a Bacillus host transformed with said nucleotide sequence or with the expression vector containing the nucleotide sequence of the Bacillus strain coding for said pullulanase.



   To this end, the invention relates to a pullulanase produced by a Bacillus, and more particularly by an aerobic and non-thermophilic microorganism, and preferably by the Bacillus

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 deramificans T 89. 117D or by a derivative or mutant of this strain of Bacillus deramificans.



   The invention also relates to a mutated pullulanase obtained by modification of the nucleotide sequence of the gene which codes for the pullulanase defined above.



   Preferably the isolated and purified pullulanase consists of a single type of polypeptide, having a molecular weight of approximately 100 (10) kDa.



   In a particularly preferred manner, said pullulanase has an isoelectric point between 4.1 and 4.5.



   The pullulanase according to the invention is thermostable and active over a wide temperature range. Pullulanase is active at acidic pH.



   Said pullulanase is capable of catalyzing the hydrolysis of the glucosidic bonds α-1,6 present both in amylopectin and in pullulan. It is therefore an enzyme called debranching or debranching.



   In addition, the N-terminal sequence (SEQ ID NO: 1) of said pullulanase is as follows in the amino-carboxy direction and from left to right:
Asp Gly Asn Thr Thr Thr Ile Ile Val His
1 5 10
Tyr Phe Cys Pro Ala Gly Asp Tyr Gln Pro
15 20
Preferably, the pullulanase according to the invention degrades the pullulan to maltotriose and the amylopectin to amylose.



   The invention relates to a thermostable and active pullulanase at acidic pH, having a half-life of approximately 55 hours measured at a temperature of approximately 60 C in a buffered solution at a pH of approximately 4.5 and the absence of substrate. Preferably said pullulanase is capable of hydrolyzing the glucosidic bonds of the a-1, 6 type in amylopectin.



  By half-life duration is meant that the pullulanase shows a relative enzymatic activity of at least 50% measured after

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 an incubation of 55 hours at a temperature of approximately 60 C in a buffered solution at a pH of approximately 4.5 and in the absence of substrate.



   The pullulanase according to the invention is thermostable at acidic pH. Indeed, the pullulanase according to the invention shows a relative enzymatic activity of at least 55% measured after an incubation of 40 hours at a temperature of 60 C in a solution buffered to a pH of about 4.5 and in absence of substrate. It shows a relative enzymatic activity of at least 70% measured after an incubation of 24 hours under these same conditions.



   By relative enzymatic activity is meant the ratio between the enzymatic activity, measured during a test carried out under given conditions of pH, temperature, substrate and duration, and the maximum enzymatic activity measured during this same test, the enzymatic activity being measured from the hydrolysis of pullulan and the maximum enzymatic activity being arbitrarily fixed at the value of 100.



   The pullulanase according to the invention is moreover stable over a wide range of acidic pHs. Under the conditions described below, it is active at a pH between 3 and 7. In fact, said pullulanase shows a relative enzymatic activity of at least
 EMI4.1
 minus 85% measured after an incubation of 60 minutes at a temperature of approximately 60 C in the absence of substrate and in a pH range between approximately 3.5 and approximately 5.8. Preferably, it shows a relative enzymatic activity greater than 90% measured in a pH range of between approximately 3.8 and approximately 5 under these same conditions.



   The pullulanase according to the invention develops an optimal enzymatic activity measured at a temperature of around 60 C in a pH range between 4.0 and 4.8. Preferably said pullulanase develops an optimal enzymatic activity measured at a temperature of approximately 60 C at a pH of approximately 4.3.



   The pullulanase according to the invention also develops an optimal enzymatic activity, measured at a pH of around 4.3, in a temperature range between 55 and 65 C, and more particularly at 60 C.



   The pullulanase according to the invention develops an activity

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 more than 80% of the maximum enzymatic activity (the maximum enzymatic activity being measured at a temperature
 EMI5.1
 at 60 C and at a pH of 4.3) in a pH range of between about 3.8 and about 4.9 for a temperature of around 60 C.



   The pullulanase according to the invention also has all the suitable properties compatible with the real industrial conditions for saccharification of starch. These properties are an optimum pH below 5, an optimum temperature around 60 C and good stability of the enzyme under these conditions of acidic pH and high temperature. The acid medium is imposed by the simultaneous use of glucoamylase and pullulanase during the industrial saccharification of starch. In fact, the glucoamylase used for the saccharification of starch is generally produced by a fungus and in particular by a strain of Aspergillus, such as Aspergillus niger, Aspergillus awamori or Aspergillus foetidus.

   The ideal conditions suitable for the saccharification of liquefied starch in the presence of a glucoamylase are a temperature of approximately 60 ° C. and a pH of approximately 4.0 to 4.5.



  This is particularly the case for the glucoamylase sold under the brands DIAZYHE L-200 by SOLVAY ENZYMES (Elkhart, United States) and OPTIDEX by SOLVAY ENZYMES (Hannover, Germany). Furthermore, the saccharification stage lasts several hours, generally from 40 to 60 hours, it is essential that the enzymes used are stable, active and effective throughout this stage, these enzymes must therefore exhibit high thermostability in an acid medium. and the longest half-life possible.



  This is why the pullulanase of the present invention is more effective than the known pullulanases.



   The present invention also relates to a process for the production of a pullulanase comprising the cultivation of an aerobic (and not thermophilic) bacterium capable of producing the pullulanase in a suitable nutritive medium containing sources of carbon and nitrogen and mineral salts under aerobic conditions and the harvest of the pullulanase thus obtained.



   The present invention also relates to a process for the production of a pullulanase comprising the culture of the strain of Bacillus deramificans T 89.117D (LMG P-13056) or a derivative of

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 this strain capable of producing pullulanase in an appropriate nutritive medium containing carbon and nitrogen sources and mineral salts under aerobic conditions and the harvest of the pullulanase thus obtained.



   The culture conditions of these bacteria such as components of the culture medium, culture parameters, temperature, pH, aeration, agitation, are well known to those skilled in the art.



   The carbon sources of the culture medium are usually chosen from starch, partially hydrolyzed starch, soluble starch, oligosaccharides, glucose, amylose, amylopectin or a mixture of two or more of these. this. The carbon sources of the culture medium are preferably chosen from partially hydrolyzed starch, glucose or a mixture of these. Good results have been obtained with glucose. The sources of nitrogen in the culture medium are usually chosen from yeast extract, soy flour, cottonseed meal, fish meal, gelatin, potato flour or a mixture of two. or more of these. The sources of nitrogen in the culture medium are preferably chosen from yeast extract, soy flour or a mixture of these.

   Good results have been obtained with yeast extract. The mineral salts of the culture medium are generally chosen for the anions from chloride, carbonate, phosphate, sulfate and for the cations from potassium, sodium, ammonium, magnesium, calcium or a mixture of two or more of these. Good results have been obtained
 EMI6.1
 with a mixture of the following salts KH2P04, K2HP04. 3H20, (NH4) 2SO4, MgClHO and Caca2. 2H20.



  The culture is generally carried out at a temperature between 20 and 45 C. and preferably between 25 and 40 C.



   The culture is generally carried out at a pH of between 3.5 and 6 and preferably between 4 and 6.



   The culture is carried out under aerobic conditions in the presence of air or oxygen and with stirring.



   The techniques for harvesting the produced pullulanase are well known to those skilled in the art. Usually, centrifugation, ultrafiltration, evaporation, precipitation are used.

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 tation, filtration, microfiltration, crystallization or a combination of either of these techniques such as centrifugation followed by ultrafiltration.



   The pullulanase can then be purified, if necessary, enzyme purification techniques are known to those skilled in the art.



   Pullulanase can also be spray dried or freeze dried.



   The present invention also relates to the identification and supply of a new isolated aerobic bacterium producing pullulanase. Generally they belong to the family of Bacillaceae. Preferably they belong to the genus Bacillus. In a particularly preferred manner, said Bacillus is the strain of Bacillus deramificans T 89.117D or a derivative or mutant of this strain.



   By derivative or mutant of this strain is meant any naturally or artificially modified bacteria. The derivatives of this strain can be obtained by known modification techniques such as ultraviolet radiation, X-rays, mutagens or genetic engineering.



   The strain of Bacillus deramificans T 89.117D was deposited in the collection named BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS (LMG culture collection, University of Ghent, Laboratory of Microbiology-KL Ledeganckstraat 35, B-9000 Ghent, Belgium) in accordance with the Budapest Treaty under LMG number P-13056.



   The strain of the present invention has been identified by its biochemical characteristics: Gram positive bacteria, aerobic which is in the form of rod, it forms an endospore.



   The invention also relates to the isolation and supply of a DNA molecule comprising a nucleotide sequence encoding the pullulanase of Bacillus deramificans T 89.117D (LMG P-13056). Preferably, this DNA molecule comprises the entire pullulanase gene from Bacillus deramificans T 89.117D.



   The invention also relates to the preparation and supply of an expression vector containing the DNA molecule which comprises the nucleotide sequence which codes for the pullulanase of Bacillus deramificans T 89.117D. Preferably the

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 DNA molecule comprises the structural gene which codes for the pullulanase of Bacillus deramificans T 89.117D. In a particularly preferred manner, this vector is the vector pUBDEBRA1.



   By expression vector is meant any DNA sequence which comprises a replicon and other regions of DNA (nucleotide sequences) which is functional independently of the host as a complete gene unit.



   By complete gene expression unit is meant the structural gene and the region (s) of the promoter and the regulatory region (s) necessary for transcription and translation. By structural gene is meant the coding sequence which is used as a template for the synthesis of RNA and allows the synthesis of protein by the host.



   The preferred expression vector is the vector pUBDEBRA1. This vector contains the gene which codes for the pullulanase of the strain of Bacillus deramificans T 89.117D according to the invention. This vector can be introduced into an appropriate host. Generally this host is a strain of Bacillus. Preferably this host is a strain of Bacillus licheniformis. In a particularly preferred manner, this host is a strain of Bacillus licheniformis SE2.



  Excellent results have been obtained with this vector when it is introduced into the strain of Bacillus licheniformis SE2 delapl, used as a host.



   The present invention also relates to recombinant strains in which said gene coding for pullulanase is introduced by genetic engineering techniques. The gene can be introduced onto a plasmid or integrated into the host chromosome in one or more copies, the promoter can be modified or replaced by another promoter better suited to the receiving host, the gene itself can be modified so as to produce a mutant pullulanase.



   The invention also relates to the strains of microorganisms different from the starting producer organism, in which the gene coding for pullulanase is introduced by transformation, either in form integrated into chromosomal DNA, or in self-replicating form (plasmid).



   The invention also relates to a mutated pullulanase obtained by modifying the nucleotide sequence of the gene which codes

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 for pullulanase defined above.



   The invention also relates to a process for the preparation of a recombinant pullulanase, the process comprising the isolation of a DNA fragment coding for pullulanase, the insertion of this DNA fragment into an appropriate vector, the introduction of this vector into an appropriate host or the introduction of this DNA fragment into the chromosome of an appropriate host, the culture of this host, the expression of pullulanase and the harvesting of pullulanase. The appropriate host is generally chosen from the group consisting of the microorganisms Escherichia coli, Bacillus or Aspergillus. Usually the host is chosen from Bacillus. Preferably the host is chosen from aerobic Bacillus microorganisms.

   In a particularly preferred manner, the host is chosen from the microorganisms Bacillus subtilis, Bacillus licheniformis, Bacillus alcalophilus, Bacillus lentus, Bacillus amyloliquefaciens or Bacillus deramificans T 89.117D (LMG P-13056).



   Good results have been obtained when the host for the expression of the pullulanase according to the present invention is a recombinant strain derived from Bacillus licheniformis, and preferably the strain of Bacillus licheniformis SE2 delapl.



   The Bacillus licheniformis SE2 strain was deposited on June 21, 1993 in the collection named BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS (LMG culture collection, Ghent, Belgium) in accordance with the Budapest Treaty under number LMG P-14034.



   The transformed strain (SE2 delapl) thus obtained from Bacillus licheniformis SE2 differs from the parent strain by the only fact that it does not contain in its chromosome the DNA sequence which codes for the mature protease.



   The invention also relates to a pullulanase produced, heterologously, by a microorganism of the genus Bacillus which contains a gene coding for an alkaline protease when it is in the wild state. Preferably, this microorganism is a strain of Bacillus licheniformis comprising the DNA molecule which comprises the nucleotide sequence which codes for the pullulanase of Bacillus deramificans T 89.117D. In a particularly preferred manner, the gene coding for the alkaline protease has been deleted from this strain of Bacillus. This strain is preferably the

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 Bacillus licheniformis SE2 delapl.



   By heterologously produced is meant a production which is not carried out by the natural microorganism, that is to say the microorganism which contains in the wild state the gene which codes for pullulanase.



   The pullulanase according to the invention has multiple outlets in various industries, such as, for example, the food industries, the pharmaceutical industries or the chemical industries.



   Pullulanase can in particular be used in baking as an "anti-staling", that is to say as an additive to prevent the bread from becoming stale during its conservation or in brewery during the manufacture of beers with low calorie content .



   Pullulanase can also be used when preparing low-calorie foods in which amylose is used as a fat substitute.



   For food applications, pullulanase can be immobilized on a support. Enzyme immobilization techniques are well known to those skilled in the art.



   The pullulanase according to the invention is particularly suitable for the treatment of starch and pullulan.



   The invention relates to the use of pullulanase for the saccharification of liquefied starch.



   The present invention also relates to the use of pullulanase during a process for degrading starch or partially hydrolyzed starch comprising a step of saccharification of starch or partially hydrolyzed starch in the presence of a pullulanase. Generally this process is carried out in the presence of one or more other enzymes such as glucoamylase, a-amylase, S-amylase, a-glucosidase or other saccharifying enzymes.



   Given its biochemical properties, the pullulanase of the present invention makes it possible to carry out the saccharification step under strongly acid conditions, that is to say up to at least a pH of 3.9. This pH is more acidic than that acceptable by known pullulanases.



   The present invention also relates to enzyme compositions comprising the pullulanase according to the invention.

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   The compositions comprising the pullulanase of the present invention can be used in solid or liquid form.



   Pullulanase is formulated according to the intended uses.



  Stabilizers or preservatives can also be added to the enzyme compositions comprising the pullulanase according to the invention. For example, the pullulanase can be stabilized by the addition of propylene glycol, ethylene glycol, glycerol, starch, pullulan, a sugar such as glucose, a salt such as sodium chloride or a mixture of two or more of these products. Good results have been obtained with the addition of propylene glycol.



   The enzymatic compositions according to the invention can also comprise, in addition to the pullulanase, one or more other enzymes. Such enzymes are in particular carbohydrate hydrolases, such as, for example, glucoamylase, a-amylase, $ -amylase, a-glucosidase, isoamylase, cyclomaltodextrin-glucotransferase, p-glucanase, (x-glucosidase, glucoseisomerase, saccharifying enzymes, enzymes that cut carbohydrate links, or a mixture of two or more of these.



   The present invention preferably relates to an enzyme composition comprising a glucoamylase and a pullulanase.



   FIG. 1 represents the restriction map of the plasmid pUBDEBRA1.



   FIG. 2 represents the restriction map of the plasmid pLD1.



   FIG. 3 represents the restriction map of the plasmid pUBCDEBRAllDNSI.



   The meaning of the symbols and abbreviations used in these figures is collated in the following table.
 EMI11.1
 
 <tb>
 <tb>



  Symbol <SEP> Meaning
 <tb> Abbreviation
 <tb> ORIEC <SEP> origin <SEP> from <SEP> replication <SEP> in <SEP> E. <SEP> coli
 <tb> REP <SEP> protein <SEP> required <SEP> for <SEP> the <SEP> replication
 <tb> ORI + <SEP> origin <SEP> from <SEP> replication <SEP> from <SEP> strand <SEP> +
 <tb>
 

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 EMI12.1
 
 <tb>
 <tb> ORI-origin <SEP> from <SEP> replication <SEP> from <SEP> brinKMR <SEP> gene <SEP> bringing <SEP> the <SEP> resistance <SEP> to <SEP> the <SEP> kanamycin
 <tb> BLMR <SEP> gene <SEP> bringing <SEP> the <SEP> resistance <SEP> to <SEP> the <SEP> bleomycin
 <tb> AMPR <SEP> gene <SEP> bringing <SEP> the <SEP> resistance <SEP> to <SEP> ampicillin
 <tb> PP <SEP> pre-sequence
 <tb> BLIAPR <SEP> sequence <SEP> encoding <SEP> for <SEP> the <SEP> protease <SEP> alkaline <SEP> from <SEP> B.
 <tb> licheniformis
 <tb> 5'BLIAPR <SEP>

  sequence <SEP> 5'slocated <SEP> in <SEP> upstream <SEP> from <SEP> the <SEP> sequence <SEP> encoding
 <tb> for <SEP> the <SEP> protease <SEP> alkaline <SEP> from <SEP> B. <SEP> licheniformis
 <tb> 3'BLIAPR <SEP> sequence <SEP> 3'slocated <SEP> in <SEP> downstream <SEP> from <SEP> the <SEP> sequence <SEP> encoding
 <tb> for <SEP> the <SEP> protease <SEP> alkaline <SEP> from <SEP> B. <SEP> licheniformis
 <tb>
 
The present invention is illustrated by the following examples.



  Example 1
The Bacillus deramificans T89.117D strain was isolated from the soil on an agar nutrient medium and selected for its ability to degrade a colored derivative of pullulan known as AZCL-pullulane and sold by the company MEGAZYME.
 EMI12.2
 



  This strain was cultured at 37 ° C. in the MYE growth medium, the composition of which is as follows: KHZP04 33 mM; K2HP04. 3H20 6 mM; (NHSO 45 mM; MgCl2. 6H20 1 mM; Cal2. 2H20 1 mM; Yeast extract 0.5% (weight / volume); Glucose 0.5% (weight / volume). The pH of the medium is adjusted to pH 4 , 5 with H3P04.



   The agar medium (MYE / agar) contains 2 X (weight / volume) additional agar.



   The strain of the present invention has been identified by its biochemical characteristics: Gram positive bacteria, aerobic which is in the form of rod, it forms an endospore.



  It therefore belongs to the genus Bacillus.



   The vegetative cells of this strain in culture on the
 EMI12.3
 MYE medium at 37 C have a bacillus shape of size 0.7 x 3.0-3.5 pm. The mobility of vegetative cells is low.



  After growing for three days at 37 ° C. on the MYE medium, microscopic observation reveals the presence of (sub) terminal sporangia slightly deformed and in the shape of an ellipse.



   The catalase test is weakly positive in the presence of

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 10% hydrogen peroxide. The oxidase test is positive in the presence of 1% tetramethyl-1,4-phenylenediammoniumdichloride.



   This strain is aerobic, i.e. it develops aerobically. It does not develop anaerobically, that is to say under an atmosphere of 84% (v / v) N2, 8% (v / v) C02,
 EMI13.1
 8% (v / v) H at 37 C, on the other hand it develops in microanaerobiosis, that is to say under an atmosphere of 82. 5% (v / v) N2, 6% (v / v) 22 7. 5% (v / v) H, 4% (v / v) C02 to 37 C.% (v / v) represents a percentage expressed in volume by volume.



   This strain is not thermophilic. It presents a normal development after incubation in MYE medium at 20 C, 30
 EMI13.2
  C, 37 C and 45 C, on the other hand it does not develop at 50 C and 55 C. It exhibits normal development after incubation in MYE medium buffered with phosphate buffer at the following pH pH 4.0, pH 4.5, pH 5.0 and pH 5.5, however it does not develop at pH 7.0. It exhibits normal development after incubation in MYE medium in the presence of NaCl at concentrations of 2.0% (weight / volume) and 3.5% (weight / volume), exhibits poor development in the presence of 5.0% (weight / volume) of NaCl and does not develop in the presence of 7.0% (weight / volume) of NaCl.



   This strain does not hydrolyze casein: indeed no lysis zone could be observed after more than 2 weeks of incubation at 37 C. It decomposes tyrosine weakly, does not produce acetoin from pyruvate and does not reduce nitrate to nitrite or N2.



   The strain of Bacillus deramificans T89.117D according to the invention is taxonomically different from the strain of Bacillus acidopullulyticus described in European patent 0 063 909 and of the strain of Bacillus naganoensis described in US patent 5,055,403. The strain of Bacillus deramificans T89.117D shows growth at pH 4.7 to 5.5, shows no growth at pH 7.0, develops in the presence of 3.5% (weight / volume) NaCl, breaks down tyrosine and does not reduce nitrate to nitrite.



   The strain of Bacillus deramificans T89.117D has been deposited in the collection named BELGIAN COORDINATED COLLECTIONS OF MICRO-

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 ORGANISMS (LMG culture collection) under number LMG P-13056.



  Example 2
The strain of Bacillus deramificans T89.117D is cultured in a liquid medium (MYA) whose composition is identical to that of MYE medium with the exception of the content of yeast extract and glucose, that is to say
Yeast extract 2.5% (weight / volume)
Potato starch 2.5% (weight / volume).



   The culture is carried out with stirring, with effective aeration, at a temperature of 37 C.



   After 68 hours of culture, the pullulanase and the cell biomass are separated by centrifugation (5000 revolutions per minute for 30 minutes), the pullulanase produced by the strain of Bacillus deramificans T89.117D is extracellular.



   Then the pullulanase is concentrated by ultrafiltration, in order to obtain a concentrated aqueous solution of pullulanase.



   The enzymatic activity of the solution obtained is measured.



   A pullulanase enzyme unit (PUN) is defined as the amount of enzyme which, at a pH of 4.5, at a temperature
 EMI14.1
 of 60 C and in the presence of pullulan, catalyzes the release of reducing sugars at the rate of 1 μM of glucose equivalent per minute.



   The measurement of the pullulanase enzymatic activity is carried out according to the following protocol. 1 ml of a 1% pullulan solution in a 50 mM acetate buffer at pH 4.5 is incubated at 60 ° C. for 10 minutes. 0.1 ml of a pullulanase solution corresponding to an activity between 0.2 and 1 PUN / ml is added thereto. The reaction is stopped after 15 minutes by adding 0.4 ml of 0.5 M NaOH. The determination of the reducing sugars released is carried out by the method of SOMOGYI-NELSON (J. Biol. Chem. 153 (1944) 375-380 ; J. Biol. Chem. 160 (1945) 61-68), and also in the other examples of this application.



   A second method is used to perform the pullulanase assays. The enzymatic reaction in the presence of pullulan is carried out according to the conditions of the test, then is stopped by the addition of sulfuric acid (0.1 N). The pullulan hydrolysis products are then subjected to HPLC chromatography (HPX-87H column from BIO-RAD; the mobile phase is

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 consisting of 10 mM H2SO4) in order to separate the different constituents. The amount of maltotriose formed is estimated by measuring the area of the peak obtained.



   The so-called debranching activity, that is to say the hydrolysis of the glucosidic bonds a-1, 6 present in amylopectin, can be quantified by the increase in the blue coloration caused, in the presence of iodine, by the release of amylose from amylopectin.



   The measurement of the disconnecting enzymatic activity is carried out according to the following protocol. 0.4 ml of a solution
 EMI15.1
 of 1% amylopectin containing a 50 mM acetate buffer at pH 4.5 is incubated at 60 ° C. for 10 minutes. The reaction is started by the addition of 0.2 ml of pullulanase and it is stopped after 30 minutes by the addition of 0.4 ml of 0.3 M HCl 0.8 ml of an iodine solution at 0 0.0025% is then added to 0.2 ml of this reaction mixture and the optical density is measured at 565 nm.



   In order to purify the pullulanase, the concentrated aqueous pullulanase solution is diafiltered with 6 times 500 ml of a NaCl solution at 9 g / l and the pH of the aqueous solution thus obtained is adjusted to pH 3.5 by addition of 25% HCl at room temperature.



   The precipitate obtained is removed by centrifugation (5000 revolutions per minute for 30 minutes), the centrifugation supernatant is recovered. The pH of this supernatant is adjusted to pH 6.0 by addition of 5 M NaOH. The precipitate obtained is removed by centrifugation.



   The centrifugation supernatant is recovered, which is heated to 55 ° C. for 15 minutes.



   The precipitate formed is again removed by centrifugation.



  The centrifugation supernatant is recovered.



   Acetone is added to this supernatant at a concentration
 EMI15.2
 final 60% (volume / volume), the suspension formed is brought to 4 ° C. for 2 hours. The precipitate formed at 4 C is dissolved in a MES buffer (acid 2 (N-morpholino) ethanesulfonic) 20 mM, CaCl 2 lmM (pH 6.0).



  This pullulanase solution is called solution A.



   This solution A is again purified by ion exchange chromatography. A column of approximately 20 ml in volume

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 internal, sold under the brand S-SEPHAROSE HP HI LOAD 16/10, is previously balanced with a 50 mM CH3COONa buffer, 100 mM NaCl (pH 4.0) at a flow rate of 5 ml / minute. Solution A is diluted 10 times in the acetate buffer and 15 ml of this diluted solution are deposited on the column. An isocratic phase is ensured by elution of 80 ml of the acetate buffer (100 mM NaCl), followed by elution with 200 ml of the 50 mM acetate buffer (pH = 6.0) containing a linear gradient of NaCl (100-500 mM ).



   The pullulanase activity is measured in each fraction.



   The most active fractions are combined into a solution called B.



   From this solution B, precipitation is carried out with acetone at a final concentration of 80% (volume / volume). The precipitate obtained is dissolved in a volume of 0.6 ml of the 20 mM MES buffer, 1 mM CaCl 2 (pH 6.0).



   This pullulanase solution is called solution C.



   The results are collated in Table 1.



   TABLE 1
 EMI16.1
 
 <tb>
 <tb> Fractions <SEP> Volume <SEP> Proteins <SEP> Activity <SEP> pullulanase <SEP> Activity
 <tb> specific
 <tb> 1 <SEP> 1 <SEP> ml <SEP> Img / mllTotall <SEP>% <SEP> 1 <SEP> PUN / ml <SEP> Total <SEP>% <SEP> PUn / mg
 <tb> 1 <SEP> 1 <SEP> 1 <SEP> 1 <SEP> 1 <SEP> 1
 <tb> 1 <SEP> Solution <SEP> AI <SEP> 1.5 <SEP> 6.48 <SEP> 9.7 <SEP> 100 <SEP> 17.5 <SEP> 26.3 <SEP> 100 <SEP> 2.7
 <tb> @
 <tb> ISolution <SEP> BI <SEP> 12 <SEP> 10, <SEP> 0251 <SEP> 0.3 <SEP> 1 <SEP> 31 <SEP> 0.7 <SEP> 1 <SEP> 8, <SEP> 4 <SEP> 1 <SEP> 321 <SEP> 28 <SEP> 1
 <tb> @
 <tb>
 
Table 1 shows that this purification step increased by a factor of 10 the specific pullulanase activity of the enzyme solution.



   The debranching activity, that is to say the hydrolysis activity of the alpha-1,6 bonds in amylopectin, of pullulanase was also measured as described above, by iodine staining after hydrolysis of amylopectin. The results show that the unplugging activity has also been enriched.



  Example 3 Determination of the isoelectric point
IEF electrophoresis is carried out on solution C (iso

  <Desc / Clms Page number 17>

 electro focusing) in a pH gradient varying from 4.0 to 6.5.



   A volume corresponding to 0.12 units of pullulanase is deposited in triplicate on the gel. After migration, one third of the gel is stained with Coomassie blue.



   The other two parts of the gel are covered with agar gels (1% w / v) buffered with 100 mM CH3COONa, 1 mM CaCl2, 1 mM MgCl2 (pH 4.5) containing 0.1% (weight / volume) respectively. ) of AZCL-pullulan or 1% (weight / volume) of amylopectin. The whole (acrylamide gel-agar gel) thus obtained is then incubated at 60 ° C. in a saturated atmosphere in relative humidity for 16 hours. The gel coated with the amylopectin overlay is then incubated at room temperature in a solution containing 3 mM 12'50 mM KI in order to reveal the unplugging activity by the appearance of the blue coloration.



   The revelation with iodine of the amylopectin gel reveals a dark blue halo, sign of an unplugging activity, at an isoelectric point comprised between 4.1 and 4.5 for the enzyme of the present invention. The revelation of pullulanase activity indicates the same result.



   This demonstrates that the pullulanase of the present invention exhibits pullulanase activity and debranching activity.



   This demonstrates that the pullulanase of the present invention is capable of hydrolyzing the a-1, 6 type bonds, both in pullulan and in amylopectin. This demonstrates a low specificity of the pullulanase of the present invention vis-à-vis its substrate.



  Example 4 pH and temperature of enzymatic activity
The enzymatic activity of pullulanase is measured at different temperatures (55.60 and 65 ° C.) and at different pHs (from 3.25 to 7) in 50 mM citrate-phosphate buffer by measuring the reducing sugars released. Solution C of pullulanase is used, as obtained in Example 2, diluted to approximately 1 PUN / ml.



   The results are collated in Table 2.



   During this test, the maximum enzymatic activity was measured, by the measurement of the reducing sugars released, for the sample placed at a pH of approximately 4.3 and at a temperature

  <Desc / Clms Page number 18>

 around 60 C. By definition, this sample was therefore assigned a relative enzymatic activity of 100%.



   This example shows that the pullulanase according to the invention has an optimal enzymatic activity measured at a temperature of around 60 ° C. in a pH range between 4.0 and 4.8.



   This example also shows that the pullulanase according to the invention has an optimal enzymatic activity, measured at a pH of about 4.3, in a temperature range between 55 and 65 C.



   In addition, this example shows that the pullulanase according to the invention develops an enzymatic activity of more than 80% of the maximum enzymatic activity in a pH range of between approximately 3.8 and approximately 4.9.
 EMI18.1
 TABLE 2
 EMI18.2
 
 <tb>
 <tb> @
 <tb> Activity <SEP> relative <SEP> from <SEP> the enzyme
 <tb> @
 <tb> pH <SEP> Temperature <SEP> C
 <tb> 55 <SEP> 60 <SEP> 65
 <tb> @
 <tb> 3, <SEP> 25 <SEP> 1 <SEP> 5, <SEP> 7 <SEP> 1 <SEP> 2.2 <SEP> 1 <SEP> 4, <SEP> 3 <SEP> 1
 <tb> 3, <SEP> 75 <SEP> 1 <SEP> 80, <SEP> 8 <SEP> 1 <SEP> 83, <SEP> 7 <SEP> 1 <SEP> 11, <SEP> 5
 <tb> 4, <SEP> 30 <SEP> 87, <SEP> 9 <SEP> j <SEP> 100 <SEP> j <SEP> 84, <SEP> 1
 <tb> 4, <SEP> 90 <SEP> 82, <SEP> 4 <SEP> 87, <SEP> 1 <SEP> 68
 <tb> 5.50 <SEP> 50.6 <SEP> 39, <SEP> 6 <SEP> 1 <SEP> 13, <SEP> 5
 <tb> 6, <SEP> 00 <SEP> 1 <SEP> 7, <SEP> 5 <SEP> 1 <SEP> 2, <SEP> 9 <SEP> 0
 <tb> 6,

  40 <SEP> 0 <SEP> 0 <SEP> 0
 <tb>
 Example 5 Determination of the half-life of the enzyme
The pullulanase solution A is diluted, as obtained in Example 2, so that it develops an enzymatic activity of approximately 0.7 PUN / ml in a 100 mM sodium acetate buffer at a pH of 4.5. The diluted solution containing the pullulanase is incubated at 60 ° C. and samples are taken at different times.

  <Desc / Clms Page number 19>

 



   A measurement of the enzymatic activity by the reducing sugar method (SOMOGYI method described above) is then carried out. The results are collated in Table 3.



   TABLE 3
 EMI19.1
 
 <tb>
 <tb> Time <SEP> Activity <SEP> relative
 <tb> hours <SEP>%
 <tb> 0 <SEP> 100
 <tb> 16 <SEP> 76
 <tb> 24 <SEP> 74
 <tb> 40 <SEP> 57
 <tb> 48 <SEP> 54
 <tb> 64 <SEP> 47
 <tb>
 
 EMI19.2
 This example shows that pullulanase is thermostable at acidic pH.



  This example shows that the half-life of pullulanase is approximately 55 hours under these conditions. Indeed, pullulanase has a relative enzymatic activity of at least 50% measured after an incubation of 55 hours at a temperature of approximately 60 C in a buffered solution at a pH of approximately 4.5, and in the absence of substrate .



  This example further shows that the pullulanase according to the invention has a relative enzymatic activity of at least 55 X measured after an incubation of 40 hours at a temperature of approximately 60 C in a solution buffered at a pH of approximately 4, 5 and in the absence of substrate. This example also shows that it has a relative enzymatic activity of at least 70% measured after an incubation of 24 hours under these same conditions.



  Example 6 Stability of the Pullulanase with Respect to the pH The solution A of the pullulanase, as obtained in Example 2, is diluted so that it develops an enzymatic activity of approximately 0.7 PUN / ml, in different 100 mM citratephosphate buffers at pH varying between pH 3.0 and 8.0.

  <Desc / Clms Page number 20>

 incubate the various dilute solutions containing pullulanase for 60 minutes at 60 C.



   Then the enzymatic activity of these different solutions is measured after incubation for 60 minutes at pH 4.2 at 60 ° C. in the presence of 1.6% (weight / volume) of pullulan. The amount of maltotriose formed is measured by HPLC chromatography. The results are collated in Table 4.



   This example shows that the pullulanase according to the invention is stable over a wide range of acid pH, in fact it has
 EMI20.1
 a relative enzymatic activity of at least 85 X measured after an incubation of 60 minutes at a temperature of approximately 60 C in the absence of substrate and in a pH range between approximately 3.5 and approximately 5.8. This example also shows that it has a relative enzymatic activity greater than 90% measured in a pH range of between approximately 3.8 and approximately 5 under these same conditions and that it is inactivated only at a lower pH or equal to 3 or greater than or equal to 7.



   TABLE 4
 EMI20.2
 
 <tb>
 <tb> 1 <SEP> 1 <SEP> 1
 <tb> 1 <SEP> pH <SEP> 1 <SEP> Activity <SEP> relative <SEP>% <SEP> 1
 <tb> 3 <SEP> 0
 <tb> 3.5 <SEP> 90
 <tb> 1 <SEP> 4 <SEP> 1 <SEP> 98 <SEP> 1
 <tb> 1 <SEP> 4, <SEP> 5 <SEP> 1 <SEP> 100 <SEP> 1
 <tb> 1 <SEP> 5 <SEP> 1 <SEP> 96 <SEP> 1
 <tb> 1 <SEP> 5, <SEP> 5 <SEP> 1 <SEP> 92 <SEP> 1
 <tb> 1 <SEP> 6 <SEP> 1 <SEP> 89 <SEP> 1
 <tb> 6.5 <SEP> 75
 <tb> 7 <SEP> 0
 <tb>
 Example 7 and Example 8R (comparison)
A saccharification medium is prepared by suspending corn starch at a concentration of 35% (weight / weight) by weight of starch dry matter and calcium chloride at a concentration of 0.02% (weight /volume).

  <Desc / Clms Page number 21>

 



   This corn starch suspension is liquefied in the presence of α-amylase, sold under the brand name TAKATHERM L-340 by SOLVAY ENZYMES, at 105 ° C. for 5 minutes at pH 6.0.



   The liquefied starch thus obtained is rapidly cooled to a temperature of 95 ° C. and the hydrolysis is continued for 120 minutes at 95 ° C. with stirring. At this stage the degree of hydrolysis is between 10 and 12 DE (DE represents the unit of "Dextrose Equivalents", that is to say the number of reducing ends expressed in glucose equivalent)? ? ? ?



   The liquefied starch thus obtained is diluted to a final concentration of 32 g of dry weight per 100 g of saccharification medium.



   The saccharification medium obtained is cooled to a temperature of 60 C.



   The pH of this saccharification medium is adjusted to different values with acetic acid, from 3.9 to 4.8 and is kept constant during saccharification.



   A quantity of glucoamylase corresponding to 0.176 DU / g is added to the saccharification medium. ds. (glucoamylase enzyme unit per g of dry matter in the saccharification medium), the glucoamylase used is sold under the brand
DIAZYME L-200 by SOLVAY ENZYMES.



   For example 7 according to the invention, a quantity of pullulanase, corresponding to 0.075 PUN / g, is also added to the saccharification medium. ds., in the form of a concentrated aqueous solution of pullulanase, as described in Example 2.



   Comparative Example 8R is carried out, as described above for Example 7, but without the addition of pullulanase.



   After 48 hours, the saccharification is stopped and the products obtained are analyzed by chromatography. The results are collated in Table 5.



   This example shows that the pullulanase of the invention is effective in saccharification. The pullulanase of the invention therefore has all the suitable properties compatible with the real industrial conditions for starch saccharification.



   This example shows that the starch conversion rate is higher in the presence of the pullulanase according to the invention, at different pHs, and this up to a very acidic pH, that is to say at

  <Desc / Clms Page number 22>

 minus 3.9.
TABLE 5
 EMI22.1
 
 <tb>
 <tb> pH <SEP> Examples <SEP> Products <SEP> obtained <SEP> in <SEP>% <SEP>> DP3
 <tb> Glucose <SEP> DP2 <SEP> DP3
 <tb> 3.9 <SEP> 8R <SEP> 94.18 <SEP> 2.92 <SEP> 0.54 <SEP> 2.37
 <tb> 7 <SEP> 95.63 <SEP> 2.90 <SEP> 0.73 <SEP> 0.73
 <tb> 4.2 <SEP> 8R <SEP> 94.18 <SEP> 2.98 <SEP> 0.56 <SEP> 2.29
 <tb> 7 <SEP> 94.79 <SEP> 4.30 <SEP> 0.56 <SEP> 0.38
 <tb> 4.5 <SEP> 8R <SEP> 93.72 <SEP> 2.88 <SEP> 0.57 <SEP> 2.83
 <tb> 7 <SEP> 95.49 <SEP> 3.00 <SEP> 0.75 <SEP> 0.76
 <tb> 4.8 <SEP> 8R <SEP> 93.32 <SEP> 2.79 <SEP> 0.60 <SEP> 3.30
 <tb> 7 <SEP> 95.25 <SEP> 2.70 <SEP> 0.87 <SEP> 1,

  18
 <tb>
   DP2 represents the oligosaccharides containing two units of glucose (glucose dimer), DP3 the oligosaccharides containing three units of glucose (glucose trimer),> DP3 the oligosaccharides containing more than 3 units of glucose.



  Example 9 and Example 10R (comparison)
Example 7 is repeated, but by fixing the pH of saccharification medium at a pH of 4.2.



   A quantity of glucoamylase corresponding to 0.17 DU / g is added to the saccharification medium. ds. (enzymatic unit per g of dry matter of saccharification medium), the glucoamylase used is sold under the brand DIAZYME L-200 by SOLVAY ENZYMES.



   For example 9 according to the invention, various amounts of pullulanase are also added to the saccharification medium, corresponding respectively to 0.0325 PUN / g. ds., 0.050 PUN / g. ds., 0.075 PUN / g. ds. and 0.10 PUN / g. ds. (pullulanase enzyme unit per gram of dry matter of saccha-

  <Desc / Clms Page number 23>

 rification), in the form of a concentrated aqueous solution of pullulanase, as described in Example 2.



   Comparative Example 10R is carried out, as described above for Example 9, but without the addition of pullulanase.



   The results are collated in Table 6.



   This example shows that the amount of pullulanase that must be used to observe an increase in the percentage of glucose produced is less than 0.0325 PUN / g. ds.



   TABLE 6
 EMI23.1
 
 <tb>
 <tb> 1 <SEP> Examples <SEP> 1 <SEP> Pullulanase <SEP> 1 <SEP> Products <SEP> obtained <SEP> in <SEP>%
 <tb> PUN / g.ds.
 <tb>



  10 <SEP> 0 <SEP> 94.78 <SEP> 3.55 <SEP> 0.73 <SEP> 0.94
 <tb> 9 <SEP> 0.0325 <SEP> 95.16 <SEP> 3.45 <SEP> 0.78 <SEP> 0.61
 <tb> 0, <SEP> 050 <SEP> 95, <SEP> 30 <SEP> 3, <SEP> 39 <SEP> 0, <SEP> 74 <SEP> 0, <SEP> 56
 <tb> 1 <SEP> 1 <SEP> 0, <SEP> 075 <SEP> 1 <SEP> 95.25 <SEP> 3, <SEP> 47 <SEP> 0, <SEP> 74 <SEP> 0, <SEP> 55
 <tb> 0, <SEP> 10 <SEP> 95, <SEP> 27 <SEP> 3, <SEP> 49 <SEP> 0, <SEP> 70 <SEP> 0, <SEP> 53
 <tb> @
 <tb>
 
 EMI23.2
 Example 11 The plasmid pUBDEBRA1 (FIG. 1) contains the gene which codes for the pullulanase of the strain of Bacillus deramificans T 89. 117D under the control of its own transcription promoter, introduced into the vector pUB131. The construction of the plasmid pUBDEBRA1 is described below.



  The chromosomal DNA is extracted from a culture of the strain of Bacillus deramificans T 89. 117D (identified under the number LMG P-13056).



  To do this, a culture of 200 ml of this bacillus is carried out in a MYE liquid medium. When in the stationary phase, it is centrifuged at 5000 revolutions per minute for 10 minutes. The centrifugation pellet thus obtained is taken up in 9 ml of TRIS-HC1 buffer (tris (hydroxymethyl) aminomethane acidified with HC1) 0.1 M, at a pH of 8, EDTA (ethylenediaminetetraacetic acid) 0.1 M, NaCl 0, 15 M containing 18 mg of lysozyme, the suspension thus obtained is incubated 15

  <Desc / Clms Page number 24>

 
 EMI24.1
 minutes at 37 C.



  The lysate thus obtained is then treated with 200 μl of a 10 mg / ml RNAse solution for 20 minutes at 50 C. 1 ml of a 10% SDS (sodium dodecyl sulfate) solution is then added to this lysate . Then this lysate is incubated for 30 minutes at 70 C.



   Then the lysate is cooled to approximately 45 ° C., 0.5 ml of a proteinase K solution at 20 mg / ml is then added thereto (prepared immediately).



   The lysate is incubated at 45 ° C. with manual shaking until a transparent solution is obtained.



   Several phenol extractions are carried out from this transparent solution under the conditions and following the procedures described in Molecular Cloning-a laboratory manual-SAMBROOK, FRITSCH, MANIATIS-second edition, 1989, on page E. 3, up to obtaining a clean interface, as described there.



   The DNA is precipitated with 20 ml of ethanol. The precipitate is recovered by centrifugation at 5000 revolutions per minute for 5 minutes, then suspended in 2 ml of TE buffer at pH 8.0 (10 mM TRIS-HCl, 1 mM EDTA at pH 8.0).



   The DNA thus obtained is then partially cleaved by the restriction enzyme Sau3AI. The restriction conditions in this example, and in all the other examples of this application, are those described by SAMBROOK et al (page 5,28-5, 32), except that these restriction conditions are increased by a factor of 10 , in order to obtain a sufficient quantity of DNA for the following purification steps.



   The ratio between the quantity of DNA used and the quantity of enzyme is adjusted in order to obtain a maximum of fragments of size between 5 and 10 kbp (kbp: 103 base pairs).



   All the fragments thus obtained are then subjected to an agarose gel electrophoresis (0.8%) as described by SAMBROOK et al (page 6.01-6, 19) and the fragments of size between 5 and 10 kpb are isolated and purified by the GENE CLEAN method. They are then ligated with the plasmid pBR322 which is sold by the company BIOLABS, [CLONTECH LABORATORIES

  <Desc / Clms Page number 25>

   (U. S. A.) catalog no. 6210-1] cut at the BamHI site and dephosphorylated as described by SAMBROOK et al (page 1.60-1. 61).



  This same technique is used in the other examples.



   The ligation thus obtained is transformed into E. coli MC1061 cells [CLONTECH LABORATORIES, catalog no C-1070-1] by electroporation (SAMBROOK et al, page 1.75-1. 81); the transformed strains are selected on a Petri dish containing LB medium (Luria-Bertani) agar and 100 μg / ml of ampicillin, after growth at 37 ° C. for approximately 18 hours. The LB medium is described by SAMBROOK et al (page A. 4). This medium contains 10 g / l of tryptone, 5 g / l of yeast extract and 10 g / l of sodium chloride.



   The colonies obtained on these dishes are then replicated on two dishes of the same medium.



   One of the two boxes is covered with an agar medium
 EMI25.1
 containing 1% (weight / volume) of agar, 100 mM sodium acetate (pH 4.5) and 0.1% (weight / volume) of AZCL-pullulan. After incubation at 60 oC for 18 hours, the colony showing the most significant area of AZCL-pullulan hydrolysis is identified, and the corresponding colony is isolated on the other replicated dish.



   The 4.5 kbp EcoRI-BamHI fragment of the plasmid pBRDEBRA3 is obtained by double digestion of the plasmid pBRDEBRA3 with BamHI and EcoRI, then purification by agarose gel electrophoresis (0.8 × weight / volume). This fragment is then ligated with the vector pUB131, (described in European patent application 0 415 296), which has previously been the subject of a double digestion with BamHI and EcoRI, at the BamHI and EcoRI sites, using the strain of Bacillus subtilis PSL1 as host.



   The plasmid pUBDEBRA1, thus obtained, is isolated and purified from the transformed PSL1 cells by the technique of alkaline lysis (SAMBROOK et al, pages 1.25-1. 28). This same technique is used in the other examples.



   All transformed Bacillus subtilis strains are capable of expressing the pullulanase gene and secreting pullulanase.



   The transformed PSL1 strains containing the plasmid pUBDEBRA1 are subcultured on a Petri dish containing the

  <Desc / Clms Page number 26>

 LB medium with 25 μg / ml of kanamycin.



   The colonies obtained are covered with an agarose overlay (1% weight / volume) containing AZCL-pullulan (0.1% weight / volume) and sodium acetate (100 mM, pH 4.5 ). After an incubation at 60 ° C. for 18 hours, it is observed that all the colonies of the transformed strains are surrounded by a hydrolysis halo of AZCL-pullulan.



  Example 12 Identification of the terminal parts of the alkaline protease gene of the host strain of Bacillus licheniformis SE2.



   This example relates to the identification of the terminal parts of the alkaline protease gene of the Bacillus licheniformis host strain, in order to prepare the deletion plasmid to delete said Bacillus licheniformis SE2 gene.



   1. Extraction of chromosomal DNA from B. licheniformis SE2.



   In order to isolate the alkaline protease gene from the chromosomal DNA of Bacillus licheniformis SE2, the chromosomal DNA is first extracted, using the method described in Example 11 for the extraction of the chromosomal DNA , with the exception of the culture medium consisting of LB medium, and it is purified.



   2. Identification of the C-terminal part of the alkaline protease gene.



   The extracted chromosomal DNA is subjected to a restriction analysis, analysis described in Molecular Cloning-SAMBROOK et al. (page 1.85) and Molecular Cloning, a laboratory Manual.



  MANIATIS et al, 1982 Cold Spring Harbor Laboratory, pages 374-379). The DNA fragments obtained from these digests are separated according to their size on an agarose gel at 0.8 X (weight / volume).



   The agarose gel is then subjected to an analysis by the SOUTHERN BLOT technique (technique described by SAMBROOK et al - page 9.31), in order to identify the fragments which contain the nucleotide sequences of the C-terminal part of the gene alkaline protease.



   The constructed probe which is used for hybridizations,

  <Desc / Clms Page number 27>

 is a synthetic oligonucleotide corresponding to the part
C-terminal of the alkaline protease gene. The technique used to construct the synthetic oligonucleotide is described in BEAUCAGE, S. L. et al. (1981), Tetrahedron Letters,
22: 1859-1882 and using p-cyanoethyl phosphoramidites in a BIOSEARCH CYCLONE SYNTHESIZER apparatus. The sequence of the synthetic oligonucleotide which has been constructed is as follows (SEQ ID NO: 2):
5'-GGCGGAGCAAGCTTTGTGG-3 '
These results show that the C-terminal part of the alkaline protease gene is located on the PstI fragment of approximately
2.7 kbp.



   Hybridization with DNA probes is carried out according to the technique described in Molecular Cloning-SAMBROOK et al.- page 9.52-9. 55. This same technique is used in the other examples.



   The preparation of chromosomal DNA extracted from the strain of Bacillus licheniformis SE2 is then digested with the enzyme PstI and the fragments obtained are separated according to their size by agarose gel electrophoresis (0.8%).



   The fragments obtained of approximately 2.7 kbp are extracted from the gels and purified by the technique known as "Gene Clean" using glass beads and placed on the market by the company BI0101 (U. S. A.).



  The 2.7 kbp PstI fragments are then ligated (SAMBROOK et al, pages 1.68-1. 69) with the plasmid pUC18 (CLONTECH
Laboratories, n 6110-1), which was previously digested at the site
PstI and dephosphorylated. The ligation thus obtained was then transformed into Escherichia coli MC1061 cells by the) CaCl2 technique (SAMBROOK et al-page 1.82-1. 84). The technique for dephosphorylating DNA fragments or linearizing vectors is described by SAMBROOK et al (pages 1. 60-1. 61). The ligation technique is also described by SAMBROOK et al (pages 1.68-1. 69).



  5 The transformed strains are selected from boxes of
Petri dish containing LB agar medium and supplemented with 100 ug / ml

  <Desc / Clms Page number 28>

 ampicillin. Strains transformed from E. coli MC1061 thus obtained are then selected by hybridization with the labeled synthetic oligonucleotide using the C-terminal probe used in the study of SOUTHERN and the plasmid pKC1 is isolated.



   The synthetic oligonucleotide is labeled by phosphorylation with ATP [y-32p] using the T4 phage T4 polynucleotide kinase and following the technique described by SAMBROOK et al (page 11. 31-11.33).



   3. Identification of the N-terminal part of the alkaline protease gene.



   The extracted chromosomal DNA is subjected to restriction analysis. The DNA fragments obtained from these digests are separated according to their size on a 0.8% agarose gel.



   The agarose gel is then subjected to an analysis by the SOUTHERN BLOT technique, in order to identify the fragments which contain the nucleotide sequences of the N-terminal part of the alkaline protease gene.



   The probe which is used for the hybridizations is a synthetic oligonucleotide corresponding to the N-terminal part of the alkaline protease gene. The sequence of the synthetic oligonucleotide which has been constructed is as follows (SEQ ID NO: 3): 5'-ATGGCTCCTGGCGCAGGC-3 '
These results show that the N-terminal part of the alkaline protease gene is located on the PstI fragment of approximately 5.5 kbp and also on a smaller BclI-PstI fragment of approximately 2 kbp. This fragment does not contain the XbaI, Clal, Hpal and SphI restriction sites.



   The preparation of chromosomal DNA extracted from the strain of Bacillus licheniformis SE2 is then digested with the enzyme PstI and the fragments obtained are separated according to their size by agarose gel electrophoresis (0.8%).



   The fragments obtained of approximately 5.5 kbp are extracted from the gels and purified by the so-called "Gene Clean" technique.

  <Desc / Clms Page number 29>

 



   The 5.5 kbp PstI fragments thus obtained are then subjected to a series of digestions with Bell, XbaI, Clal, HpaI and SphI. The DNA fragments thus produced are ligated with the plasmid pMK4, as described in SULLIVAN et al., (1984), Gene 29: 21-26, which has been previously linearized with BamHI and PstI. The plasmid pMK4 can be obtained from the collection B. G. S. C.



  (Bacillus Genetic Stock Center (Ohio State University) Columbus, Ohio, U. S. A.) under number 1E29.



   The ligations thus obtained were then transformed into Escherichia coli MC1061 cells by the CaCl technique.



   The transformed strains are selected on petri dishes containing the agar LB medium and supplemented with 100 μg / ml of ampicillin. Strains transformed from E. coli MC1061 thus obtained are then selected by hybridization with the labeled synthetic oligonucleotide using the N-terminal probe in the study of SOUTHERN and the plasmid pKP1 is isolated.



  Example 13
The sequences of the fragments introduced into the plasmids pKP1 and pKCl are determined from the Pst1 sites to the SacI sites, according to the technique described by SAMBROOK et al (pages 13.15 and 13.17 and FIG. 13.3B).



  Example 14
The plasmid pLD1 (FIG. 2) is constructed with the aim of preparing the strain of Bacillus licheniformis SE2 delapl. The construction of the plasmid pLD1 is described below.



   The plasmid pKP1 (as obtained in Example 12) is unstable in E. coli MC1061. This is why the chromosomal DNA fragment containing the N-terminal part of the alkaline protease gene of B. licheniformis SE2 was introduced into the vector pACYC184 (BIOLABS, U. S. A., under the number &num; 401-M). This introduction was carried out by introducing the EcoRI-EcoRI fragment of 1849 bp of the plasmid pKP1 into the EcoRI site of the plasmid pACYC184 and the ligation is used to transform the cells of E. coli MC1061. The plasmid pKPN11 is thus obtained.



   The transformed strains are selected on a box

  <Desc / Clms Page number 30>

 Petri dish containing LB agar medium and supplemented with 12.5 ug / ml of tetracycline. The orientation of the EcoRI-EcoRI fragment of 1849 bp in the plasmid pKPN11 is determined by restriction analysis (SAMBROOK et al.-page 1.85 and MANIATIS et al-page 374-379).



   The plasmid pKPN12 is obtained in the following way: the StyI-StyI fragment of 1671 bp is removed from the plasmid pKPN11 by digestion with StyI, followed by replacement of this fragment with the following synthetic double strand DNA, which was produced beforehand: 5 '-CTTG GAGCTC GTTAAC AGATCT-3' (SEQ ID NO: 4) 3'-CTCGAG CAATTG TCTAGA GTTC-5 '(SEQ ID NO: 5) (StyI) Pal PalII bag (StyI)
The digestion of the plasmids with restriction enzymes is carried out according to the technique described by SAMBROOK et al.



  - 1989-chapters 5.28-5. 32.



   The DNA fragment coming from the plasmid pUB131 which codes for the resistance to kanamycin and to bleomycin or to phleomycin was obtained as follows:
The 2666 bp PstI-TaqI fragment, which carries the genes which code for resistance to kanamycin and bleomycin or to phleomycin, is obtained by double PstI-TaqI digestion of the plasmid pUB131. This fragment is introduced into the PstI-AccI sites of the plasmid pBS- (STRATAGENE, U. S. A., under the number 211202). The plasmid pBSKMPM is thus obtained.



   During the preparation of the plasmid pBSKMPM, a small deletion in the region of the binding with the plasmid pBS appears, which causes the loss of the SphI and PstI sites in the plasmid pBSKMPM. The plasmid pBSKMPM is used to produce a single-stranded DNA used to carry out site-directed mutagenesis with a view to introducing the two synthetic nucleotides whose SmaI sites are identified below, one is located upstream and the other in downstream of genes for resistance to kanamycin and phleomycin.



   The technique of site-directed mutagenesis is described by SAMBROOK et al-page 15,74-15, 79. It uses the mutagenesis kit sold by BIO-RAD (n 170-3576).

  <Desc / Clms Page number 31>

 
 EMI31.1
 



  The sequences of the synthetic oligonucleotides used for the mutagenesis are the following (respectively SEQ ID NO: 6 and SEQ ID NO: 7): 5'-CATCTAATCTTCAACACCCGGGCCCGTTTGTTGAAC-3 'SmaI 5'-CAAAATAAAAAAGATACAACCCGGGTCTCTCGTATCTA -AT plasmid obtained by SmtAtta mutagenesis in the presence of the two oligonucleotides is the plasmid pBSKMPM1. This plasmid contains two SmaI restriction sites which allow the isolation of the DNA fragment containing the genes which code for resistance to kanamycin and phleomycin.



  The 1597 bp SmaI-SmaI fragment of the plasmid pBSKMPM1 is then introduced into the SmaI site of the plasmid pKPN12, the plasmid pKPN14 is thus obtained.



  The correct orientation of the fragment introduced into the plasmid pKPN14 is verified by making a selection on the plasmid DNA preparations by restriction analysis (SAMBROOK et al-page 1, 85).



  The DNA fragment, present on the plasmid pKC1 and located downstream of the N-terminal sequence of the alkaline protease, is isolated on the SacI-HindIII fragment of 1.2 kbp from the plasmid pKC1 (as obtained from Example 12) by digestion, first with HindIII.



  The 5 'protruding end of HindIII is rendered stretched by treatment with the Klenow fragment of DNA polymerase (SAMBROOK et al-page F, 2-F, 3). The SacI restriction is then performed in order to produce the desired SacI-HindIII stretch fragment. This fragment is introduced into the HpaI and SacI sites of the plasmid pKPN14, producing the plasmid pLID1.



  All these contructions are carried out by transformation of the strain of E. coli MC1061 in the presence of tetracycline (12 pg / ml) for the selection of the transformed strains.



  A plasmid capable of multiplying in B. subtilis and

  <Desc / Clms Page number 32>

 in B. licheniformis, is constructed from the plasmid pLIDI by replacing the replication functions of E. coli, which are carried by the BglII-BglII fragment of 3623 bp of the plasmid pLIDI, by the fragment which carries the replication functions of Bacillus: BglII-BamHI fragment of 2238 bp isolated from the plasmid pUB131.



   This replacement of the replication functions of E. coli by those of Bacillus was carried out by first isolating the 3.6 kbp BglII-BglII fragment from the plasmid pLIDI by digestion of the plasmid pLID1 with BglII and BamHI. An additional BamHI digestion was necessary, indeed a BglII digestion alone would result in fragments of identical size which could not be separated by agarose gel electrophoresis.



  The 3.6 kbp BglII-BglII fragment is then cloned, into the Bacillus subtilis SE3 strain, into the 2238 bp BglII-BamHI fragment which was isolated from the plasmid pUB131, producing the plasmid pLD1 (FIG. 2).



   The Bacillus subtilis SE3 strain was deposited on June 21, 1993 in the collection named BELGIAN COORDINATED COLLECTIONS OF MICROORGANISM in accordance with the Budapest Treaty under number LMG P-14035.



  Example 15
The desired modifications in the chromosomal DNA of the strain of Bacillus licheniformis SE2 are carried out by techniques based on homologous recombination. The modifications are made to produce the Bacillus licheniformis SE2 delapl strain.



   The plasmid pLD1 is transformed into B. licheniformis SE2 by the protoplast technique described by MANIATIS et al (page 150-151) and under the conditions defined with the exception of the following modifications: the lysozyme powder is added at 5 mg / ml in the SMP, instead of 1 mg / ml as defined in step 7 of the procedure described, the incubation to obtain maximum lysis with lysozyme is 60 minutes, the regeneration is carried out in DM3 medium (described by Molecular Biological Methods
 EMI32.1
 for Bacillus (Harwood et al, eds) John Wiley and Sons (1990) (pages 150-151) containing 200 ug / ml kanamycin.



  A transformed strain is isolated and the restriction card

  <Desc / Clms Page number 33>

 of the plasmid is verified (pLD1).



   The transformed strain is cultured in 50 ml of LB medium supplemented with 2 g / l of glucose and 25 μg / ml of kanamycin, for 18 hours at 37 C.



   A culture sample (0.1 ml) is taken and used to inoculate an Erlenmeyer flask containing 50 ml of the same LB medium. The
 EMI33.1
 culture is incubated at 37 ° C for 18 hours. A sample of this culture is taken and tested on a petri dish containing the agar LB medium and supplemented with 25 ug / ml of kanamycin and 1% (weight / volume) of skimmed milk (DIFCO), to detect the presence of protease.



   The absence of a hydrolysis halo around the colonies which grow on these petri dishes indicates that these colonies are unable to produce an alkaline protease.



   The cultures and the tests are repeated until a strain (apr-, Kmr) is obtained, that is to say both no longer producing alkaline protease (apr-) and resistant to kanamycin (Kmr) .



   The plasmid pLD1 present in this strain of Bacillus licheniformis SE2 delapl is then removed from the latter by culture on a growth medium at 37 Oc in the absence of antibiotic.



   This strain is cultured in 50 ml of LB medium supplemented with 2 g / 1 of glucose, for 18 hours at 37 ° C. A volume of 0.1 ml of this culture is taken and used to inoculate another Erlenmeyer flask also containing 50 ml of the same medium, the culture lasts 18 hours at 37 C. A sample is then taken and spread on a Petri dish LB medium. The isolated colonies are subcultured on a second dish of LB medium supplemented with 25 μg / ml of kanamycin. A strain sensitive to kanamycin (KmS) is isolated. We confirm its phenotype (apr-, KmS).



   The chromosomal DNA of this strain is then isolated and purified and the structure of the chromosomal deletion is verified by the technique of SOUTHERN BLOT. The deletions identified are correct as to their position, having taken place by a homologous double recombination in the sequences located upstream (5 ') and downstream (3') of the alkaline protease gene.



   The strain obtained is called B. licheniformis SE2 delapl.

  <Desc / Clms Page number 34>

 



  It does not produce an alkaline protease.



  Example 16
The plasmid pUBDEBRA1 (Figure 1) is extracted from its host, isolated and purified.



   A culture of the B. licheniformis SE2 delapl strain is prepared, then this strain is transformed with this plasmid.



   The transformation of the B. licheniformis SE2 delapl strain by the plasmid pUBDEBRA1 is carried out according to the protoplast technique.



   The transformed strain is selected, isolated and purified.



  Then it is cultivated.



  Example 17
The strain of B. licheniformis SE2 delapl transformed by the plasmid pUBDEBRA1, as obtained in Example 16, is brought into use.
 EMI34.1
 culture for 17 hours at 37 ° C. in an LB preculture medium supplemented with 0.5% (weight / volume) of glucose and 20 μg / ml of kanamycin. This preculture is transferred (5% volume / volume) into 50 ml of M2 medium supplemented with 20 μg / ml of kanamycin.



  The culture is incubated with shaking for 80 hours at 37 C. After 80 hours, the biomass is eliminated by centrifugation at 5000 revolutions per minute for 10 minutes. The centrifugation supernatant is kept. The enzymatic activity is measured on this supernatant and the presence of pullulanase activity is noted.



  Example 18
This example concerns the integration of the gene coding for pullulanase into the chromosome of the Bacillus licheniformis SE2 delapl strain.
 EMI34.2
 



  To do this, the 4.5 kb EcoRI-BamHI fragment of the plasmid pBRDEBRA3 is cloned into the EcoRI and BamHI sites of the vector pUBC131 by transformation of the E. coli strain MC1061, thus generating the plasmid pUBCDEBRA11.



   The integration vector pUBCDEBRA11DNSI (FIG. 3) is then constructed by deleting the NsiI-Nsil fragment of 886 bp from the plasmid pUBCDEBRA11. The plasmid thus obtained lost the possibility of replicating in Bacillus, by the loss of the NsiI fragment of 886 bp.



  * To carry out this construction, the plasmid pUBCDEBRA11

  <Desc / Clms Page number 35>

 is cleaved by the restriction enzyme NsiI, and the NsiI-NsiI fragment of 9368 bp is purified by agarose gel electrophoresis. This fragment is then subjected to a ligation in order to recircularize it. The ligation is transformed into E. coli MC1061, and the plasmid pUBCDEBRA11DNSl1 is obtained.



   In order to integrate the plasmid pUBCDEBRA11DNSl1 into the B. licheniformis SE2 delapl strain, it is necessary that this plasmid carry a DNA fragment homologous to the chromosomal DNA. We therefore cloned in the BamHI site of the integration vector pUBCDEBRAUDNSIl, chromosomal fragments Sau3AI from B. licheniformis.



   To do this, the chromosomal DNA extracted from the Bacillus licheniformis SE2 delapl strain is partially cleaved by the restriction enzyme Sau3AI. The DNA fragments of size between 1.5 and 3 kb are then purified by agarose gel and ligated with the plasmid pUBCDEBRA11DNSI cleaved by the restriction enzyme BamHI and dephosphorylated. The ligation thus obtained is transformed into MC1061 cells by electroporation. After selection on agar LB medium containing 100 μg / ml of ampicillin, approximately 3000 colonies are obtained.



  All of these colonies are suspended in LB medium and an extraction of the plasmids is carried out by the technique of alkaline lysis.



   The plasmid preparation thus obtained is then introduced by transformation by the protoplast technique into the strain of Bacillus licheniformis SE2 delapl. The transformed cells are selected on the regeneration medium DM3 (Molecular Biological Methods for Bacillus (Harwood, CR and Cutting, SM, Eds) J. Wiley and sons, 1990,150-151) for their resistance to phleomycin (17 ug / ml), this can only be conferred on them by chromosomal integration of one of the plasmids constructed above.



   The colonies thus obtained are subcultured on agar LB medium supplemented with 5 μg / ml of phleomycin and 0.06% of AZCL-pullulan. The colony exhibiting the largest hydrolysis halo of AZCL-pullulan is then isolated and subcultured on agar LB medium.



   An extraction of the plasmid content of this strain is

  <Desc / Clms Page number 36>

 then performed. The preparation thus obtained is subjected to an analysis by agarose electrophoresis gel, which shows the absence of plasmid.



   The chromosomal DNA is extracted and purified as described in Example 8 and subjected to an analysis by the SOUTHERN technique, which shows that the plasmid pUBCDEBRAllDNSI integrated into the chromosomal DNA by homologous recombination in a Sau3AI fragment of about 3 kb.



   This shows that the gene coding for the pullulanase of B. deramificans is expressed in B. licheniformis, in the integrated state in the chromosome.


    

Claims (16)

CLAIMS 1-Pullulanase characterized in that it is produced by the strain of Bacillus deramificans T 89.117D (LMG P-13056) or a derivative or mutant of this strain.  2-thermostable and active pullulanase at acidic pH, capable of hydrolyzing the glucosidic bonds of type a-1, 6 in amylopectin, characterized in that its N-terminal sequence (SEQ ID NO: 1) is the following in the amino-carboxy direction and from left to right: Asp Gly Asn Thr Thr Thr Ile Ile Val His 1 5 10 Tyr Phe Cys Pro Ala Gly Asp Tyr Gln Pro 15 20 3-Pullulanase according to claim 2, characterized in  EMI37.1  what it has a half-life of about 55 hours measured at a temperature of about 60 C in a buffered solution at a pH of about 4.5 and in the absence of substrate.  4-Pullulanase, characterized in that it is produced, heterologously, by a microorganism of the genus Bacillus which contains a gene coding for an alkaline protease when it is in the wild state.  5-Pullulanase according to claim 4 characterized in that  <Desc / Clms Page number 38>  that the gene encoding the alkaline protease has been deleted from the microorganism of the genus Bacillus.  6-A method for the production of a pullulanase according to any one of claims 1 to 3 characterized in that it comprises the culture of the strain of Bacillus deramificans T 89.117D (LMG P-13056) or a derivative of this strain capable of producing pullulanase in a suitable nutrient medium containing carbon and nitrogen sources and mineral salts under aerobic conditions and the harvest of the pullulanase thus obtained.  7-A method for the preparation of a pullulanase according to any one of claims 1 to 3 characterized in that it comprises the isolation of a DNA fragment coding for pullulanase, the insertion of this fragment of DNA in an appropriate vector, the introduction of this vector into an appropriate host or the introduction of this DNA fragment into the chromosome of an appropriate host, the culture of this host, the expression of pullulanase and the harvest pullulanase.  8-Use of a pullulanase according to any one of claims 1 to 5 for the treatment of starch.  9-DNA molecule comprising the nucleotide sequence which codes for the pullulanase of Bacillus deramificans T 89.117D (LMG P-13056).  10-DNA molecule according to claim 9 characterized in that it comprises the pullulanase gene from Bacillus deramificans T 89.117D.  <Desc / Clms Page number 39>    11-Expression vector containing the DNA molecule according to claim 9.  12-Expression vector pUBDEBRA1.  13-A transformed strain of Bacillus licheniformis comprising the DNA molecule according to claim 9.  14. A transformed strain of Bacilus licheniformis comprising the expression vector according to claim 12.  15-Pullulanase produced by the transformed strain of Bacillus licheniformis according to claim 13 or 14.  16-An isolated and purified culture of Bacillus deramificans T 89.117D and culture derived or mutated therefrom.