BE1006483A3 - Pullulanase, micro-organisms producing same, methods for preparing saidpullulanase, uses and formulations comprising same - 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.<IMAGE>

Description

       

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   Pullulanase, microorganisms producing it, methods for preparing this pullulanase, uses and compositions comprising it
The invention relates to a new pullulanase. The invention also relates to strains of microorganisms producing this pullulanase and methods of preparing this pullulanase. The invention also relates to the uses and compositions comprising it.



   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 . During the liquefaction stage, the starch is degraded to maltodextrins by a short-term treatment at high temperature at neutral pH in the presence of a thermostable α-amylase.



    The oc-amylase practically does not hydrolyze the glucosidic bonds K-1, 6, the bonds of this type present in amylopectin remain intact during the stage of liquefaction of the starch. During the saccharification step, the liquefied starch is degraded by a treatment at a temperature of approximately 60 ° C. and at a pH of between approximately 4 and 5 in the presence of a glucoamylase. This saccharification stage generally lasts from 40 to 60 hours.

   Glucoamylase degrades the polysaccharides composing the maltodextrins produced in the previous step, mainly by catalyzing the sequential removal of the monomeric glucose units from the non-reducing end, essentially by hydrolysis of the glucosidic bonds <x-1.4; it is also capable of hydrolyzing the glucosidic bonds oc-1, 6 but at a markedly lower speed. In addition, when high concentrations of dextrose are reached in the saccharification medium, the glucoamylase catalyzes the reverse reaction, that is to say the synthesis, from glucose, of oligosaccharides containing ci-1, 6 bonds. All these facts limit the conversion rate of starch.

   In order to remedy these drawbacks

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 and to achieve a high conversion rate of the 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, an enzyme known as debranching, that is to say capable of hydrolyzing the glucosidic bonds, is described. <xl, 6 in amylopectin, which exhibits pullulanase activity and has an optimum of activity at a pH of 4-5 to 60 C. This enzyme which derives from a strain of Bacillus acidopullulyticus is marketed by the NOVO NORDISK company under the PROMOZYME brand.



   Furthermore, in US Pat. No. 5,055,403, a pullulanase has been proposed having an enzymatic activity in an 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 OC 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.



   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.



   To this end the invention relates to a pullulanase, thermostable and active at acidic pH, capable of hydrolyzing the glucosidic bonds of type a-1, 6 in amylopectin, having an isoelectric point of approximately 4.5 and a duration of 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 in the absence of substrate.

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   This means that the pullulanase of the invention shows a relative enzymatic activity of at least 50% measured after an incubation of 55 hours at a temperature of approximately 60 C in a solution buffered to a pH of approximately 4.5 and the absence of substrate.



   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 thermostable at acidic pH, as it appears in FIG. 1 which represents the evolution of the relative enzymatic activity of pullulanase as a function of the incubation time, the incubation being carried out at a temperature of 60 OC and at a pH of 4.5, the maximum enzymatic activity being the initial activity in this case. 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 OC in a solution buffered at a pH of about 4.5 and in absence of substrate. It shows a relative enzymatic activity of at least 70 X measured after an incubation of 24 hours under these same conditions.



   The pullulanase according to the invention is moreover stable over a wide range of acidic pHs, as it appears in FIG. 2 which represents the evolution of the relative enzymatic activity of pullulanase as a function of the incubation pH, the incubation being carried out at a temperature of 60 ° C. for 60 minutes, the maximum enzymatic activity being measured at a pH of 4.5. Indeed, the pullulanase according to the invention shows a relative enzymatic activity of at least 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 around 5.8.

   It shows a relative enzymatic activity greater than 90% measured in a pH range between approximately

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 3.8 and around 5 under these same conditions. Under these same conditions, it is completely inactivated only at a pH less than or equal to 3 or greater than or equal to 7.



   The pullulanase according to the invention is active over a wide range of temperature and pH as it appears in FIG. 3 which represents the evolution of the relative enzymatic activity of pullulanase as a function of pH and temperature, the activity maximum enzymatic being measured at a pH of 4.3 and at a temperature of 60 C.



   The pullulanase according to the invention has an optimal enzymatic activity measured at a temperature of around 60 OC in a pH range of between 4.0 and 4.8.



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



   The pullulanase according to the invention develops an enzymatic activity of more than 80% of the maximum enzymatic activity in a range of pH between approximately 3.8 and approximately 4.9 for a temperature of approximately 60 C, the enzymatic activity maximum being measured at a temperature of 60 C and a pH of 4.3.



   The molecular weight of the pullulanase according to the invention is approximately 100 (10) kDa.



   The pullulanase according to the invention consists of a single type of polypeptide.



   The pullulanase according to the invention is capable of catalyzing the hydrolysis of the glucosidic bonds a-1, 6 present both in amylopectin and in pullulan. It is therefore an enzyme called debranching or debranching. The pullulanase according to the invention degrades the pullulan to maltotriose and the amylopectin to amylose. 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 OC and good stability of the enzyme under these conditions of acid pH and temperature

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 high. 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 notably the case for the glucoamylase sold under the brands DIAZYKE L-200 and OPTIDEX by SOLVAY ENZYMES. 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 the aerobic bacteria producing the pullulanase according to the invention. Generally they belong to the family of Bacillaceae. Preferably they belong to the genus Bacillus. Good results have been obtained with the Bacillus deramificans LMG P-13056 strain or a derivative of this strain.



   The strain of Bacillus deramificans LMG P-13056 was deposited in the collection named BELGIAN COORDINATED COLLECTIONS 0F MICROORGANISMS (LMG culture collection, University of Ghent, Belgium) in accordance with the Budapest Treaty.



   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.



   By derivative 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.

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   The present invention also relates to recombinant strains in which the gene coding for the pullulanase according to the invention 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 present invention also relates to a process for the production of a pullulanase comprising the cultivation of an aerobic bacterium capable of producing the pullulanase in an appropriate nutritive medium containing sources of carbon and nitrogen and mineral salts under aerobic conditions and harvesting 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 LMG P-13056 or a derivative of this strain capable of producing pullulanase in an appropriate nutritive medium containing sources of carbon and d nitrogen 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 sources of nitrogen in the culture medium are usually chosen from yeast extract, soy flour, flour

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 cottonseed, fish meal, gelatin, potato flour or a mixture of two or more of these. 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.



   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, filtration, microfiltration are used. 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 invention also relates to a mutated pullulanase obtained by modification of the nucleotide sequence of the gene which codes for the 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 microorganisms

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 Escherichia coli, Bacillus or Aspergillus. Usually the host is chosen from Bacillus.

   Preferably the host is chosen from the microorganisms Bacillus subtilis, Bacillus licheniformis, Bacillus alcalophilus, Bacillus lentus, Bacillus amyloliquefaciens or Bacillus deramificans LMG P-13056.



   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, P-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.

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



   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.



   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, 0-amylase, <x-glucosidase, isoamylase, cyclomaltodextrin-glucotransferase, p-glucanase, a-glucosidase, glucoseisomerase, saccharifying enzymes, enzymes which cut glucosidic 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 relative enzymatic activity of pullulanase as a function of the incubation time, for an incubation carried out at a temperature of 60 ° C. and at a pH of 4.5.



   FIG. 2 represents the relative enzymatic activity of pullulanase as a function of the incubation pH, for an incubation carried out at a temperature of 60 ° C. for 60 minutes.



   FIG. 3 represents the evolution of the relative enzymatic activity of pullulanase as a function of the pH and the temperature.



   FIG. 4 represents the restriction map of the plasmid pBRDEBRA 3.



   FIG. 5 represents the restriction map of the plasmid pUBC131.

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   FIG. 6 represents the restriction map of the plasmid pUBCDEBRA1.



   The present invention is illustrated by the following examples.



  Example 1
The Bacillus deramificans LMG P-13056 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 under the brand MEGAZYME.
 EMI10.1
 



  This strain was cultured at 37 ° C. in the growth medium MYE, the composition of which is as follows: MYE: H2PO4 33 mM KHPOHO 6 mM (NH4) 2SO4 45 mM MgCl2. 6H20 1 mM CaCl2. 2H20 1 mM Yeast extract 0.5%
Glucose 0.5%
The pH of the medium is adjusted to pH 4.5 with H3PO4
The agar medium (MYE / agar) contains 2% more 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
 EMI10.2
 MYE medium at 37 Oc have a bacillus size of 0.7 x 3.0, 3.5-3 µm. The mobility of vegetative cells is low.



  After a growth of three days at 37 oc 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 10 X of hydrogen peroxide. The oxidase test is positive in the presence of 1% tetramethyl-1,4-phenylenediammoniumdichloride.

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 EMI11.1
 



  This strain does not develop anaerobically, i.e. under an atmosphere of 84% N2'8% C02'8% H2 at 37 C, on the other hand it develops in microanaerobiosis, that is to say under a atmosphere of 82. 5% N2'6% 02, 7.5% H2'4% C02 at 37 OC.



  This strain presents a normal development after incubation in MYE medium at 20 C, 30 C, 37 C and 45 C, on the other hand it does not develop at 50 C and 55 C. It presents a 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 shows normal development after incubation in MYE medium in the presence of NaCl at concentrations of 2.0% and 3.5 X, present
 EMI11.2
 weak development in the presence of 5.0% NaCl and does not develop in the presence of 7.0% 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 LMG P-13056 according to the invention is taxonomically different from the strain of Bacillus acidopullulyticus described in European patent 0 063 909 and the strain of Bacillus naganoensis described in US patent 5,055,403. Certain biochemical characteristics described in these patents are collected in table 1 for comparison.

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 TABLE 1
 EMI12.1
 
 <tb>
 <tb> Test <SEP> B. <SEP> acidopul-B. <SEP> naganoensis <SEP> B.

    <SEP> deralulyticus <SEP> mificans
 <tb> Growth <SEP> to <SEP> pH <SEP> + <SEP> + <SEP> +
 <tb> 4,7-5, <SEP> 5
 <tb> Growth <SEP> to <SEP> pH <SEP> nd <SEP> nd
 <tb> 7.0
 <tb> Growth <SEP> in-nd <SEP> +
 <tb> 3.5 <SEP>% <SEP> NaCl
 <tb> Decomposition <SEP> from <SEP> - <SEP> - <SEP> +
 <tb> tyrosine
 <tb> Reduction <SEP> from
 <tb> nitrate <SEP> in <SEP> nitrite <SEP> + -
 <tb>
 + represents a positive response to the test, -represents a negative response to the test, nd means that the test has not been described in the patent under reference.



  Example 2
The Bacillus deramificans LMG P-13056 strain is cultured in a liquid medium (MYA), the composition of which is as follows:
KH2PO4 33 mM K2HP04. 3H20 6 mM (NH4) 2SO4 45 mM
 EMI12.2
 MgCl2. 6H20 1 mM CaCl2. 2H20 1 mM Yeast extract 2.5% Potato starch 2.5% The pH of the medium is adjusted to pH 4.5 with H3PO4. The culture is carried out with stirring, with aeration

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 effective, at a temperature of 37 C.



   After 68 hours of culture, the pullulanase and the cell biomass are separated by centrifugation, the pullulanase produced by the strain of Bacillus deramificans LMG P-13056 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
 EMI13.1
 of 60 OC 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 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).



   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 consists of 10 mM H 2 SO 4) in order to separate the various 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 oc-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.

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   The measurement of the disconnecting enzymatic activity is carried out according to the following protocol. 0.4 ml of a solution
 EMI14.1
 1 x amylopectin containing 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 , 0025 X 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, 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 final concentration of 60%, the suspension formed is brought to 4 ° C. for 2 hours. The precipitate formed at 4 OC is dissolved in a MES buffer (acid 2 (N-morpholino) ethanesulfonic) 20 mM, CaCl 2 1 mM (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 of internal volume, sold under the brand S-SEPHAROSE HP HI LOAD 16/10 by the company PHARMACIA, is previously equilibrated with a 50 mM CHgCOONa 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 acetate buffer

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 50 mM (pH = 6.0) containing a linear NaCl gradient (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%. 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 2.



   TABLE 2
 EMI15.1
 
 <tb>
 <tb> Fractions <SEP> Volume <SEP> Proteins <SEP> Activity <SEP> pullulanase <SEP> Activity
 <tb> specific
 <tb> ml <SEP> mg / ml <SEP> Total <SEP>% <SEP> PUN / ml <SEP> Total <SEP>% <SEP> PUN / mg
 <tb> Solution <SEP> A <SEP> 1.5 <SEP> 6.48 <SEP> 9.7 <SEP> 100 <SEP> 17.5 <SEP> 26.3 <SEP> 100 <SEP> 2.7
 <tb> Solution <SEP> B <SEP> 0.025 <SEP> 0.3 <SEP> 3 <SEP> 0.7 <SEP> 8.4 <SEP> 32 <SEP> 28
 <tb>
 
Table 2 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 a) Determination of the isoelectric point
An electrophoresis IEF (iso electro focusing) is carried out on solution C 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.

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   The other two parts of the gel are covered with agar gels (1%) buffered with CH3COONa 100 mM, CaC12 1 mM, MgC12 1 mM (pH 4.5) containing 0.1% of AZCL-pullulan sold respectively. the brand MEGAZYME, or 1 X 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 I2, 50 mM KI in order to reveal the debranching activity by the appearance of the blue coloration.



   The revelation with iodine of the amylopectin gel reveals a dark blue halo, sign of a disconnecting activity, at an isoelectric point of 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 out-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. b) pH and temperature of enzymatic activity
The enzymatic activity of pullulanase is measured at different temperatures (55.60 and 65 OC) 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 3.



   A graphical representation of the results obtained is given in FIG. 3. The abscissa represents the pH of the test and the ordinate represents the relative enzymatic activity in X. The curve * represents the test carried out at 60 oc, the curve + represents the test carried out at 55 oc and the curve &num; represents the test carried out at 65 C.



   During this test, the maximum enzymatic activity was measured for the sample placed at a pH of approximately 4.3 and at a

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 EMI17.1
 temperature of approximately 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.



   TABLE 3
 EMI17.2
 
 <tb>
 <tb> Activity <SEP> relative <SEP> from <SEP> the enzyme <SEP>%
 <tb> pH <SEP> Temperature <SEP> C
 <tb> 55 <SEP> 60 <SEP> 65
 <tb> 3.25 <SEP> 5.7 <SEP> 2.2 <SEP> 4.3
 <tb> 3.75 <SEP> 80.8 <SEP> 83.7 <SEP> 11.5
 <tb> 4.30 <SEP> 87.9 <SEP> 100 <SEP> 84.1
 <tb> 4.90 <SEP> 82.4 <SEP> 87.1 <SEP> 68
 <tb> 5.50 <SEP> 50.6 <SEP> 39.6 <SEP> 13.5
 <tb> 6.00 <SEP> 7.5 <SEP> 2.9 <SEP> 0
 <tb> 6.40 <SEP> 0 <SEP> 0 <SEP> 0
 <tb>
 c) 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 pullulanase is incubated at 60 ° C. and samples are taken at times

  <Desc / Clms Page number 18>

 different.



   A measurement of the enzymatic activity by the reducing sugar method is then carried out. The results are collated in Table 4.



   TABLE 4
 EMI18.1
 
 <tb>
 <tb> Time <SEP> Activity <SEP> relative
 <tb> hours
 <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>
 
A graphical representation of the results obtained is given in FIG. 1. The abscissa represents time in hours and the ordinate represents the relative enzymatic activity in X.



   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% 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
 EMI18.2
 that it has a relative enzymatic activity of at least 70% measured after an incubation of 24 hours under these same conditions. d) Stability of pullulanase with respect to pH

  <Desc / Clms Page number 19>

 
The pullulanase solution A, as obtained in Example 2, is diluted so that it develops an enzymatic activity of approximately 0.7 PUN / ml, in different 100 mM citrate phosphate buffers at pH varying between pH 3.0 and 8.0.

   The various dilute solutions containing the pullulanase are incubated for 60 minutes at 60 C.



   Then we measure the enzymatic activity of these different
 EMI19.1
 solutions after incubation for 60 minutes at pH 4.2 to 60 ° C. in the presence of 1.6% of pullulan. The amount of maltotriose formed is measured by HPLC chromatography. The results are collated in Table 5.



   A graphical representation of the results obtained is given in FIG. 2. The abscissa represents the pH and the ordinate represents the relative enzymatic activity in%.



   This example shows that the pullulanase according to the invention is stable over a wide range of acid pH, in fact it has
 EMI19.2
 a relative enzymatic activity of at least 85 X measured after an incubation of 60 minutes at a temperature of approximately 60 OC 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 X measured in a pH range 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.

  <Desc / Clms Page number 20>

 



  TABLE 5
 EMI20.1
 
 <tb>
 <tb> pH <SEP> Activity <SEP> relative <SEP>%
 <tb> 3 <SEP> 0
 <tb> 3.5 <SEP> 90
 <tb> 4 <SEP> 98
 <tb> 4.5 <SEP> 100
 <tb> 5 <SEP> 96
 <tb> 5.5 <SEP> 92
 <tb> 6 <SEP> 89
 <tb> 6.5 <SEP> 75
 <tb> 7 <SEP> 0
 <tb>
 
 EMI20.2
 Example 4 and Example 5R (comparison)
A saccharification medium is prepared by suspending corn starch at a concentration of 35% by weight of starch solids and calcium chloride at a concentration of 0.02%.



   This corn starch suspension is liquefied in the presence of α-amylase, sold under the brand TAKATBERK 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.

  <Desc / Clms Page number 21>

 



   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.



   For example 4 according to the invention, a quantity of pullulanase is also added to the saccharification medium, corresponding to 0.075 PUN / g. ds., in the form of a concentrated aqueous solution of pullulanase, as described in Example 2.



   Comparative Example 5R is carried out 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 6.



   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 conversion rate of starch 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 least 3.9 .

  <Desc / Clms Page number 22>

 
 EMI22.1
 TABLE 6
 EMI22.2
 
 <tb>
 <tb> pH <SEP> Examples <SEP> Products <SEP> obtained <SEP> in <SEP>% <SEP>> DP3
 <tb> Glucose <SEP> DP2 <SEP> DP3
 <tb> 3.9 <SEP> 5R <SEP> 94.18 <SEP> 2.92 <SEP> 0.54 <SEP> 2.37
 <tb> 4 <SEP> 95, <SEP> 63 <SEP> 2.90 <SEP> 0.73 <SEP> 0.73
 <tb> 4.2 <SEP> 5R <SEP> 94.18 <SEP> 2.98 <SEP> 0.56 <SEP> 2.29
 <tb> 4 <SEP> 94.79 <SEP> 4.30 <SEP> 0.56 <SEP> 0.38
 <tb> 4.5 <SEP> 5R <SEP> 93.72 <SEP> 2.88 <SEP> 0.57 <SEP> 2.83
 <tb> 4 <SEP> 95.49 <SEP> 3.00 <SEP> 0.75 <SEP> 0.76
 <tb> 4.8 <SEP> 5R <SEP> 93.32 <SEP> 2.79 <SEP> 0.60 <SEP> 3.30
 <tb> 4 <SEP> 95.25 <SEP> 2.70 <SEP> 0.87 <SEP> 1,

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



  Example 6 and example 7R (comparison)
Example 4 is repeated, but by fixing the pH of saccharification medium to 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.



   For example 6 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 enzymatic unit per gram of dry matter of saccharification medium), in the form of a concentrated aqueous solution of pullulanase, as described in Example 2.

  <Desc / Clms Page number 23>

 



   Comparative Example 7R is carried out without the addition of pullulanase.



   The results are collated in Table 7.



   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 7
 EMI23.1
 
 <tb>
 <tb> Examples <SEP> Pullulanase <SEP> Products <SEP> obtained <SEP> in <SEP>
 <tb> PUN / g. <SEP> ds.
 <tb>



  Glucose <SEP> DP2 <SEP> DP3 <SEP>> DP3
 <tb> 7R <SEP> 0 <SEP> 94.78 <SEP> 3.55 <SEP> 0.73 <SEP> 0.94
 <tb> 6 <SEP> 0, <SEP> 0325 <SEP> 95.16 <SEP> 3.45 <SEP> 0.78 <SEP> 0, <SEP> 61
 <tb> 0.050 <SEP> 95.30 <SEP> 3.39 <SEP> 0.74 <SEP> 0.56
 <tb> 0.075 <SEP> 95.25 <SEP> 3.47 <SEP> 0.74 <SEP> 0, <SEP> 55
 <tb> 0, <SEP> 10 <SEP> 95.27 <SEP> 3.49 <SEP> 0.70 <SEP> 0.53
 <tb>
 Example 8
This example concerns the cloning of the pullulanase gene.



   Chromosomal DNA is extracted from a culture of Bacillus deramificans LMG P-13056.



   To do this, a culture of 200 ml of this bacillus in the stationary phase is centrifuged. The centrifugation base as well
 EMI23.2
 obtained is taken up in 9 ml of TRIS-HCl buffer (tris (hydroxymethyl) aminomethane acidified with HCl) 0.1 M, at a pH of 8, EDTA (ethylenediaminetetraacetic acid) 0.1 M, 0.15 M NaCl containing 18 mg of lysozyme, the suspension thus obtained is incubated for 15 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 OC.



   Then the lysate is cooled to about 45 C, there

  <Desc / Clms Page number 24>

 then add 0.5 ml of a 20 mg / ml proteinase K solution (prepared immediately).



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



   Several phenol extractions are carried out from this transparent solution until a clean interface is obtained.



   The DNA is then precipitated using isopropanol.



   The DNA thus obtained is then partially cleaved by the restriction enzyme Sau3AI. The restriction conditions are those described in Molecular Cloning-a laboratory manual - (SAMBROOK, FRITSCH, MANIATIS-second edition, 1989 / 9.24-9. 28). 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 electrophoresis in agarose gel (0.8%), in sodium acetate buffer as described in Molecular Cloning-a laboratory manual - (SAMBROOK, FRITSCH, MANIATIS-second edition, 1989 / 6.22-6.35). The fragments of size between 5 and 10 kbp are isolated and purified by electroelution. They are then ligated with the plasmid pBR322 which is sold by the company BIOLABS, cut at the BamHI site and dephosphorylated as described in Molecular Cloning-a laboratory manual- (SAMBROOK, FRITSCH, MANIATIS-second edition, 1989 / 1.63-1. 73) .



   The ligation thus obtained is transformed into E. coli MC1061 cells (CASADABAN et al., 1980, J. Mol. Biol.



  138: 179-207) by electroporation (Molecular Cloning-a laboratory manual-SAMBROOK, FRITSCH, MANIATIS-second edition, 1989 /1.75); the transformed strains are selected on a Petri dish containing the LB medium (Luria-Bertani) agar and 100 μg / ml
 EMI24.1
 ampicillin, after growth at 37 C for approximately 18 hours. The LB 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

  <Desc / Clms Page number 25>

 on two boxes of the same medium.



   One of the two dishes is covered with an agar medium containing 1% agar, 100 mM sodium acetate (pH 4.5) and 0.1% AZCL-pullulan. After incubation at 60 ° C. overnight, the colonies showing a zone of hydrolysis of AZCLpullulane are identified, and the corresponding colony is isolated on the other replicated dish.



   The plasmid (pBRDEBRA 3) present in this strain is isolated by the alkaline lysis technique as described in Molecular Cloning-a laboratory manual- (SAMBROOK, FRITSCH, MANIAISsecond edition, 1989 / 1.25-1. 28), and its restriction map is determined. Analysis shows that the inserted fragment has a size of approximately 9 kbp (Figure 4).



   When the plasmid pBRDEBRA3 is introduced into the strain of E. coli MC1061 by transformation, all the transformed strains obtained are capable of hydrolyzing AZCL-pullulan. They are also capable of hydrolyzing the oc-1, 6 bonds of amylopectin. In fact, the colonies are covered with an agar medium containing 1% agar, 100 mM sodium acetate (pH 4.5) and 1% amylopectin, and incubated for 18 hours at 60 C. A coloration blue is visible around the transformed strains, when the agar is impregnated with a solution of I2-KI 3/50 mM.



   The 8.5 kb BamHI [375] -BamHI [8843] fragment of the plasmid pBRDEBRA3 was then purified by agarose gel electrophoresis and subcloned into the BamHI site [5759] of the shuttle vector (E. coli-B subtilis) pUBC131 (FIG. 5), described in European patent application 0 415 296 (FIG. 8 of this European patent application), by transformation into the strain of E. coli MC1061. The resulting plasmid (pUBCDEBRA1, FIG. 6) was then extracted from E. coli and introduced by transformation into the strain of B. subtilis 1A510.



   The B. subtilis 1A510 strain can be obtained from BACILLUS GENETIC STOCK CENTER (The OHIO STATE UNIVERSITY, Department of Biochemistry, 484 West 12th Avenue, Columbus, Ohio 43210).



   The transformed strains obtained are covered with a

  <Desc / Clms Page number 26>

 agarose overlay containing AZCL-pullulan or amylopectin. The transformed strains are isolated, which are capable of hydrolyzing AZCL-pullulan as well as of hydrolyzing amylopectin in the alpha-1, 6 position.



   This clearly indicates that the gene encoding pullulanase is expressed in B. subtilis.


    

Claims (13)

 CLAIMS 1-thermostable and active pullulanase at acidic pH, capable of hydrolyzing the glucosidic bonds of type al, 6 in amylopectin, characterized in that it has an isoelectric point of approximately 4.5 and a half-life duration approximately 55 hours measured at a temperature of approximately 60 Oc in a buffered solution at a pH of approximately 4.5 and in the absence of substrate.  2-Pullulanase according to claim 1, characterized in that it shows a relative enzymatic activity of at least 85% measured after an incubation of 60 minutes at a temperature of approximately 60 C in the absence of substrate and in a range pH between about 3.5 and about 5.8.  3-Pullulanase according to claim 1, characterized in that it has an optimal enzymatic activity measured at a temperature of about 60 C in a pH range between 4.0 and 4.8.  4-Pullulanase according to claim 1, characterized in that it has an optimal enzymatic activity, measured at a pH of about 4.3, in a temperature range between 55 and 65 Oc.  5-Pullulanase according to claims 1, characterized in that it 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 for a temperature of approximately 60 oc, the maximum enzymatic activity being measured at a temperature of 60 C and a pH of 4.3.  6-Pullulanase according to any one of the preceding claims, characterized in that it is produced by an aerobic bacterium.  7-Pullulanase according to claim 6 characterized in that the aerobic bacterium is the strain of Bacillus deramificans LMG P-13056 or a derivative of this strain.  <Desc / Clms Page number 28>    8-Bacillus deramificans LMG P-13056.  9-A method for the production of a pullulanase according to any one of claims 1 to 7 characterized in that it comprises the culture of the strain of Bacillus deramificans 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.  10-A method for the preparation of a pullulanase according to any one of claims 1 to 7 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.  11-Use of a pullulanase according to any one of claims 1 to 7 for the treatment of starch.  12- Use according to claim 11 characterized in that the pullulanase is used for the treatment of starch comprising a step of saccharification of starch or partially hydrolyzed starch.  13-Enzymatic composition comprising a pullulanase according to any one of claims 1 to 7.