EP0541676A1 - Nouvelles pullulanases thermostables - Google Patents

Nouvelles pullulanases thermostables

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Publication number
EP0541676A1
EP0541676A1 EP91914447A EP91914447A EP0541676A1 EP 0541676 A1 EP0541676 A1 EP 0541676A1 EP 91914447 A EP91914447 A EP 91914447A EP 91914447 A EP91914447 A EP 91914447A EP 0541676 A1 EP0541676 A1 EP 0541676A1
Authority
EP
European Patent Office
Prior art keywords
pullulanase
preparation
pyrococcus
dsm
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91914447A
Other languages
German (de)
English (en)
Inventor
Garabed Antranikian
Per Lin J Rgensen
Mogens WÜMPELMANN
Steen Troels J Rgensen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DK183390A external-priority patent/DK183390D0/da
Priority claimed from DK235890A external-priority patent/DK235890D0/da
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP0541676A1 publication Critical patent/EP0541676A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)

Definitions

  • thermostable enzymes More specifically, the present invention relates to novel thermostable pullulanases obtainable from members of the genus Pyrococcus and to processes for the preparation of these enzymes.
  • the invention also relates to recombinantly produced pullulanase preparations consisting essentially of a homogenous pullulanase component, the DNA encoding the pullulanase being derived from the genome of a member of the genus Pyrococcus. Moreover, the invention relates to high expression processes for the preparation of the pullulanase components.
  • the invention further relates to the use of the pul- lulanases in starch converting processes, and to liquefaction and/or saccharification processes.
  • Thermostable pullulanases are known and isolated from e.g. Bacillus acidopullulyticus, and their use in industrial saccharification processes has been described, vide EP Patent Publication No. 63,909. To comply with the demands for more thermostable enzymes extensive search has proceeded. It is the purpose of this invention to provide novel pullulanases with improved thermostability.
  • the present invention provides a pullulanases having immuno ⁇ hemical proper ⁇ ties identical or partially identical to those of the pul- lulanases derived from Pyrococcus woesei, DSM No. 3773, or Pyrococcus furiosus. DSM No. 3638.
  • the invention provides a process for the preparation of the pullulanases of the invention, which process comprises cultivation of a pullulanase producing strain of Pyrococcus in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
  • the present invention provides a recombinantly produced pullulanase preparation consisting essentially of a homogenous pullulanase component, the DNA encoding the pullulanase being derived from the genome of a member of the genus Pyrococcus.
  • the present invention provides a pullulanase preparation consisting essentially of a homogenous pullulanase component, which has immunochemical properties identical or partially identical to those of the pullulanases derived from Pyrococcus woesei. DSM No. 3773, or Pyrococcus furiosus, DSM No. 3638.
  • the invention provides a high expression process for the preparation of the enzyme comprising isolating a DNA fragment encoding the pullulanase; combining the DNA fragment with an appropriate expression signal in an appropriate plasmid vector; introducing the plasmid vector into an appropriate host either as an autonomously replicating plasmid or integrated into the chromosome; cultivating the host organism under conditions leading to expression of the pullula ⁇ nase; and recovering of the pullulanase from the culture medium.
  • the invention is directed towards the use of a pullulanase of the invention in starch converting processes.
  • the present invention provides a process for converting starch into syrups containing glucose and/or maltose, which process comprises conducting the saccharification of starch hydrolysates in the presence of a pullulanase of the invention and one or more enzymes selected from the group consisting of glucoamylase, ⁇ -glucosidase, ⁇ - amylase or other saccharifying enzymes.
  • the invention provides a process for converting starch into syrups containing glucose and/or maltose, which process comprises conducting a simultaneous liquefaction/debranching in the presence of a pullulanase of the invention and a thermos ⁇ table ⁇ -amylase, and subsequent saccharification in the presence of one or more enzymes selected from the group consisting of glucoamylase, ⁇ -glucosidase, 3-amylase or other saccharifying enzymes, optionally together with a pullulanase.
  • Fig. 1 shows the relation between temperature and the enzymatic activity of an enzyme according to the invention
  • Fig. 2 shows the relation between pH and the en ⁇ zymatic activity of an enzyme according to the invention
  • Fig. 3 shows the time coarse of the enzymatic activity of an enzyme of the invention at various temperatures (• 100 ⁇ C; A ⁇ o°C; ⁇ 120"C) ; and Fig. 4 shows a restriction map of plasmid pSJ933.
  • the present invention provides novel pullulytic enzymes obtainable from members of the genus Pyrococcus. or mutants or variants thereof, or pullulytic enzymes having immunochemical properties identical or partially identical to those of a pullulanase obtainable from a strain of Pyrococcus.
  • an enzyme variant or mutated enzyme is meant an enzyme obtainable by alteration of the DNA nucleotide sequence of the parent gene or its derivatives.
  • the enzyme variant or mutated enzyme may be expressed and produced when the DNA nucleotide sequence encoding the enzyme is inserted into a suitable vector in a suitable host organism.
  • the host organism is not necessarily identical to the organism from which the parent gene originated.
  • the enzymes of the invention are valuable for use in starch converting processes, especially in combination with other enzymes for industrial conversion of starch into various sugars.
  • the pullulytic enzymes of the invention can be described by the following characteristics.
  • the enzymes of the invention are active in a very broad temperature and pH range.
  • the enzymes possess pullulytic activity in a temperature range of from 40 to above lSO'C, showing temperature optimum in the range of 85 to 115°C, more specifically in the range of 100 to 110°C.
  • the enzymes possess pullulytic activity in a pH range of from pH 3.5 to above 8, showing pH optimum in the range of from pH 5 to 7, more specifically pH 5.5 to pH 6.5. At pH 8 approximately 55% of pullulytic activity are detectable.
  • After 4 hours at 100°C the pullulanases show essentially no loss of pullulytic activity.
  • After 24 hours at 100°C a residual activity of 65% is detect ⁇ able.
  • After 1 hour at 110°C a residual activity of approxi ⁇ mately 10% is detectable. Addition of metal ions is not required for catalytic activity.
  • the enzyme is inhibited by ⁇ - and 0-cyclodextrins.
  • the pullulanase of the invention has immunochemical properties identical or partially identical to those of the pullulanases derived from Pyrococcus woesei, DSM No. 3773, or Pyrococcus furiosus, DSM No. 3638.
  • the immunochemical properties can be determined by immunological cross-reaction identity tests.
  • the identity tests can be performed by the well-known Ouchterlony double im- munodiffusion procedure or by tandem crossed immunoelectro- phoresis according to N. H. Axelsen; Handbook of I muno- precipitation-in-Gel Techniques; Blackwell Scientific Publica ⁇ tions (1983), chapters 5 and 14.
  • the terms "antigenic identity” and "partial antigenic identity” are described in the same book, chapters 5, 19 and 20.
  • Monospecific antiserum is generated, according to the above mentioned method, by immunizing rabbits with the purified pullulanase of the invention.
  • the immunogen is mixed with Freund*s adjuvant and injected subcutaneously into rabbits every second week.
  • Antiserum is obtained after a total immuniz ⁇ ation period of 8 weeks, and immunoglobulin is prepared therefrom as described by N. H. Axelsen, supra.
  • the pullulanases of the invention can be prepared by cultivation of a pullulanase producing strain of Pyrococcus in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
  • the species P. woesei and P. furiosus are represen ⁇ tative members of the genus Pyrococcus.
  • a representative strain of P. woesei is available from DSM, No. 3773, and a represen ⁇ tative strain of P. furiosus is available from DSM, No. 3638.
  • Members of the hyperthermophilic archaebacteria Pyrococcus exhibit growth optimum between 80 and 105°C, and at pH values between 4.5 and 7.5, and are capable of growing on various complex media containing polysaccharides such as starch, glycogen, dextrin and oligosaccharides. It has been demonstrated that during growth of these bacteria, extremely thermoactive intracellular and extracellular pullulanases are produced.
  • the present invention provides recombinantly produced pullulanase preparations consisting essentially of a homogenous pullulanase component.
  • the DNA encoding the pullulanase component can be derived from any member of the genus Pyrococ ⁇ cus.
  • the DNA encoding the pullulanase component is derived from a strain of P. woesei or P. furiosus.
  • a represen ⁇ tative strain of P. woesie is available from DSM, with No. 3773, and a representative strain of P. furiosus is available from the same institute with No. 3638.
  • the pullulanase preparations of the invention consist essentially of a homogenous pullulanase component, which has immunochemical properties (vide e.g. N. H. Axelsen, op. cit.) identical or partially identical to those of the pullulanases derived from P. woesei, DSM No. 3773, or P. furiosus. DSM No. 3638.
  • immunochemical properties vide e.g. N. H. Axelsen, op. cit.
  • the pullulanase preparation of the invention has a pullulytic activity of at least 6 pullulanase units (PU)/mg of protein, more preferred at least 20 PU/ g of protein.
  • PU pullulanase units
  • a method for determining the pullulytic activity and a definition of PU are cited in Example 8.
  • the pullulanase preparation of the invention is a mono-component preparation. Furthermore, the pullulanase com ⁇ ponent has an apparent molecular weight of 95 kD. These features are determined in Example 9 of the present specifi ⁇ cation.
  • the pullulanase preparation of the invention has a pH optimum in the range of from pH 4.5 to 6.0, more specifically of from pH 4.5 to 5.5, determined as in Example 7.
  • the pullulanase preparation of the invention has a temperature optimum in the range of from 85 to 115°C, more specifically 95 to 115°C, determined as in Example 7.
  • the pullulanase preparation of the invention has a thermal stability, measured as residual activity, after 30 minutes at 100°C of more than 80%, preferably more than 90%; after 2 hours at 100°C of more than 70%, preferably more than 80%; and after 4 hours at 100°C of more than 60%, preferably more than 70%, yet more preferably more than 80%; as determined in Example 7.
  • a process of the invention preferably is a high expression process comprising isolating a DNA fragment encoding the pullulanase; combining the DNA fragment with an appropriate expression signal in an appropriate plasmid vector; introducing the plasmid vector into an appropriate host either as an autonomously replicating plasmid or integrated into the chromo- some; cultivating the host organism under conditions leading to expression of the pullulanase; and recovering of the pul ⁇ lulanase from the culture medium.
  • the pullulanase component of the pullulanase prepara ⁇ tion of the invention is producible by a species of Pyrococcus.
  • Preferred species are P. woesei and P. furiosus.
  • a DNA fragment encoding the pullulanase component or a precursor thereof may for instance be isolated by establishing a cDNA or genomic library of a pullulanase producing organism, such as e.g. P. woesei. DSM No. 3773, or P. furiosus, DSM No.
  • the DNA sequence may be inserted into a suitable replicable expression vector comprising appropriate promotor, operator and terminator sequences permitting the pullulanase to be expressed in a particular host organism, as well as an origin of replication enabling the vector to replicate in the host organism in question.
  • the resulting expression vector may then be trans- formed into a suitable host cell, such as an Escherichia coli, or a member of the genii Bacillus. Asper illus, or Strep- tomyces.
  • a suitable host cell such as an Escherichia coli, or a member of the genii Bacillus. Asper illus, or Strep- tomyces.
  • E. coli is used as a host organism, suitable plasmids are pBR322 and pACYC, or derivatives thereof.
  • a Bacillus sp. is used as a host organism, a suitable plasmid will be pUBllO; pC194; or pE194.
  • a suitable Bacillus sp. will be B. subtilis, B. licheniformis, B. amyloliquefaciens or B. lentus.
  • the medium used to cultivate the transformed host may be any conventional medium suitable for growing the cells in question.
  • the invention provides a process for the preparation of the pullulanase, the process comprising purification of the expressed pullulanase by boiling the fermentation broth, causing denaturation of the native proteins, followed by centrifugation of the fermentation broth, and recovery of the pullulanase from the supernatant. While the culture medium is heated to boiling, the native proteins, i.e. the proteins and polypeptides originating from the host organism other than the pullulanase component of the invention, are denatured.
  • the pullulanase component is an extremely thermostable enzyme, easily tolerates such treatment. While the native proteins are denatured in a few seconds, above all in a few minutes, e.g. after 2 to 5 minutes, the pullulanase component of the inven ⁇ tion is stable to boiling for several hours, cf. the determina- tion of the thermal stability in Example 7.
  • the vast majority of the proteins present in the culture medium including intracellular proteins as a consequence of lysation, can easily be separated off by centrifugation, and in terms of enzymatic activity the purification procedure has been completed, i.e. recombinant pullulanase has been recovered without any contaminating enzyme activities.
  • the pullulanases of the invention are suitable for use in combination with other enzymes for the industrial conversion of starch into various sugars. Being a debranching enzyme, pullulanases from Pyrococ ⁇ cus are especially advantageous for use in saccharification processes, following liquefaction of starch by thermostable ⁇ - amylases that retain substantial parts of their activity at saccharification conditions.
  • thermostable ⁇ - amylases that retain substantial parts of their activity at saccharification conditions.
  • ⁇ -amylase activity is present during saccharification, accumulation of low molecular weight, branched oligosaccharides can occur, lowering the final yield in the saccharification process.
  • Pullulanase with sufficient thermostability hitherto known in the art does not have any activity towards these low molecular weight substances (Promo- zy e ® , Novo Nordisk) .
  • Saccharification processes can be carried out by conventional means and essentially as described in EP Patent Publication No. 63,909.
  • Preferred enzyme dosages are in the range of 1-100 ⁇ g of pullulanase per g dry substance, more preferred 1-20 ⁇ g/g dry substance.
  • the high thermostability of pullulanases of the invention also makes these suitable for use in simultaneous liquefaction/debranching processes, the advantage of this unusual combination of enzyme activities ( ⁇ -amylolytic and pullulytic) being that a further reduction of fluid viscosity in the starch slurry can be achieved hereby, allowing for an increase in the starch concentration during saccharification.
  • preferred enzyme dosages are in the range of 1-100 ⁇ g of pul ⁇ lulanase per g dry substance, more preferred 1-20 ⁇ g/g dry substance.
  • Other conditions are as for conventional liquefac ⁇ tion and saccharification processes, vide e.g. US Patent No. 3,912,590, EP Patent Publications Nos. 252,730 and 63,909, and International Patent Application WO 90/11352.
  • pullulanase activity is also detectable in the culture supernatant. No significant difference is observed between the intracellular and the extracellular pullulanases.
  • the supernatant was concentrated using 20,000 cut-off membranes.
  • the sample (60 ml) contains around 102 U of pullulanase (1700 U/l) and 59 U of amylolytic activity (990 U/l) .
  • the specific activity of the enzymes was therefore 0.07 U/mg of pullulanase and 0.04 U/mg of amylase. The presence of oxygen did not influence the activity of these enzymes.
  • Pullulanase activity was measured according to the following procedure: 50 ⁇ l of enzyme sample were added to 200 ⁇ l of a solution containing 0.5% pullulan and 50 mM sodium acetate, pH 6.0. The mixture was incubated at 100"C for 5, 15 or 30 minutes. Incubation was terminated by transferring the mixture to an ice/water bath (0°C). After further addition of 250 ⁇ l of reagent A (see below) , the mixture was incubated at 100°C for 5 minutes. 2.5 ml deionized water were added and absorbance at 546 nm was measured.
  • 1 unit (U) is defined as the amount of enzyme which under the conditions described above liberates 1 ⁇ mol/min. of reducing sugar measured against maltose as standard. ⁇ -amylolytic activity
  • ⁇ -amylase activity was measured according to the following procedure: To 250 ⁇ l of sodium acetate buffer (50 mM pH 5.5) containing 1% (w/v) starch, up to 100 ⁇ l of enzyme solution was added and incubation was conducted at 95°C for 30 and 60 min. The activity of 1 U of ⁇ -amylase is defined as that amount of enzyme which liberates 1 ⁇ mol of reducing sugar per min with maltose as a standard.
  • O-Sepharose chromatography 20 ml of cell-free extract (46 mg of proteins) , obtained according to Example 1, were applied onto a Q-Sepharose Fast Flow column (5 x 40 cm) and proteins were eluted using 20 mM tris;HCl buffer, pH 8.0, at a flow rate of 3 ml/min. , and using a NaCl gradient (0-500 mM) . 10 ml fractions were collected. The major fraction containing pullulytic activity was eluted with 360-400 mM NaCl. A minor peak was also eluted with 470-485 mM NaCl. After three runs the fractions containing pullulytic activity were pooled and concentrated using 10,000 Da cut-off membranes (total volume 16.5 ml containing 1.4 mg/ml) . Cf. Table 4 for further data.
  • Mono 0 chromatography FPLC
  • the concentrated sample was then applied onto a Mono Q column (FPLC; HR 5/5) at a flow rate of 1 ml/min. 2 ml were used per run.
  • the buffer used for equilibration and elution was 20 mM potassium phosphate, pH 7.0.
  • Fractions containing pullulytic activity were eluted after applying a NaCl gradient (200-500 mM) .
  • the major fraction was eluted with 360-420 mM NaCl.
  • the fractions containing pullulytic activity were pooled and concentrated. After this step the pullulanase was purified 15 fold. Cf. Table 4 for further data.
  • the enzyme activity was measured between 40 and 25130 ⁇ C.
  • the buffer used was 50 mM sodium acetate, pH 5.5.
  • Enzyme assay was performed for 5 and 15 minutes. The result is presented in Fig. 1.
  • the enzymes possess pullulytic activity in a tempera ⁇ ture range of from 40 to above 130°C, showing temperature 30 optimum in the range of 85 to 115°C, more specifically in the range of 100 to 110°C. pH optimum
  • the enzymes were incubated in a 50 mM sodium acetate buffer, pH 5.5, without substrate and metal ions. After 6 hours of incubation at 100°C no loss of enzyme activity was observed. After 12 hours, 24 hours and 48 hours at 100°C around 91%, 65% and 35% of enzyme activity was measured, respectively. At 110°C 44% of enzyme activity was measured after 20 minutes and 10% of enzyme activity was measured after 1 hour. At 120°C 10% of enzyme activity was detected after 5 minutes. The results are presented in Fig. 3.
  • 1% (w/v) of substrate was incubated in 50 mM sodium acetate buffer, pH 6.0, in the presence of 1.3 U/ml of a pul ⁇ lulanase preparation. Incubation was conducted at 100°C. Samples of 500 ⁇ l were taken and treated with an ion exchanger (Serdolit MB) , and analyzed by HPLC (carbohydrate column Aminex HPX-42A) .
  • the pullulanases were also capable of attacking branched oligosaccharides that were prepared in the laboratory by incubation of starch with ⁇ - amylase. The branched oligosaccharides formed were then separated using a Bio-Gel column. More than 80% of the branched oligosaccharides were degraded to DP5 or lower.
  • Pyrococcus woesei chromosomal DNA was isolated according to Pitcher et al. (1989); Lett. Appl. Microbiol., 8_, 151-156; and partially digested with Sau3A. 100 ⁇ g of P. woesei DNA was digested with 20 units of Sau3A for 10 min. at 37 ° C. The digestion was terminated by phenol:chloroform extraction and the DNA was ethanol precipitated.
  • Ligation was performed by using chromosomal DNA: pSJ933 (digested by BamHI, the larger fragment of 5.8 kb was isolated) with a ratio of 1:3, using 4 ⁇ g of DNA/10 ⁇ l and adding 2 units of T4 ligase and incubating at room temperature (25°C) for 4 hours.
  • the plasmid pSJ933 is deposited in the E. coli strain SJ989 at NCIMB, a map of the plasmid is shown in Fig. 4) .
  • E. coli strain MC1000 was trans ⁇ formed with the ligated DNA and plated on Luria broth plus 2% agar containing 10 ⁇ g/ml chloramphenicol and incubated at 37 ⁇ C.
  • the dyed pullulan is thereafter harvested by centrifuga- tion, and the pullulan is washed 3 times with distilled water and resuspended in an appropriate volume of distilled water) .
  • the plates were then incubated at 60°C for 4 hours, and around one of the colonies a halo appeared resulting from degradation of the dyed pullulan.
  • the corresponding colony on the first set of Luria broth plates was isolated and analyzed for plasmid content.
  • the isolated colony PL2118 was grown in 10 ml Luria broth, and the plasmid was isolated by the method described by Kieser et al. (Plasmid 12:19, 1984).
  • the plasmid was analyzed by restriction mapping and showed an insert of Pyrococcus DNA of approximately 4.5 kb.
  • the strain according to Example 4 was cultivated in a 2 1 fermenter of Porton type; pH was maintained at 6.5-7.0, temperature at 37°C, aeration at 1.1 1/min., and agitation at 1000 rpm.
  • Inoculation was made by resuspending culture in physiological salt solution grown on LB agar slant containing 6 mg/1 chloramphenicol overnight at 37°C. Harvest was made at 50 hours from inoculation.
  • the cultivation was conducted as a batch fermentation on the following substrate: Glycerol Tryptone (Bacto) Yeast extract (Bacto) NaCl 5 K 2 HP0 4
  • the harvested culture broth prepared according to Example 5 was centrifuged for 30 min. at 4000 rpm/servall H 306000A in a Servall RC-3B centrifuge.
  • the pellet was resuspended with 50 mM Tris puffer, pH 7.0 to a final volume of 10% of culture broth volume. Lysozyme was added to 2 g/1 final concentration, upon which incubation at 40°C for 1 h followed by treatment of the suspension with an Ultratorrax/type TP18/10;18N;170W, submerged into the suspen ⁇ sion, for 3 minutes.
  • the suspension was subsequently set to boil for 1 and centrifuged at 9000 rpm/Servall GS3 at 4"C for 30 minutes in a Servall RC-5B centrifuge.
  • the pullulanase activity in the supernatant was concentrated 2.8 times by ultrafiltration on a DDS/GR 81PP membrane at room temperature (5mM NaN 3 added prior to ultrafil ⁇ tration) .
  • the pullulanase sample prepared according to Example 6 was submitted to characterization of the pullulanase activity in terms of pH-optimum, temperature optimum, thermal stability and substrate specificity.
  • the pullulanase sample was incubated as follows:
  • Puffer 50 mM NaAc, pH 5.0; final concentration.
  • Substrate 2% w/v pullulan, panose or soluble starch (Merck) ; final concentration.
  • Amount of pullu ⁇ lanase sample added to incuba ⁇ tion mixture 5-50% v/v.
  • Pullulanase activity at 60°C was determined as described in Example 8, except that various incubation pH/buffers were used.
  • Buffers pH 3.5-5.5: 0.05 M NaAc (final concentration): A-buffer pH 5.5-7.5: 0.025 M Na 3 citrate and 0.025 M KH 2 P0 4 (final concentration) : CP-buffer
  • Pullulanase activity at pH 5.0 was determined as described in Example 8, except that various incubation tempera ⁇ tures were used. Incubation temperature (°C) 60 70 80 90 100 105 121 Activity (%) 18 38 41 54 98 100 48
  • One ml of the incubation mixture volume is incubated at 60°C for 30 minutes.
  • One ml of the incubation mixture volume is incubated at 0°C for 30 minutes.
  • To each portion 1.5 ml of 150.5 M Na 2 C0 3 buffer, pH 10.0, are added, and the mixture is rapidly cooled.
  • the amount of reducing carbohydrate is determined in both samples by the Nelson/Somogoi method (Nelson,N. (1944) ; J.
  • the molecular weight of the pullulanase of the invention was determined by SDS PAGE conducted according to Pharmacia Phast System file No. 110, on Phastgel Gradient 8-25. A molecular weight of 95,000 (+/- 10,000) was determined.

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Abstract

Cette invention se rapporte au domaine d'enzymes thermostables. Plus particulièrement, elle se rapporte à de nouvelles pullulanases thermostables pouvant être obtenues à partir de membres de l'espèce Pyrococcus et à des procédés de préparation de ces enzymes. L'invention se rapporte aussi à des préparations de pullulanases produites par recombinaison et composées essentiellement d'un constituant de pullulanase homogène, l'ADN codant la pullulanase étant dérivé du génome d'un membre de l'espèce Pyrococcus. De plus, l'invention se rapporte à des procédés à forte expression destinés à la préparation de constituants de pullulanase dépourvus de toute activité enzymatique contaminante. L'invention se rapporte en outre à l'utilisation de pullulanases dans des procédés de conversion d'amidon et dans des procédés de liquéfaction et/ou de saccharification.
EP91914447A 1990-08-01 1991-07-31 Nouvelles pullulanases thermostables Withdrawn EP0541676A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DK1833/90 1990-08-01
DK183390A DK183390D0 (da) 1990-08-01 1990-08-01 Termostabil pullulanase
DK2358/90 1990-09-28
DK235890A DK235890D0 (da) 1990-09-28 1990-09-28 Termostabil pullulanase

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EP0541676A1 true EP0541676A1 (fr) 1993-05-19

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EP (1) EP0541676A1 (fr)
JP (1) JPH05508997A (fr)
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FI930381A0 (fi) 1993-01-29
WO1992002614A1 (fr) 1992-02-20
CA2088592A1 (fr) 1992-02-02
FI930381A (fi) 1993-03-26
JPH05508997A (ja) 1993-12-16

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