THERMOCOCCUS AMYLASE AND PULLULANASE

Abstract: The present invention relates to Thermococcus amylase and pullulanase preparations and their use in producing 5 sweeteners and ethanol from starch. In particular, the enzymes are derived from Thermococcus celer.

Description: THERMOCOCCUS AMYLASE AND PULLULANASE FIELD OF INVENTION The present invention relates to a novel thermostable amylase and a novel thermostable pullulanase and their use in 5 the production of sweeteners and ethanol from starch. BACKGROUND OF THE INVENTION The production of sweeteners from starch has been largely improved by application of different microbial enzymes to obtain better quality and yields, but the necessity of 10 performing several steps of the starch-hydrolysing process at elevated temperatures means that there is still a need for new starch-hydrolysing enzymes with increased thermal stability. It is known that Pyrococcus. e.g. Pyrococcus wosei and Pyrococcus furiosus. for reference see Arch. Microbiol. 15155. 1991, pp. 572-578, and Appl . Env. Microbiol. 56, 1990, pp.1985-1991, can produce highly thermostable amylases. It is the object of this invention to provide an amylase and a pullulanase with temperature optimum at 80°C or above 80°C. 20 SUMMARY OF THE INVENTION We have unexpectedly found that a novel thermostable amylase and a novel thermostable pullulanase can be obtained from the genus Thermococcus. a genus not previously reported to produce thermostable amylase and pullulanase; these new enzymes 25 have temperature optimum around 90°C. Accordingly, the invention provides an amylase preparation, characterized by being producible by cultivation of an amylase producing strain of the genus Thermococcus. and a pullulanase preparation, characterized by being producible by 30 cultivation of a pullulanase producing strain of the genus Thermococcus. BRIEF DESCRIPTION OF DRAWINGS The present invention is further illustrated by reference to the accompanying drawings, in which: Fig. 1 shows the relative activity (% rel . ) of an amylase (°) and a pullulanase (■) of the invention at various temperatures (determined at pH 5.5 with starch and pullulan, respectively, as substrate) . Fig. 2 shows the relative activity (% rel.) of an amylase (°) and a pullulanase (■) of the invention at various pH, determined at 90°C with starch and pullulan, respectively, as substrate. DETAILED DISCLOSURE OF THE INVENTION The Microorganism According to the invention, amylase is derived from an amylase producing strain of the genus Thermococcus, in particular Thermococcus celer. and pullulanase is derived from a pullulanase producing strain of the genus Thermococcus, in particular Thermococcus celer. A strain representative of Thermococcus celer has been made publicly available under Accession No. DSM 2476. The number is published in the DSM Catalogue of Strains, 1993. Production of Amylase and Pullulanase Amylase and pullulanase of the invention may be produced by anaerobic cultivation of the above mentioned strain on a nutrient medium containing suitable carbon and nitrogen sources, such media being known in the art. Anaerobic con¬ ditions may be achieved during the preparation of media by sparging with N 2 and following the anaerobic techniques as described by Balch and Wolfe in Appl . Env. Microbiol. 32, 1976, pp. 781-791. Alternatively, amylase and pullulanase of the invention can be produced by aerobic cultivation of a trans- formed host organism containing the appropriate genetic information from the above mentioned strain. Such transformants can be prepared and cultivated by methods known in the art. The amylase and the pullulanase may be recovered by removing the cells from the fermentation medium (e.g. by centrifugation or filtration) and then concentrating the broth (e.g. by ultrafiltration) . If desired, the amylase and the pullulanase may be further purified by known methods. Immunochemical Properties The enzymes of the invention have immunochemical properties identical or partially identical (i.e. at least partially identical) to those of an enzyme derived from the strain Thermococcus celer, DSM 2476. The immunochemical properties can be determined immunologically by cross-reaction identity tests. The identity tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectro- phoresis according to Axelsen N.H. ; Handbook of Immunopre- cipitatioή-in-Gel Techniques; Blackwell Scientific Publications (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 antisera are generated according to the above mentioned method by immunizing rabbits with the purified enzymes of the invention. The immunogens are mixed with Freund's adjuvant and injected subcutaneously into rabbits every second week. Antisera are obtained after a total im¬ munization period of 8 weeks, and immunoglobulins are prepared therefrom as described by Axelsen N.H. , supra. The Enzymes An amylase of the invention can be characterized by having amylase activity at temperatures of from below 60°C to approximately 120°C, having activity optimum at temperatures in the range 85-95°C, determined at pH 5.5 with starch as sub- strate. The amylase can also be characterized by having amylase activity at pH values of from below pH 4.5 to approximately pH 9.0, having optimum in the range pH 5.0 to pH 6.0, determined at 90°C with starch as substrate. A pullulanase of the invention can be characterized by having pullulanase activity at temperatures of from below 60°C to above 120°C, having activity optimum at temperatures in the range 85-95°C, determined at pH 5.5 with pullulan as substrate. The pullulanase can also be characterized by having pullulanase activity at pH values of from below pH 4.5 to approximately pH 9.8, having optimum in the range pH 5.0 to pH 6.0, determined at 90°C with pullulan as substrate. Determination of Amylase Activity Amylase activity is determined by measuring the amount of reducing sugar released during the incubation with starch. One unit (U) of amylase activity is defined as the amount of amylase that releases 1 μmole of reducing sugar (as maltose standard) per min. under the following assay con¬ ditions: A 0.05 ml volume of 1% soluble starch is added to 0.05 ml of 0.1 M sodium acetate buffer pH 5.5. 25 μl of enzyme solution are added to this mixture and the sample is incubated at 90°C for 30 min. The reaction is stopped by cooling on ice, and the amount of reducing sugar is determined by dinitro- salicylic acid. Sample blanks are used to correct for non- enzymatic release of reducing sugar. Determination of Pullulanase Activity Pullulanase activity is determined by measuring the amount of reducing sugar released during the incubation with pullulan. One unit (U) of pullulanase activity is defined as the amount of pullulanase that releases 1 μmole of reducing sugar (as maltose standard) per min. under the following assay conditions: A 0.05 ml volume of 1% pullulan is added to 0.05 ml of 0.1 M sodium acetate buffer pH 5.5. 25 μl of enzyme solution are added to this mixture and the sample is incubated at 90°C for 30 min. The reaction is stopped by cooling on ice, and the amount of reducing sugar is determined by dinitrosalicylic acid. Sample blanks are used to correct for nonenzymatic release of reducing sugar. Industrial Applications The enzymes of this invention possess valuable properties allowing for various industrial applications. In particular the enzymes, in being thermostable, find potential application in the production of sweeteners and ethanol from starch. Conditions for conventional starch converting processes and liquefaction and/or saccharification processes are de- scribed in for instance US Patent No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909. The following example further illustrates the present invention, and it is not intended to be in any way limiting to the scope of the invention as claimed. EXAMPLE 1 Cultivation The strain Thermococcus celer, DSM 2476, was re- cultured from glycerol-preserved cells using the medium recommended by the Deutsche Sammlung von Mikroorganismen (DSM) . The microorganisms were grown in 1 liter of batch cultures under the following conditions: Medium: DSM266 (DSM266 is described in DSM Catalogue of Strains, 1993) , pH 5.8, temp. 85°C; in the medium sulphur and tryptone were omitted and starch (0.5% w/v) was added as the only carbohydrate; yeast extract concentration was 0.1% (w/v) . The cell density achieved in this medium was ≥ 10 8 cells/ml. Anaerobic conditions were achieved during the preparation of media by sparging with N 2 and following the techniques as described by Balch in Appl . Env. Microbiol. 32, 1976, pp. 781-791. After cultivation the culture fluid was centrifuged at 12.000 x g for 30 min. at 4°C, and the cell free supernatant was concentrated up to 100-fold using an Amicon Ultrafiltration System. The cell pellet was resuspended in 50 mM sodium acetate buffer pH 5.5 and sonicated three times for 3 min. at 50% duty cycle by a BRANSON 450 sonifier. The cell debris was separated from the supernatant after centrifugation at 10.000 x g for 30 min. at 4°C. The following total activity (U) in both supernatant and cell extract was found: Amylase activity: 1.0 U/l Pullulanase activity: 1.3 U/l The half-life, determined as the incubation time (h) at 95°C after which 50% of the original activity are detected, gave the following results (the values in parentheses are the half-life at 105°C) : Amylase activity: >6h (3h) Pullulanase activity: >6h (lh) Temperature Optima Temperature optima were determined by incubation of samples for 30 minutes at pH 5.5 at temperatures from 60°C to 120°C. The ' incubation was conducted in closed Hungate tubes in order to prevent boiling of the solution. Fig. 1 shows the result (Amylase (°) and pullulanase ( ■ ) ) . pH Optima To determine pH optima, Universal buffer (Britten and Robinson) was used to obtain values from pH 4.0 to pH 10.0. Samples were incubated for 30 minutes at 90°C at the pH in question. Fig. 2 shows the result (Amylase (°) and pullulanase ( ■ ) ) .

Claims: CLAIMS 1. An amylase preparation, characterized by being producible by cultivation of an amylase producing strain of the genus Thermococcus. 2. An amylase preparation according to claim 1, wherein said amylase producing strain belongs to Thermococcus celer. 3. An amylase preparation according to claim 2, wherein said amylase producing strain is Thermococcus celer, DSM 2476. 4. An amylase according to claim 3, further charac¬ terized by: (a) Activity optimum in the range pH 5.0 to pH 6.0, determined at 90°C with starch as substrate; (b) Activity optimum at temperatures in the range 85- 95°C, determined at pH 5.5 with starch as substrate. 5. A pullulanase preparation, characterized by being producible by cultivation of a pullulanase producing strain of the genus Thermococcus. 6. A pullulanase preparation according to claim 5, wherein said pullulanase producing strain belongs to Thermo¬ coccus celer. 7. A pullulanase preparation according to claim 6, wherein said pullulanase producing strain is Thermococcus celer. DSM 2476. 8. A pullulanase preparation according to claim 7, further characterized by: (a) Activity optimum in the range pH 5.0, to pH 6.0, determined at 90°C with pullulan as substrate; (b) Activity optimum at temperatures in the range 85- 95°C, determined at pH 5.5 with pullulan as substrate. 9. The use of the amylase according to any of claims 1-4 in a process of producing sweeteners from starch. 10. The use of the amylase according to any of claims 1-4 in a process of producing ethanol from starch. 11. The use of the pullulanase according to any of claims 5-8 in a process of producing sweeteners from starch. 12. The use of the pullulanase according to any of claims 5-8 in a process of producing ethanol from starch.

Inventors: ANTRANIKIAN GARABED, SJOEHOLM CARSTEN, ANTRANIKIAN, GARABED, SJOEHOLM, CARSTEN
Applicants: ANTRANIKIAN GARABED, NOVONORDISK AS, SJOEHOLM CARSTEN, ANTRANIKIAN, GARABED, NOVO NORDISK A/S, SJOEHOLM, CARSTEN
Application year: 1995
Main classification: C12N9
IPC codes: C12N9