PYRODICTIUM XYLANASE, AMYLASE AND PULLULANASE

Abstract: The invention relates to novel thermostable amylases, pullulanases and xylanases obtainable from Pyrodictium.

Description: PYRODICTIUM XYLANASE, AMYLASE AND PULLULANASE FIELD OF INVENTION The present invention relates to a novel thermostable amylase and to a novel thermostable pullulanase and their use in the production of sweeteners and ethanol from starch, and to a novel thermostable xylanase and its use in the paper and pulp industry. « 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 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 woesei and Pyrococcus furiosus. for reference see Arch. Microbiol. 155, 1991, pp. 572-578, and Appl . Env. Microbiol. 56, 1990, pp.1985-1991, can produce highly thermostable amylases. The paper and pulp industry is using xylanase compositions in the bleaching process to enhance the brightness of bleached pulps, to decrease the amount of bleaching chemi¬ cals, e.g. chlorine, used in the bleaching stages, and to increase the freeness of pulps in the recycled paper process. Thermostable xylanases from Thermotoαa have been described, for reference see Biochem. J. 277(2) , 1991, pp. 413- 418. It is the object of this invention to provide a xylanase, an amylase and a pullulanase with temperature optimum at 100°C or above 100°C. SUMMARY OF THE INVENTION We have unexpectedly found that a novel thermostable xylanase, a novel thermostable amylase and a novel thermostable pullulanase can be obtained from the genus Pyrodictium, a genus not previously reported to produce thermostable xylanases, amylases and puUulanases; these new enzymes have temperature optimum around 110-120°C. Accordingly, the invention provides a xylanase preparation, characterized by being producible by cultivation of a xylanase producing strain of the genus Pyrodictium, and an amylase preparation, characterized by being producible by cultivation of an amylase producing strain of the genus Pyrodictium, and a pullulanase preparation, characterized by being producible by cultivation of a pullulanase producing strain of the genus Pyrodictium. 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 (o) of the invention at various temperatures (determined at pH 5.5 with starch and pullulan, respectively, as substrate) . Fig. 2a shows the relative activity (% rel.) of a xylanase of the invention at various temperatures (determined at pH 5.5 with xylan as substrate) , and Fig. 2b shows the relative activity of a xylanase of the invention at various pH (determined at 100°C with xylan as substrate) . DETAILED DISCLOSURE OF THE INVENTION The Microorganism Looking for extremely thermostable enzymes the extremely thermophilic archaebacteria may be a possible source. Very stable extracellular enzymes from archaebacteria have also been reported, for reference see J.M. Bragger et al. "Very stable enzymes from extremely thermophilic archaebacteria and eubacteria" in Appl . Microbiol . Biotechnol . 31, 1989, p. 556- 561. The genus Pyrodictium, however, has not been reported before to produce extracellular amylases, puUulanases and xylanases, in fact it is the first time a member of the family Desulfurococcaceae has been reported to produce an extracel- lular xylanase. A survey of the taxonomy of the family Desul¬ furococcaceae is described in "The Prokaryotes; A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Iden¬ tification, Applications", 2 nd Ed., Springer-Verlag, Vol I, p. 678. According to the invention, xylanase is derived from a xylanase producing strain of the family Desulfurococcaceae, in particular the genus Pyrodictium, e.g. P. abvssi, and an amylase is derived from an amylase producing strain of the genus Pyrodictium, in particular P. abvssi, and a pullulanase is derived from a pullulanase producing strain of the genus Pyrodictium. in particular P.abvssi. A strain representative of Pyrodictium abyssi has been made publicly available under Accession No. DSM 6158. The number is published in the DSM Catalogue of Strains, 1993. Production of Xylanase, Amylase and Pullulanase Xylanase, 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 conditions may be achieved during the preparation of media by sparging with H 2 /C0 2 (2 bar overpressure) and following the anaerobic techniques as described by Balch and Wolfe in Appl . Env. Microbiol. 32, 1976, pp. 781-791. Alternatively, xylanase, amylase and pullulanase of the invention can be produced by aerobic cultivation of a transformed 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 xylanase, amylase and pullulanase may be reco- vered 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 xylanase, amylase and pullulanase may be further purified by known methods. Immunochemical Properties 5 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 Pyrodictium abyssi, DSM 6158. The immunochemical properties can be determined 0 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- cipitation-in-Gel Techniques; Blackwell Scientific Publications 5 (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 0 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. 5 The Enzymes A xylanase of the invention can be characterized by having xylanase activity at temperatures of from below 70°C to approximately 120°C, having activity optimum at temperatures in the range 105-115°C, determined at pH 5.5 with xylan as sub- θstrate. The xylanase can also be characterized by having xylanase activity at pH values of from below pH 4.0 to ap ¬ proximately pH 8.5, having optimum in the range pH 5.5 to pH 6.5, determined at 100°C with xylan as substrate. An amylase of the invention can be characterized by 5having amylase activity at temperatures of from below 60°C to above 120°C, having activity optimum at temperatures in the range 110-120°C, determined at pH 5.5 with starch as substrate. A pullulanase of the invention can be characterized by having pullulanase activity at temperatures of from below 560°C to above 120°C, having activity optimum at temperatures in the range 110-120°C, determined at pH 5.5 with pullulan as substrate. Determination of Amylase Activity Amylase activity is determined by measuring the 0 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 5ml 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- 0 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 5 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 0 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. Determination of Xylanase Activity Xylanase activity is determined by measuring the amount of reducing sugar released during the incubation with xylan. One unit (U) of xylanase activity is defined as the 5 amount of xylanase that releases 1 μmole of reducing sugar (as xylose standard) per min. under the following assay conditions: A 0.05 ml volume of 1% soluble xylan 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 1030 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 15 The enzymes of this invention possess valuable properties allowing for various industrial applications. In particular the amylase and pullulanase, in being thermostable, find potential application in the production of sweeteners and ethanol from starch. Conditions for conventional starch 0 converting processes and liquefaction and/or saccharification processes are described in for instance US Patent No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909. Due to the excellent temperature stability of the xylanase, the xylanase of the invention finds potential 5 application in the paper and pulp industry. Use of xylanase in the paper and pulp industry is disclosed in e.g. WO 93/19171. 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. 0 EXAMPLE 1 Xylanase, Amylase and Pullulanase The strain Pyrodictium abvssi, DSM 6158, 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: DSM283 without citric acid (DSM283 is described in DSM Catalogue of Strains, 1993) , plus 0.5 g/1 of yeast extract plus 0.1 mg/1 of Na 2 WOX2H 2 0 plus 50.5% (w/v) starch (amylase, pullulanase) or 0.5% (w/v) xylan (xylanase) ; pH 5.5-6.0; temp. 97°C. The cell density achieved in this medium was ≥ 10 7 cells/ml. Anaerobic conditions were achieved during the preparation of media by sparging with H 2 /C0 2 (2 bar overpressure) and following the techniques as described loby 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. 15 The following total activity (U) in supernatant was found: Amylase activity: 2.2 U/l Pullulanase activity: 3.0 U/l Xylanase: 1.5 U/l 20Temperature 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. 25 Fig. 1 (Amylase (♦) and pullulanase (a ) ) and Fig 2a (xylanase) show the result. pH Optima To determine pH optima of the xylanase, Universal buffer (Britten and Robinson) was used to obtain values from pH 304.0 to pH 8.5. Samples were incubated for 30 minutes at 100°C at the pH in question. Fig. 2.b shows the result.

Claims: CLAIMS 1. A xylanase preparation, characterized by being producible by cultivation of a xylanase producing strain of the family Desulfurococcaceae. 2. A xylanase preparation according to claim 1, wherein said xylanase producing strain belongs to the genus Pyrodictium. 3. A xylanase preparation according to claim 2, wherein said xylanase producing strain belongs to Pyrodictium abvssi. 4. A xylanase preparation according to claim 3, wherein said xylanase producing strain is Pyrodictium abvssi, DSM 6158. 5. A xylanase according to claim 4, further charac- terized by: (a) Activity optimum in the range pH 5.5 to pH 6.5, determined at 100°C with xylan as substrate; (b) Activity optimum at temperatures in the range 105-115°C, determined at pH 5.5 with xylan as substrate. 6. An amylase preparation, characterized by being producible by cultivation of an amylase producing strain of the genus Pyrodictium. 7. An amylase preparation according to claim 6, wherein said amylase producing strain belongs to Pyrodictium abvssi. 8. An amylase preparation according to claim 7, wherein said amylase producing strain is Pyrodictium abyssi, DSM 6158. 9. An amylase according to claim 8, further charac¬ terized by activity optimum at temperatures in the range 110- 120°C, determined at pH 5.5 with starch as substrate. 10. A pullulanase preparation, characterized by being producible by cultivation of a pullulanase producing strain of the genus Pyrodictium. 11. A pullulanase preparation according to claim 10, wherein said pullulanase producing strain belongs to Pyrodic¬ tium abvssi. 12. A pullulanase preparation according to claim 11, wherein said pullulanase producing strain is Pyrodictium abyssi, DSM 6158. 13. A pullulanase preparation according to claim 12, further characterized by activity optimum at temperatures in the range 110-120°C, determined at pH 5.5 with pullulan as substrate. 14. The use of the xylanase according to any of claims 1-5 in a process for treatment of lignocellulosic pulp. 15. The use of the amylase according to any of claims 6-9 in a process of producing sweeteners from starch. 16. The use of the amylase according to any of claims 6-9 in a process of producing ethanol from starch. 17. The use of the pullulanase according to any of claims 10-13 in a process of producing sweeteners from starch. 18. The use of the pullulanase according to any of claims 10-13 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, C12P7, C12R1, C12S3