| Co-Investigator(Kenkyū-buntansha) |
OYAMA Hiroshi Kumamoto Institute of Technology, Applied Microbial Technology, ASSOCIATED PROF., 工学部, 講師 (50221700)
MURAO Sawao Kumamoto Institute of Technology, Applied Microbial Technology, PROFESSOR, 工学部, 教授 (00081472)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
The production of chitooligosaccharides, enzymatic hydrolysis of chitin by bacterial chitinases, is a current topic of reserach because these oligosaccharides have been evluated in various applications. Chitinases are a class of hydrolytic enzymes that commonly found in bacteria, fungi, plants, and various origins. However, these chitinases have been reported in various organisms, the reaction end products of these enzymes were N-acetylglucosamine or chitobiose. By the transglycosylation reaction using chitinase of Nocardia orientalis or Ttichoderma ressei, chitohexaose and chitoheptaose were formed from chitotetraose or chitopentaose under extremely high substrate concentration.
In the course of our screening for a novel type of chitinase efficient in the production of chitooligosaccharide, we found a chitinase in the culture filtrate of Vibrio alginolyticus TK-22. The enzyme has been purified by a series of chromatographies. The molecular weight of the enzyme was estimated to be 66,00
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0 by SDS-polyacrylamide gel electrophoresis, and 57,000 by gel filtration on a TSK-gel 2000SW column. The pI was pH 4.3 by isoelectric focusing. The E_<1cm, 1% at 280nm> value was 11.5. The optimum pH for the enzyme activity was unique, showing two optima at 4 and 9 (The relative activity at these pHs was apporoximately equal.). The optimum temperature was 45ﾟC.The enzyme was stable at pH 5-9 in 24 hr incubation at 30ﾟC,and stable up to 40ﾟC in a 30-min incubation at pH9.0.5mg of chitin was incubated with chitinase (30mug) in 1ml of 50 mM borate buffer, pH9.0 at 30ﾟC for several hrs. The reaction products from colloidal chitin were analyzed by normal-phase HPLC using a TSKgel Amide-80 column. After 24 hr incubation at 9.0 and 30ﾟC,0.13 mg N-acetylglucosamine, 0.46 mg chitobiose, 0.078 mg chitotriose, and 0.13 mg of chitopentaose were formed. After 36 hr, 0.13 mg N-acetylglucosamine, 0.34 mg chitobiose, 0.068 mg chitotriose, and 0.36 mg of chitopentaose were accumulated. At pH 4.0, another optimum pH of the enzyme, 0.015 mg N-acetylglucosamine, 0.84 mg chitobiose, 0.059 mg of chitotriose were formed after 24 hr of incubation at 30ﾟC.It is noteworthy that chitinase shows a novel activity to yield chitotriose and chitopentaose from colloidal chitin under usual hydrolytic reaction condition. Less
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