1999 Fiscal Year Final Research Report Summary
Stabilization of carbohydrate-depleted invertase by modification with comb-shpaed poly(ethylene glycol) derivative.
| Project/Area Number |
10680806
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | UNIVERSITY OF YOKOHAMA |
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Principal Investigator |
HIROTO Misao Toin University of Yokohama, Materials Science and Technology, Coprofessor, 工学部, 助教授 (00110172)
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| Co-Investigator(Kenkyū-buntansha) |
FUTAMI Ayako Toin University of Yokohama, Materials Science and Technology, Coprofessor, 工学部, 助教授 (30209142)
NISHIMURA Hiroyuki Toin University of Yokohama, Materials Science and Technology, Professor, 工学部, 教授 (60189313)
INADA Yuuji Toin University of Yokohama, Materials Science and Technology, Professor, 工学部, 教授 (40016035)
KODERA Yoh Toin University of Yokohama, Materials Science and Technology, Lecturer, 工学部, 講師 (80205426)
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| Project Period (FY) |
1998 – 1999
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| Keywords | comb-shaped PEG / chemical modification / stabilization / β-fructofranosidase / fructo-origosaccharide / heat stability / pH-dipendency |
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| Research Abstract |
The chemical modification of invertase (with CHO) with activated PM was carried out whether or not the stabilization of invertase (with CHO) could be obtained. Then invertase (without CHO) was modified with activated PM by changing the molar ratio of the modifier to amino groups in the enzyme molecule. The degree of modification of invertase (without CHO) is enhanced by increasing the molar ratio and reached up to 80%. In contrast, the enzymic activity is slowly decreased by increasing the molar ratio and tends to approach a constant level of 55% of the initial activity.
Three kinds of invertases were subjected to test the stability toward heat, pH and protease-digestion : invertase (with CHO), invertase (without CHO) and PM-invertase (without CHO) in which the degree of modification of amino group is 65% and the enzymic activity retains approximately 60% of the original activity.
The enzymic activity of invertase (without CHO) was a little higher than that of invertase (with CHO). And t
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hat of PM-invertase (without CHO) was lower than the other two invertases. Carbohydrate contents of invertase (without CHO) and PM-invertase (without CHO) treated with endo-β-N-acetylglucosaminidase H were only 0.05 mg/mg of protein in comparison with 1.0 mg/mg of protein for invertase (with CHO). In the SDS-polyacrylamide gel electrophoresis, a broad band of invertase (with CHO) was observed at molecular weight of about 120kDa.
Although the enzymic activity of invertase (without CHO) is sharply decreased with time and completely lost by 90 min-incubation, the activity of PM-invertase (without CHO) is retained by 50% of the initial activity even after 90 min-incubation. In the case of invertase (with CHO) (curves B), the activity is well retained in comparison with that of invertase (without CHO), suggesting the carbohydrate plays an important role in maintaining the conformation of the invertase molecule.
However, the heat-stability of invertase (with CHO) is rather low in comparison with that of PM-invertase (without CHO). The enzymic activity of PM-invertase (without CHO) retains the maximum level at pH range from 3.5 to 7.5 and reduces slowly at outer pHs (curve A). Less
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Research Products
(14 results)
