1996 Fiscal Year Final Research Report Summary
Protein thermostabilization by proline substitutions in accordance with the proline rule
| Project/Area Number |
07660118
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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 |
応用微生物学・応用生物化学
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| Research Institution | Kyoto Prefectural University, Department of Agricultural Chemistry |
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Principal Investigator |
SUZUKI Yuzuru Kyoto Prefectural University, Department of Agricultural Chemistry, Professor, 農学部, 教授 (50046471)
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| Co-Investigator(Kenkyū-buntansha) |
WATANABE Kunihiko Kyoto Prefectural University, Department of Agricultural Chemistry, Associate Pr, 農学部, 講師 (90184001)
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| Project Period (FY) |
1995 – 1996
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| Keywords | proline / thermostability / protein engineering / site-directed mutagenesis / differential scanning calorimetry / reversibility / thermophile |
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| Research Abstract |
Confirmation of protein thermostabilization by proline substitutions---Cumulative replacements at Lys457 (the second position of beta-turn) , Thr440 (loop) and Ile403 (the N1 position of alpha-helix) with proline residues resulted in the additive enhancement in thermostability of oligo-1,6-glucosidase from Bacillus cereus ATCC7064. The enhancement was the most effective at the second position of beta-turn and at the N1 position of alpha-herix. The mutant enzymes showed similarities in structure and function as the wild one. These observations are consistent with the results obtained before with 9 mutant enzymes. Evaluation of stability for oligo-1,6-glucosidases---The reversible stability of the mutant and wild oligo-1,6-glucosidases was analyzed by tracing the intensities of their fluorescence after denatured with different concentrations of guanidine hydrochloride. However, the reversibility of the proteins failed to be detected during the denaturation with the reagent. The mutant and wild proteins showed the irreversible resistance to guanidine hydrochloride, corresponding to the thermal stability detected before. On the other hand, a thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006, which is 72%-identical with the B.cereus enzyme in primary structure, indicated more irreversible resistance to the reagent as well as reversible stability. The system of differential scanning calorimetry could catch the reversibility of B.cereus oligo-1,6-glucosidase. This system revealed that the reversible stability for each of the wild and mutant proteins was related to the irreversible one in Tm values.
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