| Project/Area Number |
63571046
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Kyushu University |
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Principal Investigator |
IMOTO Taiji Kyushu University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (90038282)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1988: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | Chicken lysozyme / Protein engineering / Mutant lysozyme / Amino acid sequence / Protein function / Protein folding / ニワトリリゾチーム / 遺伝子工学 / 大腸菌 / 酵母 / 変異リゾチーム / 酵素機能 |
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| Research Abstract |
In order to improve the function of chicken lysozyme by protein engineering, the following experiments were carried out.
1. Various mutant chicken lysozymes which always contain an extra methionine residue at the N-terminal were expressed in E. coli, and folded to the active conformations.
2. A method to process the N-terminal sequence of lysozymes thus obtained was developed.
3. A method for the expression and secretion of lysozyme in yeast was established and various mutant lysozymes were prepared by the method.
4. From the properties of the mutant lysozymes, the following informations were obtained. (1) When catalytic carboxyls, Glu35 and Asp52, were mutated, lysozyme activity was almost completely lost. (2) Lysozyme with the mutation at Asp52 has the weak substrate binding ability. (3) The pK of dissociable residue located at position 35 was abnormal to depress the appearance of charges at this position. (4) Replacement of Trp108 with Gln make the enzyme inactive and unstable. (5) Substitution of Asp for Asn46 which is in the beta-sheet lowered the activity to less than 25%. (6) Substitution of Gly for Asn37 or Asp101 increased the lytic activity. (7) Deletion of Arg14-His15 enhanced the chitinase activity. (8) The folding of the reduced lysozyme where the calcium binding site similar to alpha-lactalbumin is constructed was controlled by the concentration of calcium.
5. In order to learn the way to improve lysozyme function for nature, various chicken type-lysozymes were isolated, and their amino acid sequences and properties were determined.
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