Improvements of Antimicrobial Activity of Lysozyme by Protein Engineering

通过蛋白质工程改进溶菌酶的抗菌活性

• 批准号: 05660142
• 负责人: KATO Akio
• 金额: $ 1.47万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05660142/
• 关键词: lysozyme; hydrophobic peptide fused lysozyme; Antimicrobial Activity; protein engineering; Gentetic engineering; リゾチームcDNA; 蛋白質工学的機能改変

The effect of the length of hydrophobic peptide (H3 ; Phe-Val-Pro, H5 ; Phe-Phe-Val-Ala-Pro, H7 ; Phe-Phe-Val-Ala-Ile-Pro) at the C-terminus of lysozyme on the bactericidial action against Escherichia coli was investgated by using the yeast harboring the expression vector inserted the cDNA of the hydrophobic fusion lysozyme. Although these hydrophobic fusion lysozyms were little secreted in the culture medium of yeast harboring the expression plasmid, they were highly experssed in the medium when the substitution of Gly with Asn at the position 49 was combined to the fusion lysozyme (H3/G49N-Lz, H5/G49N-Lz, H7/G49N-Lz) to insert the signal sequence (Asn-X-Ser/Thr) of N-linked glycosylation. Interestingly, the amount of secretion of glycosylated fusion lysozyme was much smaller than that of G49N-Lz. These results suggest that the glycosylated fusion lysozyme become unstable and then is subjected to proteolysis due to quality control in endoplasmic reticulum, because the folding dose not work smoothly. On the other hand, the folding of nonglycosylated hydrophobic fusion lysozyme (H3,5,7/G49N-Lz) works so well in ER when their secretion increase considerably. The hydrophobic fusion lysozyme retained 75-80% lytic activity of wild type-Lz. The antimicrobial activity increased in propotion to the length of hydrophobic peptide in fusion lysozyme, although H5/G49N-Lz and H7/G49N-Lz showed almost the same antimicrobial activity. These results suggested that when the hydrophobic residues form the beta-sheet conformation, the length of pentapeptide is enough to penetrate into the inner membrane through the outer LPS membrane. The denaturated fusion lysozyme without lytic activity did not show any killing action against E.coli, suggesting the importance of catalytic domein.

利用插入疏水性cDNA的表达载体的酵母，研究了溶菌酶C端疏水性肽(H3；Phe-Val-Pro、H5；Phe-Phe-Val-Ala-Pro、H7；Phe-Phe-Val-Ala-Ile-Pro)长度对大肠杆菌杀菌作用的影响。 融合溶菌酶。虽然这些疏水性融合溶菌酶在含有表达质粒的酵母培养基中分泌量很少，但当将第49位的Gly替换为Asn与融合溶菌酶(H3/G49N-Lz、H5/G49N-Lz、H7/G49N-Lz)组合以插入信号序列(Asn-X-Ser/Thr)时，它们在培养基中高度表达。 N-连接糖基化。有趣的是，糖基化融合溶菌酶的分泌量远小于G49N-Lz。这些结果表明，糖基化融合溶菌酶变得不稳定，然后由于内质网的质量控制而进行蛋白水解，因为折叠不能顺利进行。另一方面，当非糖基化疏水性融合溶菌酶 (H3,5,7/G49N-Lz) 的分泌显着增加时，其折叠在 ER 中表现良好。疏水性融合溶菌酶保留了野生型-Lz 75-80%的裂解活性。尽管H5/G49N-Lz和H7/G49N-Lz表现出几乎相同的抗菌活性，但抗菌活性与融合溶菌酶中疏水肽的长度成正比增加。这些结果表明，当疏水残基形成β-折叠构象时，五肽的长度足以穿过LPS外膜渗透到内膜。没有裂解活性的变性融合溶菌酶没有表现出对大肠杆菌的任何杀伤作用，这表明催化结构域的重要性。

项目成果

H.R.Ibrahim, M.Yamada, K.Matsushita, K.Kobayashi, and A.Kato: "Enhanced bactericidal action of lysozyme to E.coli by inserting a hydrophobic pentapeptide into its C terminus." J.Biol.Chem.269. 5059-5063 (1994)

H.R.Ibrahim、M.Yamada、K.Matsushita、K.Kobayashi 和 A.Kato：“通过在溶菌酶 C 末端插入疏水性五肽来增强溶菌酶对大肠杆菌的杀菌作用。”

A.Kato,M.Ban: "Polymannosylation to asparagine-19 in hen egg white lysozyme in yeast." FEBS Letters. 355. 76-80 (1994)

A.Kato，M.Ban：“酵母中鸡蛋清溶菌酶中天冬酰胺 19 的多甘露糖基化。”

A.Kato H.R.Ibrahim: "Food Proteins-Structure and Functionality-" K.D.Schwenke and R.Mothes,VCH, 39 (1993)

A.Kato H.R.Ibrahim：“食品蛋白质-结构和功能-”K.D.Schwenke 和 R.Mothes，VCH，39 (1993)

H.R.Ibrahim,A.Kato: "Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C terminus." J.Biol.Chem.269. 5059-5063 (1994)

H.R.Ibrahim、A.Kato：“通过在溶菌酶 C 末端插入疏水性五肽来增强溶菌酶对大肠杆菌的杀菌作用。”

H.R.Ibrahim,A.Kato: "Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C terminus" J.Biol.Chem.269. 5059-5063 (1994)

H.R.Ibrahim，A.Kato：“通过在 C 末端插入疏水性五肽来增强溶菌酶对大肠杆菌的杀菌作用”J.Biol.Chem.269。