Improvements of Antimicrobial Activity of Lysozyme by Protein Engineering

利用酵母表达载体，研究了溶菌酶C-末端疏水肽（H3 ; Phe-Val-Pro，H5 ; Phe-Phe-Val-Ala-Pro，H7 ; Phe-Phe-Val-Ala-Ile-Pro）长度对溶菌酶对大肠杆菌的杀菌活性的影响。虽然这些疏水融合溶菌酶在携带表达质粒的酵母培养基中几乎不分泌，但当将49位Gly替换为Asn组合到融合溶菌酶（H3/G49 N-Lz，H5/G49 N-Lz，H7/G49 N-Lz）中以插入N-连接糖基化的信号序列（Asn-X-Ser/Thr）时，它们在培养基中高度表达。有趣的是，糖基化融合溶菌酶的分泌量远小于G49 N-Lz。这些结果表明，糖基化的融合溶菌酶变得不稳定，然后进行蛋白水解，由于内质网中的质量控制，因为折叠不顺利工作。另一方面，非糖基化的疏水融合溶菌酶（H3，5，7/G49 N-Lz）在ER中的折叠效果很好，当它们的分泌显著增加时。疏水融合溶菌酶保留了野生型Lz的75-80%的裂解活性。H5/G49 N-Lz和H7/G49 N-Lz的抗菌活性基本相同，但抗菌活性随疏水肽长度的增加而增加。这些结果表明，当疏水残基形成β-折叠构象时，五肽的长度足以通过外LPS膜渗透到内膜中。不具有裂解活性的变性融合溶菌酶对大肠杆菌没有任何杀伤作用，提示了催化蛋白的重要性。

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.