人溶菌酶来源抗菌肽在毕赤酵母中游离表达及抑菌活性提高的研究  

Recombinant Expression of Antimicrobial Peptides Derived from Human Lysozyme in Pichia pastoris and Improvement of Their Antibacterial Activity by Directed Evolution

作　　者：白敏 任金美 温赛 BAI Min;REN Jinmei;WEN Sai(School of Light Industry Science and Engineering,Beijing Technology and Business University,Beijing 100048,China)

机构地区：[1]北京工商大学轻工科学与工程学院,北京100048

出　　处：《中国生物工程杂志》2025年第2期25-33,共9页China Biotechnology

基　　金：国家自然科学基金(22178006)资助项目。

摘　　要：人溶菌酶是c型溶菌酶的典型代表,其内部含有的抗菌肽序列可以在体外表现出多种生理活性,如杀菌、抗病毒等。尝试对人溶菌酶C端的一个抗菌性“螺旋-环-螺旋”(helix-loop-helix,HLH)结构序列进行重组表达,构建了游离型毕赤酵母表达质粒pPICZαA-HLH-pARS,经过电击转化至毕赤酵母X33(Pichia pastoris X33)中,成功获得人源抗菌肽HLH的重组毕赤酵母表达菌株。甲醇诱导表达72 h后,上清液中的分泌表达产物与人源抗菌肽HLH分子量理论值(4 kDa)一致,其表达量为58.6 mg/L,抗菌活性为260 U/mL。接下来,对人源抗菌肽HLH结构中连接两个螺旋中间loop环的5个氨基酸残基进行点饱和突变从而构建了突变基因文库,并结合酶级联荧光高通量筛选方法进行高杀菌活性突变体的筛选,得到了抗菌肽突变体2-1 A10、2-1 C10、2-2 A2。为设计改造相关位点以提升其他抗菌肽的杀菌活性,尤其是针对革兰氏阴性菌的杀菌活性提供了有效策略。Human lysozyme is a typical representative of c-type lysozymes,and its derived peptides have shown a variety of physiological activities in vitro,such as antimicrobial and antiviral activities.In this study,the structural gene of a“helix-loop-helix”(HLH)antimicrobial peptide derived from the C-terminus of human lysozyme was integrated into an episomal vector,pPICZαA-HLH-pARS,and then transformed into a host strain,Pichia pastoris X33.The positive P.pastoris clone expressing the antimicrobial peptide HLH was successfully obtained.After induction by methanol for 72 h,the yield of HLH peptide reached 58.6 mg/L and the antibacterial activity was 260 U/mL.The secreted recombinant HLH peptide in the supernatant had a molecular weight of 4 kDa,consistent with the theoretical value.Moreover,to further investigate the structure-function relationship of this HLH antimicrobial peptide,the site-saturation mutagenesis libraries of 5 amino acid residues located in the loop region in the middle of two helices of the HLH peptide were constructed,respectively,and the mutants with higher bactericidal activity were screened by the enzyme-cascade fluorescence high-throughput screening method.Three positive antimicrobial peptide mutants 2-1 A10,2-1 C10,and 2-2 A2 were obtained.This study provides new insights and mutation strategies of the loop region of HLH antimicrobial peptides to enhance their bactericidal efficacy against Gram-negative bacteria.

关 键 词：人溶菌酶 抗菌肽 毕赤酵母 游离表达 点饱和突变 

分 类 号：Q815[生物学—生物工程]