Abstract：
Lysozyme, an antimicrobial agent, is extensively employed in the food and healthcare sectors to facilitate the breakdown of peptidoglycan. However, the methods to improve its catalytic activity and secretory expression still need to be studied. In the present study, twelve lysozymes from different origins were heterologously expressed using the Komagataella phaffii expression system. Among them, the lysozyme from the European flat oyster Ostrea edulis (oeLYZ) showed the highest activity. Via a semi-rational approach to reduce the structural free energy, the double mutant Y15A/S39R (oeLYZdm) with the catalytic activity 1.8-fold greater than that of the wild type was generated. Subsequently, different N-terminal fusion tags were employed to enhance oeLYZdm expression. The fusion with peptide tag 6×Glu resulted in a remarkable increase in the recombinant oeLYZdm expression, from 2.81 × 103 U mL-1 to 2.11 × 104 U mL-1 in shake flask culture, and eventually reaching 2.05 × 105 U mL-1 in a 3-L fermenter. The work produced the greatest amount of heterologous oeLYZ expression in microbial systems that are known to exist. Reducing the structural free energy and employing the N-terminal fusion tags are effective strategies to improve the catalytic activity and secretory expression of lysozyme.
摘要：
溶菌酶,抗菌剂，广泛用于食品和医疗保健部门，以促进肽聚糖的分解。然而,提高其催化活性和分泌表达的方法仍有待研究。在本研究中,使用Komagataellaphafii表达系统异源表达了十二种来自不同来源的溶菌酶。其中,欧洲扁平牡蛎（oeLYZ）的溶菌酶活性最高。通过半理性的方法来减少结构自由能，产生了催化活性比野生型高1.8倍的双突变体Y15A/S39R（oeLYZdm）。随后，使用不同的N端融合标签来增强oeLYZdm表达。与肽标签6×Glu的融合导致重组oeLYZdm表达的显着增加，摇瓶培养从2.81×103U·mL-1到2.11×104U·mL-1，在3升发酵罐中最终达到2.05×105U·mL-1。这项工作在已知存在的微生物系统中产生了最大量的异源oeLYZ表达。降低结构自由能和使用N端融合标签是提高溶菌酶催化活性和分泌表达的有效策略。
