Screening and Characterization of a Mutant Fungal Aspartic Proteinase from Mucor pusillus
RESEARCH ARTICLE

Screening and Characterization of a Mutant Fungal Aspartic Proteinase from Mucor pusillus

Li Yuqiu Tan Hua Li Da Li Zhoulin Chi Yanping Jiang Yuanyuan Liu Xiangying Wang Jinghui, * Open Modal Li Qiyun, * Open Modal
Authors Info & Affiliations
The Open Biotechnology Journal • 10 Sep 2015 • RESEARCH ARTICLE • DOI: 10.2174/1874070701509010119

Abstract

In this study, site-directed mutagenesis was carried out to alter properties of Mucor pusillus rennet (MPR) in order to find a potential substitution of commercial chymosin. Mutant G186D/E13D screened from thousands of mutants showed a significant milk-clotting activity (MCA). Mutant G186D/E13D rennet was purified and characterized. The molecular weight was estimated to be 44 kDa by SDS-PAGE. The maximum enzyme activity was at a wide range of pH (5.0-7.0) and 60ºC. The enzyme was inhibited by metal ions (Fe2+, Fe3+, Cu+ and Zn2+), 1.10-Phenantrolin and pepstatin A. Further texture analysis of types of cheddar cheese made by non-mutant rennet, mutant (G186D/E13D) rennet and commercial rennet suggested that the soluble nitrogen content and hardness of cheddar cheese made by chimeric mutant rennet was decreased without any significant change in flavor between these cheeses. The result implicated that, to some extent, the mutant rennet could decrease hydrolysis of protein during ripening of cheese, probably as a candidate for a useful milk coagulant.

Keywords: Aspartic proteinase, Mucor pusillus rennin, Mutation, Thermostability, Proteolytic activity.