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*, Li Qiyun*
Center of Agro-food Technology, Jilin Academy of Agricultural Sciences, Changchun, Jilin, 130033, P.R. China


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Creative Commons License
© 2015 Yuqiu et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Center of Agro-food Technology, Jilin Academy of Agricultural Sciences, Changchun, Jilin, 130033, P.R. China; Tel: +86 43 87063260; Fax: +86 43 87063062; E-mail: 846812862@qq.com


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.