Combined Strategies for Improving Aflatoxin B1 Degradation Ability and Yield of a Bacillus licheniformis CotA-Laccase

Author:

Liu Yanrong1,Liu Limeng1,Huang Zhenqian1,Guo Yongpeng2,Tang Yu1,Wang Yanan1,Ma Qiugang1ORCID,Zhao Lihong1ORCID

Affiliation:

1. State Key Laboratory of Animal Nutrition and Feeding, Poultry Nutrition and Feed Technology Innovation Team, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

2. College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, China

Abstract

Aflatoxin B1 (AFB1) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from Bacillus licheniformis ANSB821 previously reported by our laboratory showed great potential to degrade AFB1 without redox mediators. However, the use of this CotA-laccase to remove AFB1 in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB1, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB1, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (kcat/Km) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into Pichia pastoris GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in P. pastoris GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB1 detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.

Funder

National Key Research and Development Program of China

National Natural Science Foundation of China

Science and Technology Project of Henan Province

Publisher

MDPI AG

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