To address the issue of mycotoxin contamination that currently constrains the development in fields such as food, a laccase BsCotA, sourced from Bacillus subtilis was selected.Through rational design, the activity and stability of the enzyme were enhanced, thereby increasing the degradation efficiency of the mycotoxins.Based on the protein structure, four mutant laccase enzymes derived from BsCotA were obtained, including three single-point mutants (CotAA344K, CotAA317T, CotAT36Y), and a mutant integrase named CotAgold.These were heterologous expressed in Pichia pastoris.Their enzymatic properties were characterized, and their efficiency in degrading mycotoxins was evaluated.The design of three single-point mutations based on the protein structure improved the stability of the laccase to varying degrees.When the three mutations were integrated into one laccase mutant, no significant antagonism effects occurred between the mutation sites, and this mutant, CotAgold, demonstrated the best stability and catalytic ability.The specific enzyme activity of CotAgold reached 99.4 U/mg, which was 10-fold higher than that of BsCotA, and the kcat/KM for ABTS cationic radicals was 7.12×105 L/(mol·s), which was 1.9-fold times that of BsCotA.The optimal pH of mutant CotAgold was 4, and the optimum reaction temperature was consistently 70 ℃, in line with the catalytic conditions of the wild-type BsCotA.CotAgold maintained over 70% of its enzyme activity after incubation for 30 min across a pH range of 2-12, displaying a noticeable improvement in stability under acidic conditions compared to the wild-type BsCotA.CotAgold retained more than 90% of its residual activity after incubation at temperatures ranging from 40-90 ℃ for 30 min, indicating a significant enhancement in stability at high temperatures compared to the wild-type.When using ABTS＋ as the mediator, the laccase CotAgold completely degraded both aflatoxin B1 and zearalenone, in contrast, the degradation rate by the wild-type laccase BsCotA was only 5%.Through rational design, a laccase mutant CotAgold was obtained, which exhibited significantly enhanced pH stability as well as thermal stability.There was also an increase in protein expression.This mutant has promising application prospects in fields such as mycotoxin degradation.
YUE Chen
,
WANG Xiao
,
XIE Ke
,
ZHENG Yanli
,
YANG Jiangke
,
LEI Lei
. Rational design of Bacillus subtilis laccase and its application in degradation of mycotoxins[J]. Food and Fermentation Industries, 2024
, 50(17)
: 1
-10
.
DOI: 10.13995/j.cnki.11-1802/ts.039316
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