In order to improve the expression level of nattokinase, the N-terminal coding sequences (NCSs) of the aprN were synonymously mutated without changing the amino acid sequence encoding nattokinase.Four mutant strains, NK1, NK2, NK3 and NK4, were constructed by transforming recombinant pHY-P43 plasmid which containing the mutated sequences into Bacillus subtilis.The enzyme activity of nattokinase was detected by fibrin plate.The expression of recombinant protein was verified by SDS-PAGE.The fermentation conditions of mutant strains were optimized by response surface.The results showed that the nattokinase activity of NK1, NK2, NK3 and NK4 strains were 94.8 IU/mL, 101.9 IU/mL, 124.2 IU/mL and 69.4 IU/mL, respectively.The NK3 strain had the highest enzyme activity, which was 50% higher than that of the original strain.The NK3 strain had the lowest enzyme activity, which was 83% of that of the original strain.The results of SDS-PAGE showed that the molecular weight of recombinant nattokinase was about 28 kDa, which was consistent with the theoretical molecular weight of 27 kDa.Response surface analysis showed that initial pH 7.56, fermentation temperature 35 ℃, and fermentation time 38 h were the optimal conditions for production of nattokinase in NK3 strain.The enzyme activity of nattokinase was 138.1 IU/mL, which was 11.2% higher than before the optimization.This study provides a new strategy to enhance the activity of nattokinase without altering the properties of nattokinase itself.
HUANG Xilin
,
HUANG Junbao
,
GAO Xuli
,
LUO Yani
,
TAO Wei
,
GUO Mingyu
,
LIU Yongyuan
,
WU Chao
,
WU Jing
,
LIU Yan
. Modification of N-terminal coding sequence of aprN promotes efficient expression of nattokinase[J]. Food and Fermentation Industries, 2025
, 51(3)
: 113
-119
.
DOI: 10.13995/j.cnki.11-1802/ts.038653
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