The mycotoxin zearalenone (ZEN) can be degraded by a lactone hydrolase ZHD， which was derived from Gliocladium roseum. Here， based on the native ZHD encoding gene zhd101， a codon optimized zhd gene was synthesized， which was used for high expression of ZHD in Pichia pastoris GS115. Meanwhile， to further improve the expression of recombinant ZHD， the plasmids containing 1 to 4 copies of the zhd expression cassette were constructed， respectively， using the biobrick method. The protein expression in the recombinant P. pastoris X3c， which was transformed with the plasmid containing 3 copies of zhd expression cassette， was the highest. In addition， the enzymatic activity of ZHD against ZEN was ... More
The mycotoxin zearalenone (ZEN) can be degraded by a lactone hydrolase ZHD， which was derived from Gliocladium roseum. Here， based on the native ZHD encoding gene zhd101， a codon optimized zhd gene was synthesized， which was used for high expression of ZHD in Pichia pastoris GS115. Meanwhile， to further improve the expression of recombinant ZHD， the plasmids containing 1 to 4 copies of the zhd expression cassette were constructed， respectively， using the biobrick method. The protein expression in the recombinant P. pastoris X3c， which was transformed with the plasmid containing 3 copies of zhd expression cassette， was the highest. In addition， the enzymatic activity of ZHD against ZEN was defined for the first time based on a standard curve of peak area vs ZEN concentration. The ZEN degradation activity of ZHD from shake flask fermentation was calculated as 22.5U/mL with the specific activity of 4976.5U/mg. Furthermore， the high-density fermentation of P. pastoris X3c strain was also performed in 5L fermenter. The maximum enzyme activity of the supernatant was 150.1U/mL， which were 6.7-fold higher than that of the shake flask fermentation.
