The Cloning And Expression Of Gene Encoding Aflatoxin-detoxifizyme

Aflatoxins are highly toxic secondary metabolites predominantly produced by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus, Aflatoxin B1 is extremely toxic and could induce the cancer, it is stability and difficult to be broken. how to detoxicate AFB1 in grain and feedstuffs were always being concerned. Comparing methods of degradating AFB1 , biological process is more moderate and efficient.Aflatoxin detoxifying enzymes can use aflatoxin B1 (AFB1) as the substrate, and degrade its toxic groups into non-toxic substances. However , the research of aflatoxin detoxifying enzymes is seldom reported at present. In this study, we cloned Aflatoxin-detoxifizyme gene from Armillariella, and identified its biochemical function. it could afford some useful information for the industrial production of aflatoxin detoxifying enzymes.The research includes the items as follows:1. A gene of 2008 bp was amplified.2.The gene was cloned into the vector pET-30a and was expressed in Rosetta (DE3). Through culture conditions optimizing, it is determined that the best-induced temperature is 20℃, IPTG concentration is 0.1mmol/L , culture time is 8h. Western blot testing results showed that , there is an significant special hybridization band in the position of 77 KD.The band, which is the expression product, is the objective protein . Analysis of the biological activities of rADTZ showed that , detoxification rate of the AFB1 is 11.185% and activity of recombinant protein is 33.53×10-3U/g .3.The gene was cloned into the vector pPIC9k-6HisEn-5'and was expressed in GS115.Western blot testing results showed that , there is a significant special hybridization band in the position of 77 KD. The band, which is the expression product, is the objective protein . Analysis of the biological activities of rADTZ showed that , detoxification rate of the AFB1 is 25.52% and activity of recombinant protein is 1.76U/g .