Screening And Validation Of Aflatoxin B1 Degrading Oxidases In Stenotrophomonas Sp.CW117

Aflatoxins are a group of secondary metabolites mian produced by Aspergillus flavus and Aspergillus parasiticus,and widely contaminated in moldy food,nuts and feed.Aflatoxins are serious hepatotoxic,carcinogenic,teratogenic and mutagenic polltants,and closely related to food safety and human health.Among the aflatoxin analogues,aflatoxin B1（AFB1）is the most toxic and the most contaminated pollutant.Therefore,the effective detoxification method on aflatoxin is of great significance to food safety and agricultural production.Biological detoxification has been received widely consideration,because of its high efficiency and specificity,and its reservation characteristics on food nutrients and flavors.At present,the biological detoxification studies mainly focus on the detoxification strains screening and degradation products identification.While,the study of biodetoxification enzymes screening and characterization are less investigated.Stenotrophomonas acidaminiphila CW117 is a difunctional mycotoxins degrading strain which was isolated by the functional microorganism enrichment technology in our laboratory.Previous studies showed the strain CW117 is a difunctional strain that can degrade Ochratoxin A and Aflatoxin B1.Based on the previous study,the serial oxidases as the AFB1 degradation candidate were screened and validated in this study by means of in vitro and in vivo.The main contents and results are outlined as follows:1)Laccase gene lc1 gene deletion and AFB1 degradation.We knocked out the laccase gene lc1（a AFB1 degrading gene）and gene lc2 from the CW117 and produced the mutant CW117^△lc1and CW117^△lc1-lc2by homologous recombination that mediated by the suicide plasmid p K18mobsacb.The degradation result showed no significant difference between the wide-type CW117 and mutant strain CW117^△lc1（p>0.05）,the result indicated that there should be other more effective degradation enzymes（degradation agents）responsible for AFB1 detoxification in CW117.2)AFB1 degradation oxidases screening and validation in vitro.Combined with the genomic information of the type species Stenotrophomonas acidaminiphila and previous studies on aflatoxin degradation,we screened out seven oxidases as the AFB1 degradation candidates.These are,peroxidase（Prx2）,dioxygenase（DIO）,acireductone dioxygenase（ARD）,tryptophan 2,3-dioxygenase（TDO）,4-hydroxyphenylpyruvate dioxygenase（HPPD117）,luciferase like monooxygenase（LLM;i.e.,flavin dependent oxidoreductase）,copper resistance system multicopper oxidase（LC2）.According to published genomic information,we designed specific primers and cloned target genes.The heterologous expression was carried out by p GEX-4T-1（or p ET-32a）in E.coli BL21 heterologous expression system.The heterologous expressed recombinant enzymes were used for AFB1degradation test in vitro.One of screened oxidases（HPPD117）showed significant degradation effect on AFB1 by the method in vitro.The recombinant protein（crude enzyme）r HPPD117 degraded 14%AFB1 with 72 hours incubation.However,the degradation activity of cell-free supernatant prepared from E.coli BL21（p GEX-4T-1/hppd117）was much higher,and the degradation ratio reached 89%within 24h.The results of p GEX-4T-1/hppd117 transformant are consistent with the degradation characteristics of parent strain CW117.That is,the AFB1 active degradation components are mainly distributed in the culture supernatant.However,the recombinant protein r HPPD117 is in bacterial cells of the host E.coli BL 21,and the degradation activity of the r HPPD117 was limited in our study.These results indicated that the degradation agents in culture supernatant of p GEX-4T-1/hppd117 transformant were mediated by gene expression of hppd117,and the degradation agents（possibly non protein substances）was the by-produt during the r HPPD117 heterologous expression.3)The lc1 and hppd117 double genes deletion and AFB1 degradation.Gene hppd117 were also knocked out by suicide plasmid p K18mobsacb.The laccase gene（lc1）mutant CW117^△lc1 was selected as the starting strain,double genes（i.e.,lc1 and hppd117）mutant CW117^△-2 was further produced by homologous recombination technology.Degradation results showed no significant difference on AFB1 degradation between the two mutants of CW117^△lc1 and CW117^△-2（p>0.05）.However,the cell-free supernatant from CW117^△-2on 12 h and 16 h culture time points showed significantly higher AFB1 degradation activity than CW117^△lc1 The statistical difference on 12 h was p<0.01,and on 16 h was p<0.05,but the culture supernatants degradation activities at other culture time-points showed no significant difference.4)Three genes（i.e.,lc1,hppd117 and llm）deletion and AFB1 degradation.On the basis of mutant CW117^△-2,the gene llm was further deleted from the genome of start strain,the three-gene deficient mutant CW117^△-3 was obtained,but the mutant strain CW117^△-3showed no significant difference with CW117^△-2 on AFB1 degradation,and the similar result was obtained from the cell-free supernatant from the two mutants.The present study showed that laccase（LC1）and 4-hydroxyphenylpyruvate dioxygenase（HPPD117）in Stenotrophomonas acidaminiphila CW117 showed degradation ability for AFB1,but the recombinant proteins of r LC1 and r HPPD117 were showed much lower degradation activity than that of strain CW117.However,the culture supernatant prepared from p GEX-4T-1/hppd117 transformant showed efficient degradation ability to AFB1.The degradation ability of the mutant which lacked the two genes of lc1 and hppd117 was only slightly affected;these results indicating that other genes（or degradation agent）in the strain CW117 can mediate AFB1 degradation.The above results found that the degradation mechanisms of Stenotrophomonas acidaminiphila CW117 on AFB1 are quite complex and multiple degradation pathways can mediate the aflatoxin degradation.