The Yield And Application Of Xylanase-glucuronidase

Xylanase(XynB)and a-glucuronidase(AguA)produced by extreme thermophilic anaerobic bacterium Thermotoga maritima MSB8 are thermostable(with XynB and AguA protein molecular weight of 43kDa,72kDa),which can degrade hemicellulose resources efficiently,having potential applications in the field of bio-transformation,food industry and feed production.Co-expression of XynB and AguA gene can increase productivity and save costs.The study constructed single promoter co-expressed XynB and AguA recombinant bacteria E.coli JM109(DE3)/pET-20b-XRA and dual promoter co-expressed XynB and AguA recombinant bacteria E.coli JM109(DE3)/pET-20b-XPA and E.coli JM109(DE3)/pET-28a-XPA,then optimized the enzymes production.Results showed that,(1)Induced for 10 h in 0.1 mM IPTG,recombinant bacteria E.coli JM109(DE3)/pET-20b-XRA reached highest production level with XynB 4.28U/mL and AguA 0.025U/mL.(2)Induced for 8 h at 42? in 10mM lactose,recombinant bacteria E.coli JM109(DE3)/pET-20b-XPA reached highest production level with XynB 4.09U/mL and AguA 0.06U/mL.(3)In LB medium,when OD600 reached about 0.7,added 0.8mM IPTG,E.coli JM109(DE3)/pET-28a-XPA produced XynB and AguA 7.6U/mL and 0.5U/mL respectively,under the same conditions,gene expression level in pET-28a vector were inhanced 3.5(XynB)and 7.3(AguA)times than in pET-20b vector.In TB medium,after response surface optimization,inducing in 0.55mM IPTG at OD600 1.93 for 8.92 h,XynB expression level was 12.21U/mL with AguA 1.76U/mL,increased by 0.6 and 2.5 times than before.From enzymatic results of XynB-AguA,Dual enzyme(XynB(10U/g)and AguA(1U/g))can hydrolyze birch xylan more thoroughly and quickly than single XynB(10U/g)with higher content and purity of xylobiose at 80 ?.Response surface optimization was used for 4.2%birch xylan hydrolysis.Optimal conditions for enzymatic hydrolysis was XynB 60U/g and AguA 9U/g in pH7.65 at 80.66 ?,the released reducing sugar was 17.91mg/mL.In addition,from reducing sugar released and electron microscopy,the good degradation can be seen on corn cob.These results not only provide a reliable reference for further amplification fermentation of recombinant bacteria,but also indicate that the recombinant strain has good prospects for industrial application.