Expression And Characterization Of An Extreme-thermostable Xylanase XYNH

Xylan as a major component of hemicellulose,it is the second largest recyclable natural resource after cellulose.Xylanases are glycosidases(O-glycoside hydrolases,EC 3.2.1.x)which catalyze the endohydrolysis of 1,4-?-D-xylosidic linkages in xylan.Among xylan degradation enzymes,endo-1,4-xylanase(EC 3.2.1.8)is the key enzyme to complete hydrolysis of xylan.As far as we know most of xylanase come from bacteria,fungi,plants and animals.Due to the excellent thermostability,thermostable xylanas has broad prospects in the biomedical engineering and bioinformatics applications.The gene of Thermotoga maritima GH10 xylanase(XynB)was synthesized(GenBank: KR078269).This gene was constructed and expressed in E.coli and P.pichia.We named this xylanase with XYNH.The purified XYNH was identified by SDS-PAGE,the results show that expressed in E.coli had a size of 38 kDa but in P.pichia had a size of about 42 kDa,indicating that XYNH in Pichia may glycosylated.After treatment with Endo H specific for N-glycosylation,there was a second protein version with the apparent molecular mass of 38 kDa,which is the same size as the enzyme expressed in E.coli,means XYNH expressed in P.pichia may contains N-glycosylation sites.Several characterizations of XYNH were carried out in this study.At 100°C and pH 5.0,the specific activity of XYNH expressed in E.coli and P.pichia was 375 IU/mg and110 IU/mg,respectively.At the same condition,the half-life times of XYNH expressed in E.coli and P.pichia were 78 and 126 min,respectively.XYNH had a wide pH stability region(pH 5.0-9.0).The low concentrations of NaCl(0.5 M)enhance activity of XYNH,while at 2 M NaCl the activity was similar to without the salt,indicated that XYNH was salinity tolerance.In 30 minutes incubation of XYNH,the decreases of temperature optimumwere 5°C,10°C and 15°C,with 15%,25% and 35% of [EMIM]OAc,respectively.XYNH retained over 80% of its activity at 90°C in 15% [EMIM]OAc during 22-h reactions.