Construction And Fermentation Process Optimization Of Recombinant Pichia Pastoris Expressing Xylanase Gene From Streptomyces Sp.FA1

Xylanase belongs to glycoside hydrolase[EC 3.2.1.x]and it can hydrolyze xylan to xylo-oligosaccharides and xylose monosaccharides in noodle products,xylo-oligosaccharide preparation,fruit and vegetable production,feed and Papermaking and other industries.In the previous study,a tenth family?GH10?xylanase?XynA?was isolated and purified from Streptomyces sp.FA1 and the gene was cloned in Escherichia coli BL21?DE3?.To further increase the production of xylanase and explore the practical use of the xylanase,the XynA gene was inserted to epissomal pGAP vector and expressed in Pichia pastoris.The expression level of episomal plasmid was compared with the traditional integration type.?1?The PARS1 and PARS2 were obtained by PCR.The pAOX1-PARS and pGAP-PARS series of episomal vectors were constructed:pPICZ?A-PARS1,pPICZ?A-PARS2,pGAPZ?A-PARS1,p GAPZ?A-PARS2.?2?The xylanase gene was respectively ligated to,episomal recombinant plasmids,and then the plasmid were electrotransformed into P.pastoris KM71 to construct episomal recombinant strains:P.pastoris KM71/pPICZ?A-PARS1-xynA,P.pastoris KM71/pPICZ?A-PARS2-xynA,P.pastorisKM71/pGAPZ?A-PARS1-xynA,P.pastoris KM71/pGAPZ?A-PARS2-xynA.After shake flask cultivation and determination of activity of recombinase XynA,the recombinant strains with high expression leve were selected.?3?The Optimization of the fermentation parameters of the recombinant strain 3.6L bioreactor was performed.The different carbon sources such as glycerol,glucose,sucrose and mixed carbon sources?sucrose:glycerol=1:2?were studied.The most suitable fermentation conditions were as follows:for BSM culture medium 50%glycerol was the most suitable feed carbon source,and the highest enzyme activity was up to 350 U·m L^-1 after 96 hours culture.The relative expression of the free vector expression recombinase increased by 81.2%compared with the relative integration expression.?4?The episomal plasmid containing the xylanase gene was transformed to the traditional integratedxylanaseexpressionstraintoconstructP.pastoris KM71/pPIC9K-xynA/pGAPZ?A-xynA.The xylanase activity was determined after shake flask induction for 144 h.The results showed that the recombinant Xyn A enzyme activity reached202 U·m L^-1,which was 1.7 times that of the traditional integrated type.The recombinant XynA crude enzyme solution was electrophoretically purified by 40%?w/v?ammonium sulfate precipitation,and purification by Mono S cation exchange chromatography.The enzyme characterization study showed that the optimum pH of XynA was 5.5,and the enzyme activity of XynA could be retained more than 80%in pH 3-11 range;the optimum temperature was60°C,and the half-life was 120 h at 55°C;10 mmoL·L^-11 Fe³⁺inhibited the activity of XynA,and almost no enzyme activity was detected;XynA retained about 85%of activity under 10mmo L·L^-1 Mn²⁺and Cr²⁺conditions.?5?The fermentation process of P.pastoris KM71/pPICK-xynA/pGAPZ?A-PARS2-xynA was optimized with induction temperature,initial induction of bacterial concentration,and methanol-induced concentration in a 3.6 L bioreactor.The induction temperature was 28°C,initial OD₆₀₀ concentration was 100 and methanol concentration was 1.5%?v/v?,after 144 h induction the XynA enzyme activity reach 3030 U·m L^-1,which was 2.1 times that of the traditional integration type.At the same time,the specific enzyme activity of Xyn A was increased by 34.4%.The expression level is the highest for the expression of Streptomyces-derived GH10 family xylanase in P.pastoris.