GH43家族β-D-木糖苷酶RuXyn1在马克斯克鲁维酵母中的表达及其应用  

Expression and Application of GH43 β-D-xylosidase RuXyn1 in Kluyveromyces marxianus

作　　者：段懿桐 李雪莹 周峻岗[1,2] 余垚 吕红[1,2] DUAN Yitong;LI Xueying;ZHOU Jungang;YU Yao;Lü Hong(State Key Laboratory of Genetic Engineering,Fudan University,Shanghai 200438,China;Shanghai Engineering Research Center of Industrial Microorganisms,School of Life Sciences,Fudan University,Shanghai 200438,China)

机构地区：[1]复旦大学遗传工程国家重点实验室,上海200438 [2]复旦大学生命科学学院上海工业菌株工程技术研究中心,上海200438

出　　处：《复旦学报（自然科学版）》2020年第5期599-607,共9页Journal of Fudan University：Natural Science

基　　金：上海市科学技术委员会项目(19DZ2282100);国家科技部项目(2014AA021301)。

摘　　要：木聚糖的降解需要内切β-木聚糖酶和β-D-木糖苷酶的协同作用,β-D-木糖苷酶能将β-木聚糖酶的水解产物木寡糖降解为木糖,因此在半纤维素的利用中具有重要的意义.本研究将中国牦牛瘤胃微生物来源的糖基水解酶GH43家族β-D-木糖苷酶基因RuXyn1克隆到含两种不同分泌信号肽(菊粉酶信号肽和α-factor信号肽)的马克斯克鲁维酵母(Kluyveromyces marxianus)分泌表达载体(pUKDN132和pUKDN129)中,转化K.marxianus Fim-1 ura3Δ,通过筛选获得两株分泌表达β-D-木糖苷酶基因RuXyn1的重组菌株Fim-1/pUKDN132-RuXyn1和Fim-1/pUKDN129-RuXyn1.重组菌株摇瓶培养72h后,上清液中β-D-木糖苷酶酶活分别为0.304U/mL和0.045U/mL.在5L发酵罐高密度发酵中,Fim-1/pUKDN132-RuXyn1分泌表达的β-D-木糖苷酶酶活为1.75U/mL,而Fim-1/pUKDN129-RuXyn1的酶活为1.6U/mL,表明在表达β-D-木糖苷酶RuXyn1时,菊粉酶信号肽介导的分泌效率优于α-factor信号肽.以玉米芯为底物,我们测试了K.marxianus重组表达的β-D-木糖苷酶RuXyn1的降解效果,发现在商业化的β-木聚糖酶和纤维素酶CTec2中,添加β-D-木糖苷酶RuXyn1能够显著提升玉米芯的降解效率,还原糖产量提高7%,木糖和葡萄糖的产量分别提高12.4%和6.1%.The degradation of xylan requires concerted action of endogenousβ-xylanase andβ-D-xylosidase.Sinceβ-D-xylosidase degrades xylooligosaccharide which is a hydrolyzed product ofβ-xylanase into xylose,it is of great significance in the utilization of hemicellulose.In this study,we cloned a GH43β-D-xylosidase gene RuXyn1 from microorganisms in Chinese yak rumen into two Kluyveromyces marxianus secretory expression vectors pUKDN132 and pUKDN129,containing two different secretory signal peptides(inulinase andα-factor signal peptide),and transformed these recombinant expression vectors into the host strain,K.marxianus Fim-1 ura3Δ.The two recombinant strains,Fim-1/pUKDN132-RuXyn1 and Fim-1/pUKDN129-RuXyn1,could efficiently secrete theβ-D-xylosidase RuXyn1 with 0.304 U/mL and 0.045 U/mL after shaking flask culture for 72 hours,respectively.Similarly,in the 5 L fermenter high-density fermentation,theβ-D-xylosidase activity in the supernatant produced by Fim-1/pUKDN132-RuXyn1 was 1.75 U/mL,while it was 1.6 U/mL in Fim-1/pUKDN129-RuXyn1.These results indicated that the expression efficiency of inulinase signal peptide is better than that ofα-factor signal peptide when expressingβ-D-xylosidase RuXyn1 in K.marxianus.The degradation effect of the recombinantly expressedβ-D-xylosidase RuXyn1 in K.marxianus was further investigated using the corn cob.Analysis data showed that the degradation efficiency of corn cob was significantly improved by addingβ-xylosidase RuXyn1 to the mixture of commercialβ-xylanase and cellulase CTec2.To be specific,the yield of reducing sugar increased by 7%,and the yield of xylose and glucose increased by 12.4%and 6.1%,respectively.

关 键 词：木聚糖 β-D-木糖苷酶 表达载体 马克斯克鲁维酵母 重组表达 

分 类 号：Q36[生物学—遗传学] Q814