产琥珀酸丝状杆菌1,3-1,4-β-葡聚糖酶基因在毕赤酵母中的表达  

Expression of Fibrobacter succinogenes 1,3-1,4-β-glucanase gene in Pichia pastoris

作　　者：杨玉霞[1] 张慧玲[1] 汪艳[1] 陈勇[1] 

机构地区：[1]新疆农业大学新疆肉乳用草食动物营养与饲料重点实验室,新疆乌鲁木齐830052

出　　处：《南京农业大学学报》2014年第4期123-130,共8页Journal of Nanjing Agricultural University

基　　金：新疆维吾尔自治区科技支疆项目(201191138);新疆维吾尔自治区高技术研究发展项目(201211104)

摘　　要：为实现瘤胃细菌产琥珀酸丝状杆菌（Fibrobacter succinogenes）1,3-1,4-β-葡聚糖酶基因（FsGLU）的高效表达,开发新的饲用β-葡聚糖酶资源,根据毕赤酵母对密码子的偏爱性、G＋C含量等特点,对FsGLU基因进行密码子优化,然后合成优化后的1,3-1,4-β-葡聚糖酶基因（FsGLUm）,转化毕赤酵母GS115,以甲醇诱导表达并对重组FsGLUm的酶学特性、底物特异性和对消化酶的耐受性进行了研究。结果表明：摇瓶水平条件下,以甲醇诱导表达48 h时,重组FsGLUm酶活性提高25.1%（P〈0.05）。在10 L发酵罐中诱导96 h后,重组FsGLUm的活性为6 424 U·mL-1,菌体湿质量和干质量达到274.6和123.6 g·L-1。酶学特性分析表明：纯化后的重组FsGLUm最适反应温度为37℃,最适反应pH值为5.0,比活性为10 397 U·mg-1。在温度低于37℃、pH 5.0~6.5时该酶具有较好的稳定性。底物特异性分析表明：重组FsGLUm可水解大麦β-葡聚糖和地衣多糖,不降解燕麦木聚糖、桦木木聚糖和羧甲基纤维素钠。在胃蛋白酶和胰蛋白酶共同作用60 min后,仍保留了50.5%的酶活性。结论：FsGLU基因在毕赤酵母GS115中实现了高效表达,重组FsGLUm作为饲用酶制剂具有潜在的应用价值。To achieve high-level expression of 1,3-1,4-β-glucanase gene（FsGLU） derived from rumen bacteria Fibrobacter succinogenes in Pichia pastoris,and develop a new feed enzyme preparation resources of β-glucanase,and according to the codon usage frequency and contents of guanine and cytosine of highly expressed genes in P.pastoris,the wild type FsGLU gene was optimized,and the modified β-glucanase gene（FsGLUm） was chemical synthesized and placed under the control of an alcohol oxidase promoter（AOX1） in the plasmid pPIC9 K.Two yeast expression vector pPIC9K-FsGLU and pPIC9K-FsGLUm were constructed and integrated into the genome of methylotrophic yeast P.pastoris GS115 by electroporation. Then,the optimized and wild type β-glucanase gene were expressed in yeast by methanol induction.The strain with highest 1,3-1,4-β-glucanase activity was selected and cultivated in shaking flasks and 10 L fermenter.Enzymatic properties,substrate specificity and tolerability of digestive enzymes of the purified recombinant FsGLUm were also investigated.By comparison with the wild FsGLU,the enzyme activity of the optimized FsGLUm increased 25.1%（P0.05） at the flask level after 48 h of methanol induction.In 10 L fermenter,enzyme activity of the FsGLUm,and wet and dry cell weight which reached the maximum after 96 h of induction,were 6 424 U·mL-1,274.6 g·L-1and 123.6 g·L-1respectively.Enzymatic properties analysis showed that,the optimum pH and reaction temperature of the purified recombinant FsGLUm was 37 ℃ and 5.0,and specific activity was 10 397U·mg-1.The FsGLUm showed relatively higher stability at 20-37 ℃ and pH 5.0-6.5.Substrate specificity analysis showed that,the FsGLUm could hydrolysis barley β-glucan and lichenan,but could not work on oat spelt xylan,birch xylan and sodium carboxymethyl cellulose.In the action of pepsin and trypsin after 60 min,the FsGLUm still retained 50.5% of the maximal activity.Conclusions： the 1,3-1,4-β-glucanase gene originated from F.succinogenes expressed with high efficien

关 键 词：产琥珀酸丝状杆菌 1 3-1 4-β-葡聚糖酶基因 毕赤酵母 基因表达 酶学性质 

分 类 号：S852.23[农业科学—基础兽医学]