不同启动子调控的两种半纤维素酶分泌表达的比较
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摘要
为提高耐热木聚糖酶DSB和耐热甘露聚糖酶ManB在毕赤酵母GS115中分泌表达的酶活力,选择了3个调控蛋白表达能力较强的启动子P_(FLD1),P_(GAP)和P_(TEF1),以P_(AOX1)为参照,分别在毕赤酵母GS115中进行分泌表达.摇瓶发酵结果表明:P_(FLD1),P_(TEF1),P_(GAP)和P_(AOX1)能有效介导DSB和ManB的分泌,但其胞外酶活相差较大.对于DSB,使用P_(AOX1)时酶活力明显高于P_(FLD1),P_(TEF1)和P_(GAP),最高酶活达846 U/mL;对于ManB,使用P_(FLD1)时酶活力高于P_(AOX1),最高酶活达222 U/mL.故在GS115中分泌表达DSB时选用P_(AOX1),而分泌表达ManB时选用P_(FLD1).
To improve the expression of thermostable xylanase DSB and thermostable mannanase ManB in Pichia pastoris GS115,three strong promoters P_(FLD1),P_(TEF) and P_(GAP) were chosen and used to regulate the expression of DSB and ManB,using P_(AOX1) as a control. The shake-flask fermentation results indicated that P_(FLD1),P_(TEF),P_(GAP) and P_(AOX1)could efficiently control the secretion of DSB and ManB,but their extracellular enzyme activity differed greatly. As for DSB,the enzyme activity of P_(AOX1) was much higher than P_(FLD1),P_(TEF) and P_(GAP),and the highest enzyme activity reached 846 U/mL. But as for ManB,the enzyme activity of P_(FLD1) was higher than P_(AOX1) and the highest enzyme activity reached 222 U/mL. Therefore,P_(AOX1) was the most efficient promoter for DSB expression and P_(FLD1)was the most efficient promoter for ManB expression in Pichia pastoris GS115,respectively.
To improve the expression of thermostable xylanase DSB and thermostable mannanase ManB in Pichia pastoris GS115,three strong promoters P_(FLD1),P_(TEF) and P_(GAP) were chosen and used to regulate the expression of DSB and ManB,using P_(AOX1) as a control. The shake-flask fermentation results indicated that P_(FLD1),P_(TEF),P_(GAP) and P_(AOX1)could efficiently control the secretion of DSB and ManB,but their extracellular enzyme activity differed greatly. As for DSB,the enzyme activity of P_(AOX1) was much higher than P_(FLD1),P_(TEF) and P_(GAP),and the highest enzyme activity reached 846 U/mL. But as for ManB,the enzyme activity of P_(FLD1) was higher than P_(AOX1) and the highest enzyme activity reached 222 U/mL. Therefore,P_(AOX1) was the most efficient promoter for DSB expression and P_(FLD1)was the most efficient promoter for ManB expression in Pichia pastoris GS115,respectively.
引文
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[3] 蒋慧慧,李丰功,陆毅,等. 三种酵母启动子在毕赤酵母中的功能比较[J]. 中国生物工程杂志,2011,31(5): 60-68.
[4] SCHOONOOGHE S,KAIGORODOV V,ZAWISZA M,et al. Efficient production of human bivalent anti-MUCI Fab-scFv antibodies in Pichia pastoris [J]. BMC Biotechnol,2009,9:70.
[5] MELLITZER A,WEIS R,GLIEDER A,et al. Expression of lignocellulolytic enzymes in Pichia pastoris [J] . Microbial Cell Factories,2012,11(1): 61.
[6] LI H,WANG Y,XU A,et al. Large-scale production,purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris [J]. FEMS Yeast Res,2011,11(2): 160-167.
[7] CEREGHINO J L,CREGG J M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris [J]. FEMS Microbiol Rev,2000,24(1): 45-66.
[8] RESINA D,SERRANO A,VALERO F,et al. Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter[J]. J Biotechnol,2004,109(1/2): 103-113.
[9] AHN J,HONG J,LEE H,et al. Translation elongation factor 1-alpha gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter[J]. Appl Microbiol Biotechnol,2007,74(3): 601-608.
[10] 暴立娟,宋庆凤,李杰. 重组木聚糖的安全高效表达与应用研究[J]. 食品工业科技,2011,1(1): 156-159.
[11] 熊海容,陈丰,王亚伟,等.组氨酸标签木聚糖酶的构建表达和酶学特性鉴定[J]. 中南民族大学学报(自然科学版),2014,33(3): 27-31.
[12] 张继泰,曾小英,付正,等. 嗜热真菌木聚糖酶与稀酸联合水解甜菜渣生产L-阿拉伯糖的初步研究[J]. 中南民族大学学报(自然科学版),2009,28(4): 50-53.
[13] WANG Y,SHI P,LUO H,et al. Cloning,over-expression and characherization of an alkali-tolerant endo-β-1,4-mannanase from Penicillium freii F63[J]. J Biosci Bioeng,2012,113(6): 710-714.
[14] 萨姆布鲁克 J,拉塞尔 D W.分子克隆实验指南精编版[M]. 黄培堂,王恒樑,周晓巍,等译. 北京:化学工业出版社,2007.
[15] 张轩薇,叶燕锐,刘晓肖,等.毕赤酵母新启动子在细胞表面展示中的应用[J].生物技术通报,2013,36(4): 129-135.
[16] ZhanR,Mu W,Jiang B,et al. Efficient secretion of inulin fructotransferase in Pichia pastoris using the formaldehyde dehydrogenase 1 promoter[J]. J Ind Microbiol Biotechnol,2014,41(12): 1783-1791.