木聚糖酶XynG1-1在毕赤酵母中的表达及发酵条件优化
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摘要
将来源于Paenibacillus campinasensis G1-1的木聚糖酶编码基因成功整合到毕赤酵母GS115基因组上,构建了高产木聚糖酶XynG1-1的毕赤酵母工程菌。采用响应面法对该工程菌的发酵条件进行优化。首先使用Design-Expert软件进行Plackett Burman实验设计筛选出影响产酶量的3个主要因素,即甲醇含量、生物素含量和培养时间。在此基础上使用Design-Expert软件进行Box-Behnken实验设计,通过响应面分析得出优化的发酵培养条件为:甲醇含量2.28%,培养时间37.29 h,生物素4 mg/L,酵母粉20 g/L,蛋白胨20 g/L,YNB 30 g/L,装液量100 m L/L,转速250 r/min、温度28℃、磷酸缓冲液pH 6.0。经实验验证,优化后的培养条件下胞外重组酶活达到707.2 IU/m L,与响应面预测结果一致,较优化前木聚糖酶酶活提高了7.9倍,较原始菌株产酶量提高了19.8倍。经10 L发酵罐扩大培养之后,重组木聚糖酶的酶活达到2 703 IU/m L。因此,该研究有效提高了木聚糖酶XynG1-1的发酵产量,并且,该重组酶保持了良好的酶学性质,可为工业化生产及应用奠定基础。
The gene encoding xylanase from Paenibacillus campinasensis G1-1 was successfully integrated into the genome of Pichia pastoris GS115,and engineered P. pastoris for high-yielding xylanase XynG1-1 was constructed.The fermentation conditions of the engineered bacteria were optimized by using the response surface method. Firstly,Plackett Burman design was carried out using Design-Expert software and three main factors influencing the yield of xylanase were screened out,which were content of methanol,content of biotin and incubation time. Then the Box-Behnken of Design-Expert software was used to design the experiment,the optimize culture conditions for fermentation were confirmed by the analysis of the response surface to be follows: yeast powder of 20 g/L,peptone of 20 g/L,YNB of 30 g/L,biotin of 4 mg/L,methanol content of 2. 28%,liquid volume of 100 m L/L,incubation time of37. 29 h,rotational speed of 250 r/min,temperature 28 ℃,phosphate buffer pH 6. 0. Verified by experiments,the extracellular recombined enzyme activity under the optimized cultivation condition was 707. 2 IU/m L,which was consistent with the prediction of the response surface method and was 7. 9-fold of the former xylanase enzyme activity and19. 8-fold of the enzyme activity produced by original strains. After enlarged culture in 10 L fermentation tank,the recombined xylanase enzyme activity reached 2 703 IU/m L. Therefore,this study effectively improved the fermentation yield of xylanase XynG1-1,and the recombinant enzyme retained better enzymology properties. It established foundation for the further industrial production and application of xylanase XynG1-1.
The gene encoding xylanase from Paenibacillus campinasensis G1-1 was successfully integrated into the genome of Pichia pastoris GS115,and engineered P. pastoris for high-yielding xylanase XynG1-1 was constructed.The fermentation conditions of the engineered bacteria were optimized by using the response surface method. Firstly,Plackett Burman design was carried out using Design-Expert software and three main factors influencing the yield of xylanase were screened out,which were content of methanol,content of biotin and incubation time. Then the Box-Behnken of Design-Expert software was used to design the experiment,the optimize culture conditions for fermentation were confirmed by the analysis of the response surface to be follows: yeast powder of 20 g/L,peptone of 20 g/L,YNB of 30 g/L,biotin of 4 mg/L,methanol content of 2. 28%,liquid volume of 100 m L/L,incubation time of37. 29 h,rotational speed of 250 r/min,temperature 28 ℃,phosphate buffer pH 6. 0. Verified by experiments,the extracellular recombined enzyme activity under the optimized cultivation condition was 707. 2 IU/m L,which was consistent with the prediction of the response surface method and was 7. 9-fold of the former xylanase enzyme activity and19. 8-fold of the enzyme activity produced by original strains. After enlarged culture in 10 L fermentation tank,the recombined xylanase enzyme activity reached 2 703 IU/m L. Therefore,this study effectively improved the fermentation yield of xylanase XynG1-1,and the recombinant enzyme retained better enzymology properties. It established foundation for the further industrial production and application of xylanase XynG1-1.
引文
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[2]CASAS G A,STEIN H H.Effects of microbial xylanase on digestibility of dry matter,organic matter,neutral detergent fiber,and energy and the concentrations of digestible and metabolizable energy in rice coproducts fed to weanling pigs[J].J Anim Sci,2016,94(5):1 933-1 939.
[3]LI Y,GE X,SUN Z,et al.Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw[J].Bioresour Techno,2015,186:316-320.
[4]COLLINS T,GERDAY C,FELLER G.Xylanases,xylanase families and extremophilic xylanases[J].FEMS Microbiol Rev,2005,29(1):3-23.
[5]OKAZAKI F,OGINO C,KONDO A,et al.Expression,crystallization and preliminary X-ray diffraction studies of thermostableβ-1,3-xylanase from Thermotoga neapolitana strain DSM 4359[J].Acta Crystallogr Sect F Struct Biol Cryst Commun,2011,67(Pt 7):7 797-81.
[6]李宁.链霉菌来源的木聚糖降解酶相关基因的克隆及酶学性质研究[D].北京:中国农业科学院博士学位论文,2009.
[7]SUNNA A,ANTRANIHIAN G.Xylanolytic enzymes from fungi and bacteria[J].Critical Reviews in Biotechnology,1997,17:39-67.
[8]SUBRAMANIYAN S,PREMA P.Biotechnology of microbial xylanases:enzymology,molecular biology,and application[J].Crit Rev Biotechnol,2002,22(1):33-64.
[9]DOS SANTOS J A,VIEIRA J M,VIDEIRA A,et al.Marine-derived fungus Aspergillus cf.tubingensis LAMAI 31:a new genetic resource for xylanase production[J].AMBExpress,2016,6(1):25.
[10]GUAN G Q,ZHAO P X,ZHAO J,et al.Production and partial characterization of an alkaline xylanase from a Novel Fungus Cladosporium oxysporum[J].Biomed Res Int,2016,2016:4575024.
[11]XU B,DAI L,LI J,et al.Molecular and biochemical characterization of a Novel Xylanase from Massilia sp.:RBM26 isolated from the feces of Rhinopithecus bieti[J].J Microbiol Biotechnol,2016,26(1):9-19.
[12]GHAFFAR A,KHAN SA,MUKHTAR Z,et al.Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115[J].Mol Biol Rep,2011,38(5):3 227-3 233.
[13]LI Y Y,ZHONG K X,HU A H,et al.High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris[J].Protein Expr Purif,2015,108:90-96.
[14]王丹丹,周晨妍,朱新术等.毕赤酵母工程菌发酵木聚糖酶条件的响应面优化[J].食品工业科技,2016,37(2):194-198.
[15]袁冬华.重组木聚糖酶的发酵及应用研究[D].无锡:江南大学硕士学位论文,2011.
[16]ZHENG Hong-chen,LIU Yi-han,LIU Xiao-guang,et al.Isolation,purification and characterization of a thermostable xylanase from a novel strain Paenibacillus campinasensis G1-1[J].Journal of Microbiology and Biotechnology,2012,22(7):930-938.
[17]ZHENG Hong-chen,LIU Yi-han,LIU Xiao-guang,et al.Overexpression of a Paenibacillus campinasensis xylanase in Bacillus megaterium and its applications to biobleaching of cotton stalk pulp and saccharification of recycled paper sludge[J].Bioresource Technology,2012,125:182-187.