摘要:
目的:筛选合成培养基中影响重组毕赤酵母高效表达木聚糖酶的关键成分。方法:首先通过单因素实验和t检验筛选重组毕赤酵母表达木聚糖酶的最佳初始诱导培养基,其次利用Plackett-Burman设计及逐步回归建立培养基成分和响应值间的多元线性回归模型,并筛选出关键培养基成分,最后利用相关系数分析,对非关键成分含量的选取做出合理的取舍。结果:筛选出甘油磷酸钠、硫酸钙、PTM1和硫酸铵是影响重组酵母表达木聚糖酶的关键培养基成分,且当培养基组合为甘油磷酸钠20 g/L,(NH4)2SO4 2.0 g/L,CaSO4·2H2O 2.0 g/L,K2SO4 20.0 g/L,MgSO4·7H2O 6.0 g/L,PTM1 8.0 mL/L,甲醇10.0 mL/L,吐温80 2.0 g/L时,木聚糖酶的酶活力和比酶活分别达到2298.4 U/mL和9926.3 U/mg,分别是优化前的3.055倍和3.889倍。结论:Plackett-Burman设计不仅可以显著提升培养基优化目标,还能筛选出关键培养基成分,从而为进一步优化奠定了基础。
Abstract:
Objectives:The aim of this study was to screen the key components in synthetic medium that affect the efficient expression of xylanase in recombinant Pichia pastoris. Methods:Firstly,the best original defined medium was selected by employing single factor method and t-test. Secondly,the key components were screened via establishing the multivariate linear regression models between medium components and responses with Plackett-Burman design and stepwise regression. Finally,the concentration of non-critical medium components were determined with correlation coefficient analysis. Results:Glycerophosphate,calcium sulfate,PTM1 and ammonium sulphate were screened as key components. Furthermore,it was found that the enzyme activity and specific enzyme activity of xylanase reached maximum values of 2298.4 U/mL and 9926.3 U/mg in Plackett-Burman design,respectively(i.e. when glycerophosphate 20 g/L,(NH4)2SO4 2.0g/L,CaSO4·2H2O 2.0 g/L,K2SO4 20.0 g/L,MgSO4·7H2O 6.0 g/L,PTM1 8.0 mL/L,Methanol 10.0 mL/L,Tween 80 2.0 g/L),which were 3.055 and 3.889 times as many as before optimization,respectively. Conclusion:Plackett-Burman design not only significantly enhance the optimization objectives,but also screen the key components,and thus lay the foundation for further optimization.
百度学术
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