食品科学 ›› 2010, Vol. 31 ›› Issue (3): 160-164.doi: 10.7506/spkx1002-6300-201003036

• 生物工程 • 上一篇    下一篇

二次回归正交组合设计与综合相关系数法对耐热纤维素酶基因工程菌发酵条件的优化与分析

杜瑞卿1,吕文平2,*,王 丽1   

  1. 1.南阳师范学院生命科学与技术学院 2.江南大学 食品科学与技术国家重点实验室
  • 收稿日期:2009-05-11 出版日期:2010-02-01 发布日期:2010-12-29
  • 通讯作者: 吕文平 E-mail:duruiqing8@163.com
  • 基金资助:

    国家自然科学基金项目(30972120);南阳师范学院生物化学与分子生物学重点学科科研资助项目

Optimization of Fermentation Medium for Thermostable Cellulase Production by Engineered Bacteria Using Quadratic Regression Orthogonal Composite Design Coupled with Integrated Correlation Coefficient Analysis

DU Rui-qing1 LU Wen-ping2,* WANG Li1   

  1. 1. School of Life Science and Technology, Nanyang Normal University, Nanyang 473061, China
    2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
  • Received:2009-05-11 Online:2010-02-01 Published:2010-12-29
  • Contact: LU Wen-ping E-mail:duruiqing8@163.com

摘要:

目的:研究耐热纤维素酶基因工程菌的规模化生产。方法:采用二次回归正交组合设计试验对内切纤维素酶重组菌的发酵条件进行优化,利用综合相关系数分析法分析因素影响程度以及相对重要性。测定菌体生长的生物量、内切纤维素酶活性。结果: 影响因素与纤维素酶活性间的二次回归方程为:Y2=45.858-1.653Z1-1.258Z2Z3-2.012Z12-1.499Z22-1.415Z32-1.719Z42,方差分析具有显著性。内切纤维素酶重组菌产酶的最佳发酵条件为:添加15.9g/L 乳糖、10g/L麸皮、15g/L硫酸铵、5g/L玉米浆、1.0g/L碳酸钙;理论产酶量最好为46.5357U/mL;在诱导时间约为4h,实际实验酶产量达70.78U/mL。综合相关系数分析表明:4个影响因素的重要性依次为:诱导剂X1(乳糖)>氮源X3(硫酸铵和玉米浆)>钙质量浓度X4(碳酸钙)>碳源X2(麸皮),相互间通过交互对纤维素酶活性的间接影响都非常大,直接影响作用较小。 结论:通过使用综合相关系数分析与二次回归正交组合分析,两者结合使用有互补性,能够较好地指导耐热纤维素酶基因工程菌发酵条件的优化。

关键词: 耐热纤维素酶, 响应面, 综合相关系数, 优化

Abstract:

Objective: To provide reference for large-scale production of thermostable cellullase by engineered bacteria fermentation. Methods: The optimal fermentation medium of recombinant Escherichia coli obtained from Clostridium thermocellum for the production of thermostable cellullase were investigated using quadratic regression orthogonal composite design coupled with integrated correlation coefficient analysis, where four factors and their relative importance were analyzed. Results: The quadratic regression equation between four experimental factors and cellulose activity was presented as follows: Y2 = 45.858-1.653Z1-1.258Z2Z3-2.012Z12-1.499Z22-1.415Z32-1.719Z42. Variance analysis exhibited a significant difference. The optimal fermentation medium of the recombinant strain for the production of thermostable cellullase consisted of 15.9 g/L lactose, 10 g/L wheat bran, 15 g/L ammonium sulfate, 5 g/L corn syrup and 1.0 g/L calcium carbonate. The theoretical cellullase activity was 46.19 U/mL after 4 h of induction time. However, the actual cellullase activity was 70.78 U/mL. Integrated correlation coefficient analysis showed the importance of four factors according to the following order of inducer X1 (lactose)>nitrogen source X3 (ammonium sulfate and corn syrup)>calcium concentration X4 (calcium carbonate)>carbon source X2 (wheat bran). The indirect effects from cross-interaction on cellulase activity were all larger than the direct effects. Conclusion: The combinatorial application of integrated correlation coefficient and quadratic regression orthogonal composite design exhibits a complementary effect, which benefits the optimization of fermentation medium of recombinant Escherichia coli obtained from Clostridium thermocellum for the production of thermostable cellullase.

Key words: Clostridium thermocellum cellullase, quadratic regression orthogonal composite design, integrated correlation coefficient, optimization

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