大斑刚毛座腔菌高产漆酶条件的响应面优化及酶学特性

doi:10.3864/j.issn.0578-1752.2015.11.008

摘要/Abstract

摘要： 【目的】优化大斑刚毛座腔菌（Setosphaeria turcica）高产漆酶的最佳发酵条件，确定其酶学性质，为进一步开发应用奠定基础。【方法】以大斑刚毛座腔菌为出发菌株，首先采用单因素试验确定影响菌株产漆酶的碳源、氮源及铜离子的种类及范围，并在单因素试验的基础上，以漆酶酶活力为响应值，采用central composite design（CCD）响应面设计试验，利用Design Expert软件对响应值进行3因素3水平下的多元二次回归拟合分析，优化大斑刚毛座腔菌产漆酶的发酵培养基成分；初步分离大斑刚毛座腔菌发酵液中的漆酶，以ABTS为反应底物，设置不同温度及pH，测定漆酶的最适反应温度、pH及热稳定性、pH稳定性，进一步测定其反应动力学常数Km值、Vm值，确定其酶学特性。【结果】建立了以漆酶酶活力为响应值的多元二次回归模型，模型差异显著（P=0.0001），可以用该模型来拟合试验；响应面分析结果表明，各因素对漆酶活力的影响大小依次为Cu²⁺>葡萄糖>尿素，而葡萄糖和尿素交互作用极显著；通过拟合求出模型极值点，对应的大斑刚毛座腔菌产漆酶的最佳培养条件为：葡萄糖50.05 g?L^-1，KH₂PO₄ 1 g?L^-1，尿素1.46 g?L^-1，MgSO₄ 0.5 g?L^-1，蛋白胨2 g?L^-1，玉米浆0.5 g?L^-1，CuSO₄ 0.07 g?L^-1，Tween80 3 mL?L^-1，28℃，150 r/min振荡培养7 d；在此条件下漆酶活力最高达（40.00±1.20）U?mL^-1。对大斑刚毛座腔菌漆酶发酵液初步分离，经SDS-PAGE检测其漆酶相对分子量约为80 kD；以ABTS为底物时，最适反应温度为50℃，最适pH为4.2，在温度较高且弱酸性条件下活性较高，并且具有良好的温度稳定性和pH稳定性，常温下pH为4.2保持14 h后酶活力基本不变，50℃保温14 h后漆酶活力仍保持在60%以上；进一步在常温、pH为4.2时测定其米氏常数Km值为0.036 mmol?·L^-1，最大反应速率Vm为28.63 mmol?L^-1?min^-1。【结论】利用大斑刚毛座腔菌液体发酵产漆酶并研究其酶学特性，表明作为玉米致病菌的大斑刚毛座腔菌产漆酶具有发酵周期短、活性高、稳定性好等特性，可进一步研究开发应用。

关键词: 大斑刚毛座腔菌, 漆酶, 响应面, 酶学性质

Abstract: 【Objective】The objective of this study is to optimize fermentation parameters for the laccase production by Setosphaeria turcica, investigate its enzymatic properties, and to develop the applications of the laccase.【Method】According to the results of single factor experiments to confirm the categories and concentration scales of the carbon source, nitrogen source and Cu²⁺ as single parameters, the response surface experiments of fermentation using S. turcica were preformed which the enzymatic activity was chosen as the response value. The experiments were designed by the central composite design (CCD), and analyzed by Multivariate Quadratic Regression Analysis of Design Expert for three factors and three levels, then the optimal fermentation medium for laccase production by S. turcica was determined. And the properties of laccase isolated from the fermentation culture were characterized using ABTS as substrate, including the optimum temperature and pH, the stability at different pH and temperatures, and the kinetics constants of Km and Vm.【Result】The multivariate quadratic regression model which the enzymatic activity was chosen as response value was significantly different (P=0.0001), which meant the established regressing equation for activity of laccase had an excellent goodness of fit and the laccase fermented by S. turcica could be analyzed by this model. According to the model, the factors influencing the activity of laccase were in the order as follows: Cu²⁺＞glucose＞urea and the interaction of glucose and urea was very significant. The optimum conditions, which were predicted by this model, were glucose 50.05 g?L^-1, KH₂PO₄ 1 g?L^-1, urea 1.46 g?L^-1, MgSO₄ 0.5 g?L^-1, peptone 2 g?L^-1, maize steep liquor 0.5 g?L^-1, CuSO₄ 0.07 g?L^-1 and Tween80 3 mL?L^-1, at 28℃, and 150 r/min. The maximal enzyme activity was (40.00±1.20) U?mL^-1 after 7 d of cultivation. The laccase of S. turcica had a molecular weight of approximately 80 kD analyzed by SDS-PAGE after preliminarily purified. It was demonstrated that the optimum pH and temperature were pH 4.2 and 50℃, respectively, and the laccase of S. turcica had higher activity at higher temperature and weak acidic condition, using ABTS as substrate. It also exhibited high thermal stability and the stability of pH, retaining its activity basically unchanged after incubation at pH 4.2 for 14 h and above 60% at 50℃ for 14 h. The Km was 0.036 mmol?L^-1 and Vm was 28.63 mmol?L^-1·min^-1, at room temperature and pH 4.2. 【Conclusion】The enzymatic properties of the laccase of S. turcica were investigated, and the fermentation medium by response surface methodology was optimized. The laccase of S. turcica has a potential value for further research due to the advantages of high activity, short fermentation period and high stability.

Key words: Setosphaeria turcica, laccase, response surface methodology, enzymatic characters

曹可可，刘宁，马双新，曹志艳，梁东旭，柴江婷，董金皋. 大斑刚毛座腔菌高产漆酶条件的响应面优化及酶学特性[J]. 中国农业科学, 2015, 48(11): 2165-2175.

CAO Ke-ke, LIU Ning, MA Shuang-xin, CAO Zhi-yan, LIANG Dong-xu, CHAI Jiang-ting, DONG Jin-gao. Optimization of Fermentation Condition for Laccase Production by Setosphaeria turcica Using the Response Surface Methodology and the Enzymatic Characters[J]. Scientia Agricultura Sinica, 2015, 48(11): 2165-2175.

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