基于响应曲面法优化贵州木霉NJAU4742产胞外木聚糖酶的研究

Study on conditions for xylanase production by Trichoderma guizhouense NJAU4742 through response surface methodology

[目的]本研究旨在通过响应曲面法优化生物肥功能菌贵州木霉NJAU4742液体发酵过程中产胞外木聚糖酶的参数,为生产木霉固体菌种及其生物有机肥提供理论依据。[方法]采用单因子试验,获得不同单因子对菌株产胞外木聚糖的影响;利用Minitab 15.0软件设计Plackett-Burman试验并对试验结果进行分析,选出核心因子;再利用响应曲面法(response surface methodology)设计Box-Behnken试验完成核心因子的优化,并结合Design Expert 10.0软件分析试验数据,最终得出菌株产木聚糖酶的最佳条件;在最佳条件下,利用凝胶电泳分析胞外蛋白条带差异并测定蛋白含量。[结果]单因子试验结果表明:尿素与氯化钙含量、培养基初始pH值、温度、培养时间对菌株产木聚糖酶影响比较大,尿素和氯化钙最佳含量分别为0.25 g·L-1、0.40 g·L-1,最佳初始pH值3.0、培养温度28℃、培养时间7 d。响应曲面优化试验确定尿素添加量、氯化钙添加量、初始pH值和磷酸二氢钾添加量为影响菌株产胞外木聚糖酶的核心因子,并确定液体发酵主要参数:尿素0.29 g·L-1、氯化钙0.35 g·L-1、磷酸二氢钾2.12 g·L-1、初始pH值3.16、培养温度28℃、培养时间7 d、孢子接种量1.0×105CFU·mL-1。凝胶电泳结果表明,发酵条件优化后部分区域蛋白含量明显增加。[结论]发酵过程参数优化后贵州木霉NJAU4742产胞外木聚糖酶活性达到19.86 U·mL-1,比没有优化前提高1.25倍,菌株胞外蛋白含量比原始培养条件培养提高1.50倍。

[Objectives]The aim of this study is to optimize the parameters of extracellular xylanase production by the biological organic fertilizer functional strain Trichoderma guizhouense NJAU4742 under liquid fermentation through the response surface method,which will provide a theoretical basis for the production of solid spawn and biological organic fertilizer.[Methods]The effects of different single factors on the production of xylanase were obtained by using the single factor experiment,and the key factors were selected by using Plackett-Burman designs based on the Minitab 15.0 software.Then the key factors for the optimization experiments were completed by using Box-Behnken designs based on the response surface methodology.The experimental data were analyzed by the Design-Expert 10.0 software.Finally,the best conditions for xylanase production of the strain were obtained.Under the best conditions,we analyzed the differences of extracellular protein bands by using polyacrylamide gel electrophoresis and determine the protein content.[Results]The results of single factor experiment showed that urea content,calcium chloride content,initial pH value,temperature and culture time owned great influence on the xylanase production,and the optimal values for these parameters were urea content 0.25 g·L-1,calcium chloride content 0.40 g·L-1,initial pH value 3.0,temperature 28℃,culture time 7 days,respectively.The response surface method optimization experiment showed that the urea content,the calcium chloride content,the initial pH value and the potassium dihydrogen phosphate content were the main factors which affected the production of extracellular xylanase,and the main parameters of liquid fermentation were:0.29 g·L-1 urea,0.35 g·L-1 calcium chloride,2.12 g·L-1 potassium dihydrogen phosphate,initial pH value 3.16,culture temperature 28℃,culture time 7 days and inoculum volume 1.0×105 CFU·mL-1 spore.Polyacrylamide gel electrophoresis result showed that the protein content in some regions increased significantly.[Conclusions]The extracellular xylanase activity of T.guizhouense NJAU4742 reached to 19.86 U·mL-1 after optimizing the fermentation parameters,which increased by 25%compared to the initial activity.And the extracellular protein content of the strain increased by 50%compared to the initial activity.