响应面法优化革耳Panus rudis FG-35菌株产漆酶培养基

生物技术通报 ›› 2014, Vol. 0 ›› Issue (4): 57-63.

响应面法优化革耳Panus rudis FG-35菌株产漆酶培养基

刘剑, 刘君昂, 周国英, 李河, 杨菁

(1.中南林业科技大学经济林培育与保护教育部重点实验室，长沙 410004；
2.中南林业科技大学林学院，长沙 410004)

收稿日期:2014-01-20 出版日期:2014-04-29 发布日期:2014-04-29
作者简介:刘剑，男，硕士研究生，研究方向：林业应用微生物；E-mail：408199303@qq.com
基金资助:
湖南省科技重大专项(2011FJ1006)，林业公益性行业科研专项经费项目(201004014)，中南林业科技大学研究生科技创新基金项目(CX2012B15)

Optimization of Components for Laccase Production by Panus rudis FG-35 Using Response Surface Methodology

Liu Jian, Liu Jun’ang, Zhou Guoying, Li He, Yang Jing

(1. The Key Laboratory for Non-wood Forest Cultivation and Conservation of Ministry of Education，Central South University of Forestry and Technology，Changsha 410004；
2. College of Forestry，Central South University of Forestry ＆ Technology，Changsha 410004)

Received:2014-01-20 Published:2014-04-29 Online:2014-04-29

摘要/Abstract

摘要： 采用Plackett-Burman 设计和响应面分析相结合的方法，对革耳Panus rudis FG-35菌株产漆酶的液体培养基配方进行优化。单因素试验结果显示，发酵培养基中的最优碳源为可溶性淀粉，最优氮源为蛋白胨；Plackett-Burman 设计筛选出影响漆酶产量的 3 个重要因素为可溶性淀粉、金属Ca²⁺离子和吐温-40，在此基础上运用最陡爬坡试验逼近最大响应值区域，最后利用Box-Behnken试验设计及响应面分析法进行回归分析，获得最佳培养基配方为：可溶性淀粉 10.040 4 g/L、蛋白胨 0.2 g/L、K₂HPO₄ 1.00 g/L、ZnSO₄?7H₂O 0.008 g/L、MgSO₄?7H₂O 0.5 g/L、CuSO₄?7H₂O 0.007 g/L、FeSO₄?7H₂O 0.005 g/L、MnSO₄0.035 g/L、CaCl₂ 0.081 6 g/L、VB₁ 0.1 g/L、吐温-40 0.428%。在优化后的条件下摇瓶发酵产漆酶酶活力为263.31 U/mL，与模型预测值接近，发酵产酶量比优化前提高1.07倍，同时优化后的发酵液对木质素降解进行试验发现，优化后漆酶对木质素降解率提高了14.34%。

关键词: 漆酶, Plackett-Burman设计, 响应面, 降解

Abstract: We optimized the liquid medium components to produce the laccase activity of Panus rudis FG-35 by using Plackett-Burman design and response surface methodology. The results of single-factor test showed that soluble starch and peptone were the best sources of carbon and nitrogen. Then the Plackett-Burman design was applied to determine the specific medium components affecting laccase activity and found that soluble starch, Ca²⁺and Tween-40 were the key factors. Based on these results, steepest ascent experiments were applied to find central points of laccase activity. These significant parameters were further optimized using Box-Behnken design, response surface methodology and regression analysis. Finally, the optimal medium was：soluble starch 10.040 4 g/L, peptone 0.2 g/L, K₂HPO₄1.00 g/L, ZnSO₄?7H₂O 0.008 g/L, MgSO₄?7H₂O 0.5 g/L, CuSO₄?7H₂O 0.007 g/L, FeSO₄?7H₂O 0.005 g/L, MnSO₄0.035 g/L, CaCl₂0.081 6 g/L, VB₁ 0.1 g/L, Tween-40 0.428%. Under these optimal conditions, the activity of laccase increased from 263.31 U/mL to 127.194 U/mL(1.0-fold increase in total yield), similar to the predictions. And the results of lignin degradation experiments indicated that the optimal medium made contribution to the degradation rate of lignin which about 14.34% improvement over before.

Key words: Laccase, Plackett-Burman design, Response surface, Degradation

刘剑, 刘君昂, 周国英, 李河, 杨菁. 响应面法优化革耳Panus rudis FG-35菌株产漆酶培养基[J]. 生物技术通报, 2014, 0(4): 57-63.

Liu Jian, Liu Jun’ang, Zhou Guoying, Li He, Yang Jing. Optimization of Components for Laccase Production by Panus rudis FG-35 Using Response Surface Methodology[J]. Biotechnology Bulletin, 2014, 0(4): 57-63.

参考文献

[1] Yoshida H. LXIII, -Chemistry of Lacquer(Urusht)part I. Comm-unication from the Chemical Society of Tockio[J]. J Chem Soc, 1883, 43：472-486.
[2] 钞亚鹏, 钱世钧.真菌漆酶及其应用[J].生物工程进展, 2001, 21：23-28.
[3] 司静, 崔宝凯, 戴玉成.栓孔菌属漆酶高产菌株的初步筛选及其产酶条件的优化[J].微生物学通报, 2011, 38：405-416.
[4] Snajdr J, Baldrian P. Temperature affects the production, activity and stability of ligninolytic enzymes in Pleurotas ostreatus and Trametes versicolor[J]. Folia Microbiol(Praha), 2007, 52：498-502.
[5] Montgomery DC. 实验设计与分析［M]. 北京：中国统计出版社, 1998：589-640.
[6] Ooijkaas LP, Wilkinson EC, Tramper J, et al. Biotechnol[J]. Bioeng, 1999, 64(1)：92-100.
[7] Montgomery DC. Design and analysis of experiments［M]. 3nd ed. New York：John Wiley & Sons, 1991.
[8] 邱爱连, 李文燕, 郑耀通, 等.黄孢原毛平革菌抗营养阻遏产漆酶诱变育种及其产酶特性[J].微生物学报, 2011, 51(3)：352-359.
[9] 吴坤, 朱显峰, 张世敏, 等.杂色云芝产漆酶的发酵条件研究[J].菌物系统, 2001, 20(2)：207-213.
[10] 刘文华, 蔡宇杰, 范晶晶, 等.毛栓菌产漆酶条件优化及该酶对合成染料脱色的特性[J].微生物学通报, 2013, 40(5)：727-738.
[11] 司静, 李伟, 崔宝凯, 戴玉成.真菌漆酶性质、分子生物学及其应用研究进展[J].生物技术通报, 2011(2)：48-55.
[12] 司静, 崔宝凯, 贺帅, 戴玉成.微酸多年卧孔菌产漆酶条件优化及其在染料脱色中的应用[J].应用与环境生物学报, 2011, 17(5)：736-741.
[13] 王华, 刘小刚, 罗华, 等.木质素降解菌株筛选及葡萄枝条木质素降解研究[J].西北农业学报, 2009, 18：302-305, 311.
[14] Childs RE, Bardsley WG. Thesteady-state kinetics of peoxidase with 2, 2^,-azino-di -(3-ethyl-benzthiazoline-6-sulphonicacid)as chromogen[J]. Biochen J, 1975, 145：93-103.
[15] Plackett RL, Burman JP. The design of optimum multifactorial experiments[J]. Biometrika, 1946, 34(4)：255-272.
[16] Davies OL, George EP, Lewis RC. The design and analysis of industrial experiments［M]. 2nd ed. London：Long-man Group Limited, 1978.
[17] 梅乐和, 胡升, 许静, 等.纳豆枯草芽孢杆菌的筛选和纳豆激酶发酵条件优化[J].浙江大学学报, 2004, 38：1355-1360.
[18] 褚以文.微生物培养基优化方法及其OPTI优化软件[J].国外医药抗生素分册, 1999, 20(2)：58-61.
[19] 王华, 于寒松, 朴春红, 等.响应面法优化重组大肠杆菌PUCRF发酵培养基[J].食品科学, 2011, 32(9)：225-230.
[20] 张超, 张克峰, 侯书国, 等.利用响应面法优化ε-聚赖氨酸发酵培养基[J].微生物学通报, 2011, 38(5)：641-646.
[21] 张亮亮, 徐曼, 汪咏梅, 等.响应面优化化香树果序中鞣花酸超声波提取的研究[J].林产化学与工业, 2011, 31：19-24.
[22] 张学林, 唐湘华, 李俊俊, 等.响应面法优化小桐子油制备生物柴油工艺的研究[J].生物技术通报, 2013(6)：183-187.