《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (5): 1353-1361.doi: 10.16431/j.cnki.1671-7236.2019.05.012

• 动物营养与饲料科学 • 上一篇    下一篇

一株高产内切纤维素酶贝莱斯芽孢杆菌的产酶条件优化及酶学性质分析

陈龙1, 吴兴利1, 李立佳1, 魏炳栋1, 于维1, 闫晓刚1, 谷巍2, 徐海燕2, 刘臣1, 刘洪亮3, 张芳毓1   

  1. 1. 吉林省农业科学院畜牧科学分院, 公主岭 136100;
    2. 山东宝来利来生物工程股份有限公司, 泰安 271000;
    3. 大连金普新区农业局, 大连 116100
  • 收稿日期:2018-11-28 出版日期:2019-05-20 发布日期:2019-05-20
  • 通讯作者: 张芳毓 E-mail:zhangfangyu610@163.com
  • 作者简介:陈龙(1989-),男,吉林吉林人,博士,研究实习员,研究方向:发酵饲料菌种筛选,E-mail:chenliang198931@163.com
  • 基金资助:

    吉林省农业科学院创新工程项目(CXGC2017JQ003);吉林省现代农业产业技术示范推广项目(吉牧201804-1);吉林省科技厅国际科技合作项目(20160414042GH);吉林省科技厅重点科技研发项目(20180201042NY);安全绿色饲料添加剂中试中心项目(20160626002NY)

Optimization of Enzyme Production Conditions and Analysis of Enzymatic Properties of a High-yield Endocellulolytic Enzyme Bacillus velezensis

CHEN Long1, WU Xingli1, LI Lijia1, WEI Bingdong1, YU Wei1, YAN Xiaogang1, GU Wei2, XU Haiyan2, LIU Chen1, LIU Hongliang3, ZHANG Fangyu1   

  1. 1. Branch of Animal Husbandry, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, China;
    2. Shandong BaoLai-LeeLai Bioengineering Co., Ltd., Tai'an 271000, China;
    3. Dalian Jinpu New Area of Agricultural Bureau, Dalian 116100, China
  • Received:2018-11-28 Online:2019-05-20 Published:2019-05-20

摘要:

试验旨在对一株杜仲树皮内生菌贝莱斯芽孢杆菌(Bacillus velezensis)的内切纤维素酶(CMCase)进行产酶条件优化及酶学性质分析,为探究贝莱斯芽孢杆菌CMCase的相关特性提供参考依据。采用单因子试验设计,分别优化pH、接种量、温度、时间、碳源和氮源等发酵条件,并探讨温度、pH、底物专一性、金属离子及表面活性剂对CMCase的影响。结果表明,以玉米秸秆为碳源(30 g/L)、豆粕为氮源(30 g/L)、3%接菌量、pH 7.0的条件下37℃培养48 h,贝莱斯芽孢杆菌157的CMCase最高可达(5.14±0.18) U/mL。经CMCase酶学性质分析发现,贝莱斯芽孢杆菌157的最适酶反应温度为60℃,最适pH为5.0;温度稳定性试验发现,在40和50℃时,相对剩余酶活力均高于80%,随着温度的升高,酶活力逐渐降低,当温度高于55℃,相对剩余酶活力约为50%;pH稳定性试验发现,在pH 5.0~10.0之间耐受性良好,相对剩余酶活力均高于90%,随作用时间的延长,相对剩余酶活力变化不大。贝莱斯芽孢杆菌157 CMCase属于典型内切型纤维素酶。Na+、Mg2+、Ca2+可促进贝莱斯芽孢杆菌157 CMCase酶活力,而Co2+、Hg2+、Fe2+、Cu2+和SDS抑制CMCase酶活力;表面活性剂Tween-80、Tween-20、Triton X-100对贝莱斯芽孢杆菌157 CMCase无影响。本试验通过对贝莱斯芽孢杆菌157进行产酶条件优化及CMCase酶学性质分析发现,贝莱斯芽孢杆菌157 CMCase具有产酶量高,耐受pH范围广的特点,在饲料添加剂、洗涤剂和造纸领域中具有一定的应用价值。

关键词: 贝莱斯芽孢杆菌157; 内切纤维素酶; 产酶; 条件优化; 酶学性质

Abstract:

This study was aimed to test the optimization and characterization of high-yield cellulase produced by an endophytic Bacillus velezensis (B.velezensis) 157 from Eucommia bark,which provided a reference for exploring the relevant characteristics of B.velezensis 157 CMCase.Single-factor experiment was designed to optimize fermentation conditions such as pH,inoculation amount,temperature,fermentation time,carbon source and nitrogen source,and to explore the effects of temperature,pH,substrate specificity,metal ions and surfactants on the enzymatic properties of CMCase.The optimized conditions of CMCase production by B.velezensis 157 were as follows:Maize straw as carbon source (30 g/L),soybean meal as nitrogen source (30 g/L),3% inoculation,and pH 7.0 under the condition of 37℃ for 48 h.After the optimization,the CMCase activity of B.velezensis 157 was up to (5.14±0.18) U/mL.The CMCase properties analysis of B.velezensis 157 found that the optimal temperature of enzyme reaction was 60℃,it could still maintain above 80% cellulase activity when temperature was from 40 to 50℃;With the increase of temperature,the relative residual enzyme activity decreased,and it decreased to 50% when the temperature was higher than 55℃.The optimal pH of cellulase was 5.0,and it could still maintain over 90% cellulase activity in a pH range of 5.0 to 10.0;The pH had little effect on the relative residual enzyme activity with the extension of the action time.The cellulase from B.velezensis 157 belonged to typical endoglueanases.Na+,Mg2+,Ca2+ could increase the activity of B.velezensis 157 CMCase,Co2+,Hg2+,Fe2+,Cu2+ and SDS inhibited the activity of CMCase;The surfactant Tween-80,Tween-20 and Triton X-100 had no effect on CMCase activity.In this study,the enzyme production conditions and enzymatic properties of B.velezensis 157 CMCase were optimized,and it was concluded that B.velezensis 157 had a high yield of CMCase and tolerated a wide range of pH,which had certain application value in feed additive,detergent and paper industry.

Key words: Bacillus velezensis 157, endocellulase, enzyme production, conditions optimization, enzymatic properties

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