Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (2/3): 63-69    DOI: 10.13523/j.cb.2011036
技术与方法     
疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计提高其活性和温度稳定性
魏子翔,张柳群,雷磊,韩正刚,杨江科()
武汉轻工大学生物与制药工程学院 武汉 430023
Improving the Activity and Thermal Stability of Thermomyces lanuginosus Lipase by Rational Design
WEI Zi-xiang,ZHANG Liu-qun,LEI Lei,HAN Zheng-gang,YANG Jiang-ke()
College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China
 全文: PDF(35509 KB)   HTML
摘要：

目的:通过对疏棉状嗜热丝孢菌(Thermomyces lanuginosus) 脂肪酶的理性设计,获得高酶活与耐高温的脂肪酶品种,为脂肪酶在饲料、油脂加工和生物柴油等领域的应用奠定基础。方法:对脂肪酶典型结构域lid和loop区域的系统发育分析,找到候选的位点,理性设计并通过实验验证,获得脂肪酶活性和耐高温特性显著提高的脂肪酶重组子,并构建多拷贝载体,完成50L发酵罐中进行产酶能力评价。结果:经过设计的脂肪酶高温稳定性显著提升。其在80℃下放置12h后仍保留78.94%的酶活。脂肪酶重组子在50L发酵罐中发酵诱导168h,其上清液酶活达到29 000U/mL。结论:成功设计并获得了一种新型的高活性、耐高温的脂肪酶品种TLL,并实现了高效表达,为其产业化和工业应用奠定了基础。
关键词： 脂肪酶;  理性设计;  高活性;  高温稳定性    
Abstract:

Objective:In order to obtain a lipase with high enzyme activity and high temperature resistance, the Thermomyces lanuginosus lipase was modified by rational design, which laid the foundation for the application of lipase in the feed industry, oil processing and biodiesel.Methods:A candidate site was found by phylogenetic analysis of the lid and loop regions of the typical domain of lipase. A lipase recombinant with significantly improved lipase activity and high temperature resistance was obtained through rational design and experimental verification, and a multi-copy vector was constructed to complete the evaluation of the enzyme production ability in a 50L fermentor.Results:The thermal stability of lipase was significantly improved after designing. The crude enzyme solution still retained 78.94% of the enzyme activity after being placed at 80℃ for 12h. After fermentation for 168h in a 50L fermentor, the enzyme activity of the supernatant reached 29 000U/mL.Conclusion:A novel high activity and high thermal stable lipase was successfully designed and obtained, which facilitates its large-scale production and industrial application.
Key words: Lipase    Rational design    High activity    Thermal stability
收稿日期: 2020-11-19 出版日期: 2021-04-08
ZTFLH:  Q814.9  
通讯作者: 杨江科     E-mail: jiangke.yang@gmail.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
魏子翔
张柳群
雷磊
韩正刚
杨江科

引用本文:

魏子翔,张柳群,雷磊,韩正刚,杨江科. 疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计提高其活性和温度稳定性[J]. 中国生物工程杂志, 2021, 41(2/3): 63-69.

WEI Zi-xiang,ZHANG Liu-qun,LEI Lei,HAN Zheng-gang,YANG Jiang-ke. Improving the Activity and Thermal Stability of Thermomyces lanuginosus Lipase by Rational Design. China Biotechnology, 2021, 41(2/3): 63-69.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2011036    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I2/3/63

图1  脂肪酶TLL HotSpot Wizard 3.0的蛋白质比对结果
图2  脂肪酶tll突变位点三维结构示意图
图3  重组子构建与优化前后脂肪酶的表达情况
图4  优化前后TLL的酶学性质结果
图5  优化后的脂肪酶在发酵罐中的表达情况
[1] Treichel H, Oliveira D, Mazutti M A, et al. A review on microbial lipases production. Food and Bioprocess Technology, 2010,3(2):182-196.
doi: 10.1007/s11947-009-0202-2
[2] Hasan F, Shah A A, Hameed A. Industrial applications of microbial lipases. Enzyme and Microbial Technology, 2006,39(2):235-251.
[3] Jaeger K E, Eggert T. Lipases for biotechnology. Current Opinion in Biotechnology, 2002,13(4):390-397.
doi: 10.1016/s0958-1669(02)00341-5 pmid: 12323363
[4] Kobayashi S. Recent developments in lipase‐catalyzed synthesis of polyesters. Macromolecular Rapid Communications, 2009,30(4‐5):237-266.
[5] Andualema B, Gessesse A. Microbial lipases and their industrial applications: review. Biotechnology(Faisalabad), 2012,11(3):100-118.
[6] 蔡海莺, 张婷, Dumba Trish, 等. 疏绵状嗜热丝孢菌脂肪酶在毕赤酵母中的高效表达和性质鉴定. 中国食品学报, 2018,18(6):262-271.
Cai H Y, Zhang T, Trish D, et al. High-efficient expression and properties characterization of Thermomyces lanuginosus lipase in Pichia pastoris. Journal of Chinese Institute of Food Science and Technology, 2018,18(6):262-271.
[7] 代敏, 纪昌涛, 汪小锋, 等. 疏棉状嗜热丝孢菌脂肪酶在毕赤酵母中的表面展示及酶学性质. 微生物学报, 2012,52(7):857-865.
Dai M, Ji C T, Wang X F, et al. Cell surface display of Thermomyces lanuginosus lipase in Pichia pastoris and its characterization. Acta Microbiologica Sinica, 2012,52(7):857-865.
[8] Sumbalova L, Stourac J, Martinek T, et al. HotSpot Wizard 3.0: web server for automated design of mutations and smart libraries based on sequence input information. Nucleic Acids Research, 2018,46(W1):W356-W362.
pmid: 29796670
[9] Pavelka A, Chovancova E, Damborsky J. HotSpot Wizard: a web server for identification of hot spots in protein engineering. Nucleic Acids Research, 2009,37(suppl_2):W376-W383.
[10] Arima K, Liu W H, Beppu T. Studies on the lipase of thermophilic fungus Humicola lanuginosa. Agricultural and Biological Chemistry, 1972,36(5):893-895.
[11] Wada K, Toya Y, Banno S, et al. 13C-metabolic flux analysis for mevalonate-producing strain of Escherichia coli. Journal of Bioscience and Bioengineering, 2017,123(2):177-182.
doi: 10.1016/j.jbiosc.2016.08.001 pmid: 27570223
[12] Secundo F, Carrea G, Tarabiono C, et al. The lid is a structural and functional determinant of lipase activity and selectivity. Journal of Molecular Catalysis B:Enzymatic, 2006,39(1-4):166-170.
[13] Santarossa G, Lafranconi P G, Alquati C, et al. Mutations in the “lid” region affect chain length specificity and thermostability of a Pseudomonas fragi lipase. FEBS Letters, 2005,579(11):2383-2386.
doi: 10.1016/j.febslet.2005.03.037 pmid: 15848176
[14] Zhang J H, Jiang Y Y, Lin Y, et al. Structure-guided modification of Rhizomucor miehei lipase for production of structured lipids. PLoS One, 2013,8(7):e67892.
doi: 10.1371/journal.pone.0067892 pmid: 23844120
[15] 邵化. 耐热脂肪酶/酯酶基因资源的挖掘与研究. 武汉. 华中科技大学, 2014.
Shao H. Study on the resources of thermostable lipases/esterases. Wuhan: Huazhong University of Science and Technology, 2014.
[16] Scorer C A, Clare J J, Mccombie W R, et al. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Bio/ Technology, 1994,12(2):181.
[17] Rabert C, Weinacker D, Pessoa A, et al. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system. Brazilian Journal of Microbiology, 2013,44(2):351-356.
doi: 10.1590/S1517-83822013005000041 pmid: 24294221
[1] 周惠颖,周翠霞,张婷,王雪雨,张会图,冀颐之,路福平. 强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能[J]. 中国生物工程杂志, 2021, 41(2/3): 53-62.
[2] 朱衡,张继福,张云,胡云峰. 环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*[J]. 中国生物工程杂志, 2020, 40(5): 57-68.
[3] 李炳娟,刘金锭,廖谊芳,韩文英,刘珂,侯晨露,张磊. 老黄酶OYE家族的蛋白质工程的研究进展 *[J]. 中国生物工程杂志, 2020, 40(3): 163-169.
[4] 苏永君,胡蝶,胡博淳,李闯,文正,章晨,邬敏辰. 定点突变提高环氧化物水解酶AuEH2催化对甲基苯基缩水甘油醚的对映选择性*[J]. 中国生物工程杂志, 2020, 40(3): 88-95.
[5] 朱衡,张继福,张云,孙爱君,胡云峰. 聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶 *[J]. 中国生物工程杂志, 2020, 40(1-2): 124-132.
[6] 朱衡,林海蛟,张继福,张云,孙爱君,胡云峰. 氨基载体共价结合固定化海洋假丝酵母脂肪酶 *[J]. 中国生物工程杂志, 2019, 39(7): 71-78.
[7] 林海蛟,张继福,张云,孙爱君,胡云峰. 添加剂对大孔吸附树脂固定化脂肪酶的影响 *[J]. 中国生物工程杂志, 2019, 39(4): 38-51.
[8] 石红璆,查代明,张炳火,李汉全. 全细胞脂肪酶研究进展 *[J]. 中国生物工程杂志, 2018, 38(11): 51-58.
[9] 唐存多,史红玲,焦铸锦,刘飞,许建和,阚云超,姚伦广. CPC乙酰化酶底物结合区域Loop上脯氨酸对其催化特性的影响*[J]. 中国生物工程杂志, 2017, 37(12): 34-39.
[10] 虞晓丹, 吴秀秀, 姚冬生, 刘大岭, 谢春芳. 基于分子结构评价的Bacillus subtilis β-1,4-内切木聚糖酶胰蛋白酶抗性的理性设计[J]. 中国生物工程杂志, 2016, 36(8): 80-88.
[11] 郭超, 王志彦, 甘一如, 李丹, 邓勇, 于浩然, 黄鹤. 技术与方法理性设计改造牛肠激酶的热稳定性[J]. 中国生物工程杂志, 2016, 36(8): 46-54.
[12] 曹莹莹, 邓盾, 张云, 孙爱君, 夏方亮, 胡云峰. 南海深海新颖低温脂肪酶的克隆、表达及酶学性质鉴定[J]. 中国生物工程杂志, 2016, 36(3): 43-52.
[13] 周勇, 徐刚, 杨立荣, 吴坚平. 信号肽优化在枯草芽孢杆菌体系中对脂肪酶LipS分泌表达的影响[J]. 中国生物工程杂志, 2015, 35(9): 42-49.
[14] 查代明, 张炳火, 李汉全, 闫云君. 假单胞菌属脂肪酶的分子生物学研究进展[J]. 中国生物工程杂志, 2015, 35(9): 114-121.
[15] 马晨露, 唐存多, 史红玲, 王瑞, 岳超, 夏敏, 邬敏辰, 阚云超. 头孢菌素C乙酰化酶的半理性改造及7-ACA的生物合成[J]. 中国生物工程杂志, 2015, 35(12): 65-71.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会