基于易错PCR定向进化扩展青霉<em> Penicillium expansum</em> FS1884碱性脂肪酶

中国生物工程杂志

2011, Vol. 31

Issue (11): 64-68

研究报告

基于易错PCR定向进化扩展青霉 Penicillium expansum FS1884碱性脂肪酶

施碧红, 陈明, 翟妙仙, 严蕾, 陈文静

福建师范大学教育部工业微生物工程研究中心福建师范大学生命科学学院福州 350108

Directed Evolution of Alkaline Lipase from Penicillium Expansum FS1884 by Error-prone PCR

SHI Bi-hong, CHEN Ming, ZHAI Miao-xian, YAN Lei, CHEN Wen-jing

Engineering Research Center of Industrial Microbiology, Ministry of Eductation, Fujian Normal University, College of Life Science, Fujian Normal University, Fuzhou 350108, China

全文: PDF(643 KB) HTML

摘要：

为改善扩展青霉FS1884碱性脂肪酶的活性及酶学性质,利用连续两轮易错PCR对扩展青霉FS1884脂肪酶基因PEL进行随机突变,在大肠杆菌JM109中构建突变文库。含突变脂肪酶基因的重组质粒电击转化巴斯德毕赤酵母GS115,经过YPOM板初筛和橄榄油检验板复筛,获得一株酶活性提高的脂肪酶突变体:PEL-ep25-GS。与野生型脂肪酶PEL-GS相比,在最适温度40℃、pH9.4时突变体的酶活力是野生型酶的1.3倍。测序结果表明:该突变体第253位氨基酸发生了突变,由赖氨酸变成蛋氨酸。

关键词： 易错PCR; 脂肪酶; 酶活

Abstract:

To improve the enzymes properties and increase its activity, two continuous rounds of error-prone PCR was introduced to Penicillium expansum FS1884 lipase (PEL) for random mutagenesis, which contributes to a library of mutated clones in E.coli JM109. The recombinant plasmid (pPIC3.5K-ep-PEL)harboring the mutated lipase genes were transformed to Pichia pastoris GS115, and screened by YPOM medium plate and olive oil plate, one mutant named as PEL-ep25-GS whose lipase activity has been increased was screened. The mutated lipase showed 1.3 times activity of that of the wild type under the reaction condition of pH9.4 and 40℃. Sequencing result indicated that the lysine in the site of 253 was changed into methionine (Lys253Met) in the mutated lipase when comparing to the wild type one.

Key words: Error-prone PCR Lipase Enzyme activity

收稿日期: 2011-07-05 出版日期: 2011-11-25

ZTFLH:

Q55

基金资助:

福建省自然科学基金资助项目(B0610027)

通讯作者: 施碧红 E-mail: shibh@fjnu.edu.cn

	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

引用本文:

施碧红, 陈明, 翟妙仙, 严蕾, 陈文静. 基于易错PCR定向进化扩展青霉 Penicillium expansum FS1884碱性脂肪酶[J]. 中国生物工程杂志, 2011, 31(11): 64-68.

SHI Bi-hong, CHEN Ming, ZHAI Miao-xian, YAN Lei, CHEN Wen-jing. Directed Evolution of Alkaline Lipase from Penicillium Expansum FS1884 by Error-prone PCR. China Biotechnology, 2011, 31(11): 64-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I11/64

[1] 施巧琴,章文贤,蒋咏梅,等.产生碱性脂肪酶的突变株FS1884育种技术体系.中国医学研究与临床,2005,3(1):9-13. Shi Q Q, Zhang W X, Jiang Y M, et al. Chinese Medical Research & Clinical, 2005, 3(1): 9-13.

[2] Tobin M B, Gustafsson C, Huisman G W. Directed evolution: the 'rational’ basis for ' irrational’ design. Current Opinion in Structural Biology, 2000, 10(4):421-427.

[3] Pritchard L, Corne D, Kell D, et al. A general model of error-prone PCR. Journal of Theoretical Biology, 2005, 234(4):497-509.

[4] Minning S, Schmidt-Dannert C, Schmid R D. Functional expression of Rhizopus oryzae lipase in Pichia pastoris: high-level production and some properties. Journal of Biotechnology, 1998, 66(2-3):147-156.

[5] 李强,张建军,蔡容华,等.扩展青霉 FS1884 脂肪酶基因的定点诱变及其活性分析.福建师范大学学报:自然科学版, 2009,25(4):105-108. Li Q, Zhang J J, Cai, R H, et al. Journal of Fujian Normal University (Natural Science Edition), 2009, 25(4): 105-108.

[6] Beisson F, Tiss A, Rivière C, et al. Methods for lipase detection and assay: a critical review. European Journal of Lipid Science and Technology, 2000, 102(2):133-153.

[7] van Loo B, Spelberg J H L, Kingma J, et al. Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling. Chemistry & Biology, 2004, 11(7):981-990.

[8] Liebeton K, Zonta A, Schimossek K, et al. Directed evolution of an enantioselective lipase. Chemistry & Biology, 2000, 7(9):709-718.

[9] Fang Y, Lu Y, Lv F, et al. Improvement of alkaline lipase from Proteus vulgaris T6 by directed evolution. Enzyme and Microbial Technology, 2009, 44(2):84-88.

[10] Moore J C, Jin H M, Kuchner O, et al. Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences. Journal of Molecular Biology, 1997, 272(3): 336-347.

[11] Quyen D T, Schmidt-Dannert C, Schmid R D. High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase. Protein Expression and Purification, 2003, 28(1):102-110.

[12] 林琳, 谢必峰,杨冠珍,等.扩展青霉PF898碱性脂肪酶基因组DNA的克隆及序列分析.中国生物化学与分子生物学报,2003, 19(1):12-16. Lin L, Xie B F, Yang G Z, et al. Chinese Journal of Biochemistry and Molecular Biology, 2003, 19(1): 12-16.

[1]	陈中伟,郑璞,陈鹏程,吴丹. 耐热植酸酶突变体的筛选及性质研究 ^*[J]. 中国生物工程杂志, 2021, 41(2/3): 30-37.
[2]	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.
[3]	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.
[4]	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.
[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]	杜立,宿玲恰,吴敬. 提高源自Bacillus circulans 251的β-CGTase对麦芽糖亲和性及其在生产海藻糖中的应用 ^*[J]. 中国生物工程杂志, 2019, 39(5): 96-104.
[8]	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.
[9]	史超硕,李登科,曹雪,袁航,张钰文,于江悦,路福平,李玉. 两个不同启动子及其组合对碱性蛋白酶AprE异源表达的影响 ^*[J]. 中国生物工程杂志, 2019, 39(10): 17-23.
[10]	陈子晗,周海胜,尹新坚,吴坚平,杨立荣. **Amphibacillus xylanus谷氨酸脱氢酶基因工程菌培养条件优化 ^***[J]. 中国生物工程杂志, 2019, 39(10): 58-66.
[11]	黄燕,孙益荣,吴敬,宿玲恰. 重组Humicola insolens角质酶的高密度发酵优化 ^*[J]. 中国生物工程杂志, 2019, 39(1): 63-70.
[12]	李佩忆,周于聪,李雅乾,陈捷. 深绿木霉中碳代谢抑制因子CRE1功能特性研究 ^*[J]. 中国生物工程杂志, 2018, 38(6): 17-25.
[13]	黄鹏,杜望春,施尉珺,饶玉良,孙庆文,张宁. 人源类溶菌酶蛋白6的功能研究及生理特性分析 ^*[J]. 中国生物工程杂志, 2018, 38(3): 1-8.
[14]	郭晓璐,龚秀芳,陈家锋,丁晨曦,胡丹,潘秀珍,王长军. 2型猪链球菌磷酸甘油酸激酶基因的克隆表达及酶活性测定 ^*[J]. 中国生物工程杂志, 2018, 38(3): 16-23.
[15]	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.

Viewed

Full text

Abstract

Cited

Shared

Discussed