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Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability
He, Dong1,2,3; Luo, Wen1,5; Wang, Zhiyuan1; Lv, Pengmei1,4; Yuan, Zhenhong1,4; Huang, Shaowei5; Xv, Jingliang1
2017-07-01
Source PublicationENZYME AND MICROBIAL TECHNOLOGY
Volume102Pages:35-40
AbstractDirected evolution has been proved an effective way to improve the stability of proteins, but high throughput screening assays for directed evolution with simultaneous improvement of two or more properties are still rare. In this study, we aimed to establish a membrane-blot assay for use in the high-throughput screening of Rhizomucor miehei lipases (RMLs). With the assistance of the membrane-blot screening assay, a mutant E47K named G10 that showed improved thermal stability was detected in the first round of error-prone PCR. Using G10 as the parent, two variants G10-11 and G10-20 that showed improved thermal stability and methanol tolerance without loss of activity compared to the wild type RML were obtained. The T-50(60)-value of G10-11 and G10-20 increased by 12 degrees C and 6.5 degrees C, respectively. After incubation for 1 h, the remaining residual activity of G10-11 and G10-20 was 63.45% and 74.33%, respectively, in 50% methanol, and 15.98% and 30.22%, respectively, in 80% methanol. Thus, we successfully developed a membrane-blot assay that could be used for the high-throughput screening of RMLs with improved thermostability and methanol tolerance. Based on our findings, we believe that our newly developed membrane-blot assay will have potential applications in directed evolution in the future.
SubtypeArticle
KeywordDirected Evolution Membrane-blot Methanol Tolerance Thermostability Rhizomucor Miehei Lipases
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
DOI10.1016/j.enzmictec.2017.03.010
WOS Subject ExtendedBiotechnology & Applied Microbiology
WOS KeywordPICHIA-PASTORIS ; DIRECTED EVOLUTION ; PROTEIN EXPRESSION ; SALT-BRIDGES ; ENZYME ; STABILITY ; RESIDUES ; LACCASE
Indexed BySCI
Language英语
Funding OrganizationSpecial funds for applied R & D projects of Guangdong Province(2015B020241002) ; Science and Technology Planning Project of Guangdong Province(2016A010104008)
WOS SubjectBiotechnology & Applied Microbiology
WOS IDWOS:000401688900005
Citation statistics
Cited Times:1[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.giec.ac.cn/handle/344007/14122
Collection中国科学院广州能源研究所
Affiliation1.Chinese Acad Sci, Guangzhou Inst Energy Convers, Key Lab Renewable Energy, Guangzhou 510640, Guangdong, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
3.Chinese Acad Sci, Guangdong Key Lab New & Renewable Energy Res & De, Guangzhou 510640, Guangdong, Peoples R China
4.Collaborat Innovat Ctr Biomass Energy, Zhengzhou 450002, Henan Province, Peoples R China
5.South China Agr Univ, Guangzhou 510642, Guangdong, Peoples R China
First Author AffilicationGuangZhou Institute of Energy Conversion,Chinese Academy of Sciences
Recommended Citation
GB/T 7714
He, Dong,Luo, Wen,Wang, Zhiyuan,et al. Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability[J]. ENZYME AND MICROBIAL TECHNOLOGY,2017,102:35-40.
APA He, Dong.,Luo, Wen.,Wang, Zhiyuan.,Lv, Pengmei.,Yuan, Zhenhong.,...&Xv, Jingliang.(2017).Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability.ENZYME AND MICROBIAL TECHNOLOGY,102,35-40.
MLA He, Dong,et al."Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability".ENZYME AND MICROBIAL TECHNOLOGY 102(2017):35-40.
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