NDLI: Directed evolution of cutinase using in vitro compartmentalization

Content Provider	Springer Nature Link
Author	Hwang, Bum Yeol
Copyright Year	2012
Abstract	High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase.
Starting Page	500
Ending Page	505
Page Count	6
File Format	PDF
ISSN	12268372
Journal	Biotechnology and Bioprocess Engineering
Volume Number	17
Issue Number	3
e-ISSN	19763816
Language	English
Publisher	The Korean Society for Biotechnology and Bioengineering
Publisher Date	2012-06-01
Publisher Place	Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	cutinase directed evolution in vitro compartmentalization flurbiprofen Biotechnology Industrial and Production Engineering
Content Type	Text
Resource Type	Article
Subject	Bioengineering Biomedical Engineering Applied Microbiology and Biotechnology Biotechnology

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