NDLI: A Semi-Rational Approach to Engineering Laccase Enzymes

Content Provider	Springer Nature Link
Author	Miele, Annalisa Giardina, Paola tomista, Eugenio Piscitelli, Alessandra Sannia, Giovanni Faraco, Vincenza
Copyright Year	2010
Abstract	In order to develop improved laccase-based bio-catalysts, semi-rational mutagenesis of the laccase POXA1b from Pleurotus ostreatus was performed through a combination of directed evolution with elements of rational enzyme modification. The R4 laccase was prepared by joining mutations of previously selected POXA1b random variants. An enhancement of stability features was thus obtained, making the novel enzyme R4 more appropriate as scaffold for directed evolution. A library of 1000 randomly mutated variants of R4 was prepared and screened for the ability of oxidising 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). One of the variants selected (V148L) for improved activity was also proved to show higher stability than R4 at pH 5, and to retain its high stability at pH 7 and 10. In comparison with the POXA1b wild-type laccase, the semi-rational approach allowed us to develop a more efficient bio-catalyst, rising specific activity on ABTS up to around 5-fold. The new variant was also proved to be both more versatile and more durable than the wild-type enzyme, exhibiting higher activity in wide temperature and pH ranges and higher stability at acidic (t $_{1/2}$ at pH 5 = 35 days), neutral (t $_{1/2}$ at pH 7 = 38 days) and alkaline (t $_{1/2}$ at pH 10 = 62 days) pH values.
Starting Page	149
Ending Page	156
Page Count	8
File Format	PDF
ISSN	10736085
Journal	Molecular Biotechnology
Volume Number	46
Issue Number	2
e-ISSN	15590305
Language	English
Publisher	Humana Press Inc
Publisher Date	2010-05-14
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Oxidases Semi-rational mutagenesis Directed evolution Enzyme stability Human Genetics Biological Techniques Protein Science Cell Biology Biochemistry Biotechnology
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biochemistry Bioengineering Applied Microbiology and Biotechnology Biotechnology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Hydrophobic Substitution of Surface Residues Affects Lipase Stability in Organic Solvents

SORS: A Universal One-Round PCR-Based Method for Site-Directed Mutagenesis

A Discussion of Molecular Biology Methods for Protein Engineering

A simple method for site-directed mutagenesis with double-stranded plasmid DNA

Overview of Regulatory Strategies and Molecular Elements in Metabolic Engineering of Bacteria

Evaluation of the Conformational Stability of Recombinant Desulfurizing Enzymes from a Newly Isolated Rhodococcus sp.

Protein and Genome Evolution in Mammalian Cells for Biotechnology Applications

Engineering of Human CYP3A Enzymes by Combination of Activating Polymorphic Variants

Cloning of Intron-Removed Enolase Gene and Expression, Purification, Kinetic Characterization of the Enzyme from Theileria annulata

A Semi-Rational Approach to Engineering Laccase Enzymes

Similar Documents

Hydrophobic Substitution of Surface Residues Affects Lipase Stability in Organic Solvents

SORS: A Universal One-Round PCR-Based Method for Site-Directed Mutagenesis

A Discussion of Molecular Biology Methods for Protein Engineering

A simple method for site-directed mutagenesis with double-stranded plasmid DNA

Overview of Regulatory Strategies and Molecular Elements in Metabolic Engineering of Bacteria

Evaluation of the Conformational Stability of Recombinant Desulfurizing Enzymes from a Newly Isolated Rhodococcus sp.

Protein and Genome Evolution in Mammalian Cells for Biotechnology Applications

Engineering of Human CYP3A Enzymes by Combination of Activating Polymorphic Variants

Cloning of Intron-Removed Enolase Gene and Expression, Purification, Kinetic Characterization of the Enzyme from Theileria annulata

A Semi-Rational Approach to Engineering Laccase Enzymes