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Improving the activity of the endoglucanase, Cel8M from Escherichia coli by error-prone PCR

Paper ID Volume ID Publish Year Pages File Format Full-Text
16798 42613 2016 7 PDF Available
Title
Improving the activity of the endoglucanase, Cel8M from Escherichia coli by error-prone PCR
Abstract

•Catalytic efficiency of Cel8M from Escherichia coli was improved by error-prone PCR.•The variant, Cel8ME15 with substitution of K353E showed 1.42 fold increased activity.•The substitution of G117S could result in 1.61 fold increased activity in Cel8ME18.•These results could be applied to improve the activities of GH8 enzymes.

Endoglucanase is a key enzyme involved in cellulose hydrolysis and can be used in multiple industrial fields. In this study, we used error-prone PCR to engineer the endoglucanase, Cel8M, from Escherichia coli. The Cel8M belongs to the glycoside hydrolase family 8 and shows 99% identity with the reported endoglucanase from E. coli K12. Through screening of approximately 10,000Cel8M variants, two variants, Cel8ME15 and Cel8ME18, respectively showing 1.42 fold and 1.61 fold increased activities, were obtained. Through sequence analysis, it was found that Cel8ME15 had two mutations, with the residues Ala9 and Glu353 respectively substituting the residues Val9 and Lys353 of Cel8M; while Cel8ME18 had one mutation with the residue Ser117 replacing the residue Gly117 of Cel8M. Based on the analysis of the predicted 3D structure of Cel8M, it was suggested that changes of K353E and G117S might directly affect the substrate binding affinity and therefore contribute to the improved activities of Cel8ME15 and Cel8ME18. Based on all the results we had, it is believed that this study should provide a useful reference for the future engineering of other endoglucanases from glycoside hydrolase family 8.

Keywords
Endoglucanase; Error-prone PCR; Congo red; 3D-Structural modelling
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Improving the activity of the endoglucanase, Cel8M from Escherichia coli by error-prone PCR
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 86, May 2016, Pages 52–58
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us