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Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method

Paper ID Volume ID Publish Year Pages File Format Full-Text
23863 43478 2012 8 PDF Available
Title
Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method
Abstract

α-Amino ester hydrolases (AEH, E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of α-amino β-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability. AEH from Xanthomonas campestris exhibits an optimal activity temperature of 25 °C, an observed half-life of 5 min at 30 °C, and a “T-50” value, the temperature at which the half-life is 30 min, of 27 °C. To improve the thermostability of AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri. A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. Independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a “T-50” value of 34 °C (7 °C improvement) and 1.3-fold activity compared to wild-type.

► Thermostability of an enzyme relevant in β-lactam antibiotic synthesis was improved. ► The AEH variant E143H/A275P/N186D/V622I was identified as the best variant. ► The combined approach was an efficient protein engineering method.

Keywords
Amino ester hydrolase; β-Lactam antibiotics; Ampicillin; Thermostability
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Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 160, Issues 3–4, 31 August 2012, Pages 214–221
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