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Protein engineering of a thermostable polyol dehydrogenase

Paper ID Volume ID Publish Year Pages File Format Full-Text
17106 42641 2012 8 PDF Available
Title
Protein engineering of a thermostable polyol dehydrogenase
Abstract

The polyol dehydrogenase PDH-11300 from Deinococcus geothermalis was cloned, functionally expressed in Escherichia coli and biochemically characterized. The enzyme showed the highest activity in the oxidation of xylitol and 1,2-hexanediol and had an optimum temperature of 45 °C. The enzyme exhibited a T5060-value of 48.3 °C. The T5060 is the temperature where 50% of the initial activity remains after incubation for 1 h. In order to elucidate the structural reasons contributing to thermostability, the substrate-binding loop of PDH-11300 was substituted by the loop-region of a homolog enzyme, the galactitol dehydrogenase from Rhodobacter sphaeroides   (PDH-158), resulting in a chimeric enzyme (PDH-loop). The substrate scope of this chimera basically represented the average of both wild-type enzymes, but surprisingly the T5060 was noticeably increased by 7 °C up to 55.3 °C. Further mutations in the active site led to identification of residues crucial for enzyme activity. The cofactor specificity was successfully altered from NADH to NADPH by an Asp55Asn mutation, which is located at the NAD+ binding cleft, without influencing the catalytic properties of the dehydrogenase.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► A polyol dehydrogenase from D. geothermalis was cloned and functionally expressed. ► Thermostability was improved by generation of a chimeric mutant. ► Active site residues identified by mutations. ► Cofactor dependency was changed from NADH to NADPH. ► The substrate scope was investigated.

Keywords
PDH, polyol dehydrogenase; SDR, short chain dehydrogenase; MD, molecular dynamicsPolyol dehydrogenase; Thermostability; Protein engineering; Deinococcus geothermalis; Cofactor dependency
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 51, Issue 4, 10 September 2012, Pages 217–224
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