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An unusual chimeric amylosucrase generated by domain-swapping mutagenesis

Paper ID Volume ID Publish Year Pages File Format Full-Text
16792 42613 2016 10 PDF Available
Title
An unusual chimeric amylosucrase generated by domain-swapping mutagenesis
Abstract

•The biochemical properties of four types of amylosucrase (ASase) were analyzed.•Chimeric Deinococcus geothermalis (DGAS)/Neisserria polysaccharea ASases were made.•DGAS-B synthesized only polymers

Amylosucrase (ASase; EC 2.4.1.4) synthesizes α-1,4-glucans using sucrose as a sole substrate. The aim of this study was to compare the enzymatic properties of four recombinant ASase genes to determine the underlying mechanisms thereof. Following cloning and expression in Escherichia coli, we determined that the ASase enzyme from Deinococcus geothermalis (DGAS) had the highest thermostability whereas ASase from Neisseria polysaccharea (NPAS) showed the greatest polymerization activity. Chimeric ASases were constructed using dgas and npas genes by overlap extension polymerase chain reaction. Two of the six chimeric ASases generated, NPAS-B′ and DGAS-B, showed ASase activity using sucrose as the sole substrate. However, DGAS-B was not able to produce longer α-1,4-glucans; the highest degree of polymerization was <12. In the kinetic study, not only the substrate binding affinity but also the production rate of DGAS-B was greater than those of DGAS. Molecular dynamic computational simulation suggested that DGAS-B could not synthesize longer glucan chains because of the change in flexibilities of loops 4, 7, and 8 as compared to those of DGAS.

Keywords
Amylosucrase; Deinococcus geothermalis; Neisseria polysaccharea; Domain swapping; Chimeric enzyme
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An unusual chimeric amylosucrase generated by domain-swapping mutagenesis
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 86, May 2016, Pages 7–16
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