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Improving maltodextrin specificity for enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid by site-saturation engineering of subsite-3 in cyclodextrin glycosyltransferase from Paenibacillus macerans

Paper ID Volume ID Publish Year Pages File Format Full-Text
23494 43443 2013 8 PDF Available
Title
Improving maltodextrin specificity for enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid by site-saturation engineering of subsite-3 in cyclodextrin glycosyltransferase from Paenibacillus macerans
Abstract

•The subsite-3 of CGTase was systematically engineered to improve maltodextrin specificity.•The AA-2G yield was improved by 85.8% by a quadruple CGTase mutant K47L/Y89F/N94P/D196Y.•The reaction kinetics of all the CGTase mutants was systematically analyzed and modeled.•The pH stability and thermal stability of all the mutants were analyzed.•The mechanism for the enhanced maltodextrin specificity was explored by structure modeling.

In this work, the subsite-3 of cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was engineered to improve maltodextrin specificity for 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) synthesis. Specifically, the site-saturation mutagenesis of tyrosine 89, asparagine 94, aspartic acid 196, and aspartic acid 372 in subsite-3 was separately performed, and three mutants Y89F (tyrosine → phenylalanine), N94P (asparagine → proline), and D196Y (aspartic acid → tyrosine) produced higher AA-2G titer than the wild-type and the other mutants. Previously, we found the mutant K47L (lysine → leucine) also had a higher maltodextrin specificity. Therefore, the four mutants K47L, Y89F, N94P, and D196Y were further used to construct the double, triple, and quadruple mutations. Among the 11 combinational mutants, the quadruple mutant K47L/Y89F/N94P/D196Y produced the highest AA-2G titer of 2.23 g/L, which was increased by 85.8% compared to that produced by the wild-type CGTase. The reaction kinetics of all the mutants were modeled, and the pH and thermal stabilities of all the mutants were analyzed. The structure modeling indicated that the enhanced maltodextrin specificity may be related with the changes of hydrogen bonding interactions between the side chain of residue at the four positions (47, 89, 94, and 196) and the substrate sugars.

Keywords
Cyclodextrin glycosyltransferase (CGTase); l-Ascorbic acid (l-AA); Maltodextrin; 2-O-Glucopyranosyl-l-ascorbic acid (AA-2G); Site-saturation mutagenesis
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Improving maltodextrin specificity for enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid by site-saturation engineering of subsite-3 in cyclodextrin glycosyltransferase from Paenibacillus macerans
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 166, Issue 4, 20 July 2013, Pages 198–205
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