fulltext.study @t Gmail

Directed evolution of a family 26 glycoside hydrolase: Endo-β-1, 4-mannanase from Pantoea agglomerans A021

Paper ID Volume ID Publish Year Pages File Format Full-Text
23501 43444 2013 7 PDF Available
Title
Directed evolution of a family 26 glycoside hydrolase: Endo-β-1, 4-mannanase from Pantoea agglomerans A021
Abstract

•Compared to the wild-type enzyme, several variants with 1.14- to 3.30-fold increased catalytic efficiency (kcat/Km) toward locust bean gum (LBG) were obtained.•The molecular modeling and the docking analysis of the substrate suggested the nature of the residue at the 267 site significantly affect the enzyme activity by regulating substrate binding affinity and product release.•The molecular modeling and the docking analysis of the substrate revealed that enhanced polar contacts or positive charges were more favorable at the 134 site.

Pantoea agglomerans endo-β-1, 4-mannanase (Man26P) hydrolyzes hemicellulose by cleaving the internal bonds in the mannan-based chains. In order to obtain improved enzymes and understand the structure-function relationship better, Man26P was engineered using a modified DNA shuffling method and a 96-well plate high-throughput screening technology. Compared to the wild-type enzyme, two mutants Gly267Ser and His134Arg/Phe141Leu with 1.14- and 3.30-fold increased catalytic efficiency (kcat/Km) toward locust bean gum (LBG) were obtained from the mutant library containing 19,700 clones. Combined with site-directed mutagenesis, more substitutions were introduced into the 134 site, among which, His134Arg and His134Lys exhibited 2.81- and 2.75-fold increased catalytic efficiency versus the wild-type enzyme. And with an increase in the pKa values of the side chains of the mutated residues at the 134 site, a decrease was observed in their Km values against LBG. The molecular modeling and the docking analysis of the substrate suggest that the enhancement of polar contacts or positive charges at the 134 site contribute to the interaction of the enzyme with the substrate. Furthermore, the nature of the residue at the 267 site could also significantly affect the enzyme activity by regulating substrate binding affinity and product release. The study provides useful information for the directed evolution of mannanases.

Keywords
β-1, 4-mannanase; DNA shuffling; Site-directed mutagenesis; Catalytic efficiency
First Page Preview
Directed evolution of a family 26 glycoside hydrolase: Endo-β-1, 4-mannanase from Pantoea agglomerans A021
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
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 167, Issue 3, 10 September 2013, Pages 350–356
Authors
, , , ,
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