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Probing the active site environment of alkaliphilic family 11 xylanase from Penicillium citrinum: Evidence of essential histidine residue at the active site

Paper ID Volume ID Publish Year Pages File Format Full-Text
17893 42704 2007 7 PDF Available
Title
Probing the active site environment of alkaliphilic family 11 xylanase from Penicillium citrinum: Evidence of essential histidine residue at the active site
Abstract

Alkaliphilic xylanases are not only important for their biotechnological applications, but also for their implications in protein structure–function. Here, we present for the first time the presence of single active site histidine residue in the microenvironment of GH-family 11 alkaliphilic xylanase from extremophilic fungus Penicillium citrinum MTCC 6489 using chemical modifications. The kinetic studies showed a time dependent inactivation of xylanase by OPTA or DEPC resulting in a pseudo-first-order kinetics with a second-order rate constant of 49.8 and 5.08 min−1 M−1, respectively. The difference spectrum of DEPC modified versus native protein exhibit an absorbance maximum at 244 nm characteristic of the formation of N-carbethoxyhistidine, which is completely reversed by neutralized hydroxylamine implying the presence of histidine residue. Moreover, the rate of inactivation shows pH dependence with an inflection point at 6.2. CD studies reveal no significant change in the DEPC modified xylanase conformation. Substrate dependent protection (0.5% xylan) from DEPC inactivation phenomenon conclusively proves the presence of histidine residue in the active site. To explore the presence of tryptophan in the active site xylanase is modified with NBS, which reveals its position in close proximity to active site, but not involved in catalysis.

Keywords
3.2.1.8GH, glycosyl hydrolase; DEPC, diethylpyrocarbonate; OPTA, o-phthalaldehyde; TNBS, 2,4,6-trinitrobenzenesulfonic acid; PHMB, p-hydroxymercuribenzoic acid; NBS, N-bromosuccinamideGH-family 11 xylanase; Penicillium citrinum; Active site histidine; Ext
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Probing the active site environment of alkaliphilic family 11 xylanase from Penicillium citrinum: Evidence of essential histidine residue at the active site
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 41, Issue 4, 3 September 2007, Pages 440–446
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