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Biophysical and biochemical studies of a major endoglucanase secreted by Xanthomonas campestris pv. campestris.

Paper ID Volume ID Publish Year Pages File Format Full-Text
16745 42609 2016 7 PDF Available
Title
Biophysical and biochemical studies of a major endoglucanase secreted by Xanthomonas campestris pv. campestris.
Abstract

•A major cellulolytic enzyme XccCel5A, secreted by the bacterium Xanthomonas campestris pv. campestris was expressed and purified.•Biophysical, biochemical and enzymatic characterization of XccCel5A was conducted.•Low-resolution SAXS molecular envelope of the endoglucanase in solution was determined.

Endoglucanases are the main cellulolytic enzymes secreted by the bacterium Xanthomonas campestris pv. campestris (Xcc). The major endoglucanase exported by this bacterium into an external milieu is an enzyme XccCel5A, which belongs to GH5 family subfamily 1 and is encoded by the gene engXCA. We purified XccCel5A using ammonium sulfate precipitation followed by size exclusion chromatography and identified it by zymogram analysis. Circular dichroism and fluorescence spectroscopy studies showed that XccCel5A is stable in a wide pH range and up to about 55 °C and denatures at the higher temperatures. The optimal conditions for enzyme activity were identified as T = 45 °C and pH = 7.0. Under the optimum conditions the catalytic efficiency (kcat/KM) of the enzyme was determined as 5.16 × 104 s−1 M−1 using carboxymethylcellulose (CMC) as a substrate. Our SAXS studies revealed extended tadpole-shape molecular assembly, typical for cellulases, and allowed to determine an overall shape of the enzyme and a relative position of the catalytic and cellulose binding domains.

Keywords
Xanthomonas campestris; Endoglucanases; Hydrolysis; SAXS
First Page Preview
Biophysical and biochemical studies of a major endoglucanase secreted by Xanthomonas campestris pv. campestris.
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 91, September 2016, Pages 1–7
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering