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Expression and characterization of dextransucrase gene dsrX from Leuconostoc mesenteroides in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
24972 43549 2008 8 PDF Available
Title
Expression and characterization of dextransucrase gene dsrX from Leuconostoc mesenteroides in Escherichia coli
Abstract

The dextransucrase gene dsrX from Leuconostoc mesenteroides CGMCC 1.544 was cloned into the vector pET-28a(+) and expressed as a N-terminal His6-tag fusion protein of 167.57 kDa in Escherichia coli BL21(DE3). DsrX with the high volumetric activity of 8.8 U ml−1 culture and the specific activity of 97.37 U mg−1 crude enzyme extracts was measured in the optimized recombinant expression system. The resultant expression level of the fusion protein amounted to 24.5% of the total cell proteins. The results of affinity chromatography and western blotting indicated that the three sensitive sites of proteolysis existed in the N-terminal catalytic domain of DsrX. Both the recombinant and native enzyme activity were slightly activated by 1 mmol l−1 Mn2+ and strongly inhibited by 1 mmol l−1 Cu2+ or Al3+, and their optimum pH values were 5.4. The optimum temperature of the recombinant enzyme for dextran synthesis was 30 °C, which was 10 °C less than that of the native one. The transglucosylation products of two enzymes were studied by using thin layer chromatography and high-performance anion exchange chromatography. It could be concluded that the better sample-pretreatment temperature in SDS-PAGE was 37 °C, which significantly improved the detection of thermal instable enzyme than that of 100 °C.

Keywords
Expression; Characterization; Dextransucrase gene; Leuconostoc mesenteroides; Escherichia coli
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Expression and characterization of dextransucrase gene dsrX from Leuconostoc mesenteroides in Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 133, Issue 4, 29 February 2008, Pages 505–512
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
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Full-text PDF Download
Online Support
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