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Cloning, functional expression and characterization of Aspergillus sulphureus β-mannanase in Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
25266 43564 2007 10 PDF Available
Title
Cloning, functional expression and characterization of Aspergillus sulphureus β-mannanase in Pichia pastoris
Abstract

Using RT-PCR and rapid amplication of cDNA ends (RACE) techniques, a 1345 bp full-length cDNA fragment was obtained from Aspergillus sulphureus. The gene, designated MANN, codes for a 383-residue with a calculated mass of 41,389 Da. MANN displayed amino acid sequence similarity to the β-mannanase of Aspergillus aculeatus and Trichoderma reesei, members of the glycoside hydrolase family 5. The recombinant β-mannanase gene was successfully expressed in a fully active form in Pichia pastoris. Southern blot analysis showed that the recombinant β-mannanase gene had successfully integrated into the P. pastoris X-33 genome. SDS-PAGE and Western blot assays demonstrated that the recombinant β-mannanase, a 48 kDa glycosylated protein, was secreted into the culture medium. The enzyme had high specific activity toward locust bean gum and an extremely broad pH range of 2.2–8.0. It showed highest activity at pH 2.4 and 50 °C and was stable at temperature below 40 °C. The Km and Vmax values for locust bean gum at 50 °C and pH 2.4 are 0.93 mg mL−1 and 344.83 U mg−1, respectively. The isoelectric point of the recombinant protein is 4.89. The enzymatic activity of recombinant A. sulphureus β-mannanase was not significantly affected by a range of ions or EDTA.

Keywords
RACE; β-Mannanase; Aspergillus sulphureus; Pichia pastoris
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Cloning, functional expression and characterization of Aspergillus sulphureus β-mannanase in Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 128, Issue 3, 20 February 2007, Pages 452–461
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
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