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

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 P-mannanase of Aspergillus aculeatus and Trichoderma reesei, members of the glycoside hydrolase family 5. The recombinant P-mannanase gene was successfully expressed in a fully active form in Pichia pastoris. Southern blot analysis showed that the recombinant P-mannanase gene had successfully integrated into the P. pastoris X-33 genome. SDS-PAGE and Western blot assays demonstrated that the recombinant beta-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 degrees C and was stable at temperature below 40 degrees C. The K-m and V-max values for locust bean gum at 50 degrees 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 beta-mannanase was not significantly affected by a range of ions or EDTA. (c) 2006 Elsevier B.V. All rights reserved.