Cloning, functional expression and characterization of Aspergillus sulphureus beta-mannanase in Pichia pastoris.
Using RT-PCR and rapid amplication of cDNA ends (RACE) techniques, a 1345bp full-length cDNA fragment was obtained from Aspergillus sulphureus. The gene, designated MANN, codes for a 383-residue with a calculated mass of 41,389Da. MANN displayed amino acid sequence similarity to the beta-mannanase of Aspergillus aculeatus and Trichoderma reesei, members of the glycoside hydrolase family 5. The recombinant beta-mannanase gene was successfully expressed in a fully active form in Pichia pastoris. Southern blot analysis showed that the recombinant beta-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 48kDa 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 0.93mgmL(-1) and 344.83Umg(-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.
Chen
Xiaoling
X
National Key Laboratory of Animal Nutrition, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100094, PR China.
Cao
Yunhe
Y
Ding
Yuhua
Y
Lu
Wenqing
W
Li
Defa
D
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics*; D003001:Cloning, Molecular; D015870:Gene Expression; D006031:Glycosylation; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D044902:beta-Mannosidase / Q000235:genetics* / Q000378:metabolism
D044902:beta-Mannosidase