Cloning and overexpression of a maltase gene from Schizosaccharomyces pombe in Escherichia coli and characterization of the recombinant maltase |
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Authors: | Zhenming Chi Xiumei Ni Shumin Yao |
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Institution: | aUNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No.5, Qingdao, Shandong 266003, China |
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Abstract: | The Schizosaccharomyces pombe maltase structural gene (SPMAL1+) was amplified from genomic DNA of S. pombe by PCR. An open reading frame of 1740 bp, encoding a putative 579 amino-acid protein with a calculated molecular mass of 67.7 kDa was characterized in the genomic DNA insert of plasmid pQE30. The specific maltase activity in the induced transformants was 21 times higher than that in wild-type. However, the estimated molecular mass of the purified recombinant maltase was 44.3 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified recombinant maltase were 40 °C and 6, respectively. The recombinant maltase was weakly activated by Mg2+, Ca2+, Na+, and Ba2+, but was strongly inhibited by Hg2+, Ag+ and Cu2+, EDTA, and PMSF. The purified maltase could actively hydrolyse ρ-nitrophenyl glucoside (PNPG), maltose, dextrin, and soluble starch. The results demonstrate that maltase from S. pombe was different from that from other yeasts, and might be usefully exploited in the future by the biotechnology industry or lead to the development of new molecular genetic tools. |
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Keywords: | Fission yeast Fungal biotechnology Fungal enzymes Fungal genomics Genetic engineering |
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