Cloning and overexpression of a maltase gene from Schizosaccharomyces pombe in Escherichia coli and characterization of the recombinant maltase.
The Schizosaccharomyces pombe maltase structural gene (SPMAL1(+)) was amplified from genomic DNA of S. pombe by PCR. An open reading frame of 1740bp, encoding a putative 579 amino-acid protein with a calculated molecular mass of 67.7kDa 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 44.3kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified recombinant maltase were 40 degrees C and 6, respectively. The recombinant maltase was weakly activated by Mg(2+), Ca(2+), Na(+), and Ba(2+), but was strongly inhibited by Hg(2+), Ag(+) and Cu(2+), EDTA, and PMSF. The purified maltase could actively hydrolyse rho-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.
UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No.5, Qingdao, Shandong 266003, China. zhenming@sdu.edu.cn
Zhenming
Z
Chi
Xiumei
X
Ni
Shumin
S
Yao
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D000520:alpha-Glucosidases