Cloning of maltase gene from a methylotrophic yeast, Hansenula polymorpha.
The Hansenula polymorpha maltase structural gene (HPMAL1) was isolated from a genomic library by hybridization of the library clones with maltase-specific gene probe. An open reading frame of 1695 nt encoding a 564 amino-acid protein with calculated molecular weight of 65.3 kD was characterized in the genomic DNA insert of the plasmid p51. The protein sequence deduced from the HPMAL1 exhibited 58 and 47% identity with maltases from Candida albicans and Saccharomyces carlsbergesis encoded by CAMAL2 and MAL62, respectively, and 44% identity with oligo-alpha-1,6-glucosidase from Bacillus cereus. The recombinant Hansenula polymorpha maltase produced in Escherichia coli hydrolyzed p-nitrophenyl-alpha-D-glucopyranoside (PNPG), sucrose, maltose and alpha-methylglucoside and did not act on melibiose, cellobiose, trehalose and o-nitrophenyl-beta-D-galactopyranoside (ONPG). The affinity of the recombinant enzyme for its substrates increased in the order maltose <alpha-methylglucoside <sucrose <PNPG. Southern analysis revealed presence of a single maltase gene in H. polymorpha. This is the first report on the maltase gene sequence from a methylotrophic yeast.
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Institute of Molecular and Cell Biology, Riia 23, University of Tartu, 51010, Tartu, Estonia.
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Alamäe
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism; D005656:Fungal Proteins; D005947:Glucose / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D004187:Disaccharides; D005656:Fungal Proteins; D005960:Glucosides; D008757:Methylglucosides; C019502:4-nitrophenyl alpha-glucoside; C027020:methylglucoside; D013395:Sucrose; D008320:Maltose; C424662:MAL1 protein, Pichia angusta; D000520:alpha-Glucosidases; D005947:Glucose