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Cloning of maltase gene from a methylotrophic yeast, Hansenula polymorpha
1Institute of Molecular and Cell Biology, Riia 23, University of Tartu, 51010, Tartu, Estonia.
Abstract:
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
Insights
Researchers isolated the Hansenula polymorpha maltase gene (HPMAL1) and characterized its protein. This methylotrophic yeast maltase gene sequence is reported here for the first time.
Area of Science:
- Molecular biology
- Yeast genetics
- Enzymology
Background:
- Maltase enzymes are crucial for carbohydrate metabolism in yeasts.
- Understanding yeast maltase genes aids in metabolic engineering and biotechnology.
- The methylotrophic yeast Hansenula polymorpha offers a unique system for studying gene function.
Purpose of the Study:
- To isolate and characterize the maltase structural gene (HPMAL1) from Hansenula polymorpha.
- To determine the biochemical properties of the recombinant Hansenula polymorpha maltase.
- To establish the presence and copy number of the maltase gene in the H. polymorpha genome.
Main Methods:
- Genomic library screening using a maltase-specific gene probe.
- DNA sequencing and analysis of the open reading frame (ORF).
- Recombinant protein expression in Escherichia coli and enzymatic activity assays.
- Southern blot analysis for gene copy number determination.
Main Results:
- The HPMAL1 gene consists of a 1695 nt ORF encoding a 564 amino acid protein.
- The deduced protein sequence shows homology to known maltases and glucosidases from other organisms.
- Recombinant H. polymorpha maltase hydrolyzed PNPG, sucrose, maltose, and alpha-methylglucoside, with varying substrate affinities.
- Southern analysis confirmed a single maltase gene in H. polymorpha.
Conclusions:
- The HPMAL1 gene sequence and its encoded maltase properties have been elucidated.
- This study provides the first report of a maltase gene sequence from a methylotrophic yeast.
- The characterized enzyme and gene offer potential for biotechnological applications.