Molecular cloning and characterization of amylase from soil metagenomic library derived from Northwestern Himalayas.
The increasing demand for novel biocatalysts stimulates exploration of resources from soil. Metagenomics, a culture independent approach, represent a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. In this study, a soil-derived metagenomic library containing 90,700 recombinants was constructed and screened for lipase, cellulase, protease and amylase activity. A gene (pAMY) of 909 bp encoding for amylase was found after the screening of 35,000 Escherichia coli clones. Amino acid sequence comparison and phylogenetic analysis indicated that pAMY was closely related to uncultured bacteria. The molecular mass of pAMY was estimated about 38 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Amylase activity was determined using soluble starch, amylose, glycogen and maltose as substrates. The maximal activity (2.46 U/mg) was observed at 40 degrees C under nearly neutral pH conditions with amylose; whereas it retains 90% of its activity at low temperature with all the substrates used in this study. The ability of pAMY to work at low temperature is unique for amylases reported so far from microbes of cultured and uncultured division.
Sharma
Sarika
S
Biotechnology Division, Indian Institute of Integrative Medicine (Council of Scientific and Industrial Research), Canal Road, Jammu, 180001, India. sharmasarika19@rediffmail.com
Khan
Farrah Gul
FG
Qazi
Ghulam Nabi
GN
D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D007194:India; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054892:Metagenome; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D012988:Soil Microbiology; D013696:Temperature
D008670:Metals; D008049:Lipase; D000681:Amylases