Cloning and expression of Lipomyces starkeyi α-amylase in Escherichia coli and determination of some of its properties
2004
Kang, H.K. | Lee, J.H. | Kim D. | Day, D.F. | Robyt, J.F. | Park, K.H. | Moon, T.W.
The Lipomyces starkeyi α-amylase (LSA) gene encoding soluble starch-degrading α-amylase was cloned and characterized from a derepressed and partially constitutive mutant for both dextranase and amylase activities. The nucleotide (nt) sequence of the cDNA fragment reveals an open reading frame of 1944 bp encoding a 619 amino acid (aa) mature protein (LSA) with a calculated molecular weight of 68.709 kDa that was estimated to be about 73 kDa, including His tag (4 kDa) based on SDS-PAGE (10% acrylamide gel), activity staining, and the Western blotting, using anti-amylase-Ab. LSA had a sequence similar to other α-amylases in four conserved regions of the α-amylase family: (I) 287DIVVNH292, (II) 372GLRIDTVKH380, (III) 399GEVFD403, (IV) 462FLENQD467. Polymerase chain reaction and sequence analysis showed one intron of 60 nucleotides in the genomic lsa at positions between 966 and 967 of cDNA. The cloned LSA amylase showed a maximum activity at pH 6 and optimum temperature of 40°C, with greater than 90% stability between pH 5 and pH 8 for 16 h. It was inhibited by Cu2+ and stimulated by Ca2+ and Mg2+. Enzyme activity was not affected by 1 mM EGTA but was inhibited by 1 mM EDTA. LSA did not hydrolyze maltodextrins of G2 to G4, yet formed G2 + G3 from G5, G2 + G4 or G3 + G3 from G6, and G3 + G4 from G7. LSA did not hydrolyze soluble starch in the present of 2% (w/v) of acarbose. Kinetics of LSA was carried out by using starch as a substrate and the inhibition type of acarbose was the mixed non-competitive type (Ki=3.4 micromolar).
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