Cloning and expression ofLipomyces starkeyiα-amylase inEscherichia coliand determination of some of its properties

The Lipomyces starkeyi alpha-amylase (LSA) gene encoding soluble starch-degrading alpha-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 alpha-amylases in four conserved regions of the alpha-amylase family: (I) (287)DIVVNH(292), (II) (372)GLRIDTVKH(380), (III) (399)GEVFD(403), (IV) (462)FLENQD(467). 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 (o)C, with greater than 90% stability between pH 5 and pH 8 for 16 h. It was inhibited by Cu(2+) and stimulated by Ca(2+) and Mg(2+). 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 microM).