Cloning and expression of Lipomyces starkeyi alpha-amylase in Escherichia coli and 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).
Kang
Hee Kyoung
HK
Laboratory of Functional Carbohydrate Enzymes and Microbial Genomics, Chonnam National University, Gwang-Ju, South Korea.
Lee
Jin Ha
JH
Kim
Doman
D
Day
Donal F
DF
Robyt
John F
JF
Park
Kwan-Hwa
KH
Moon
Tae-Wha
TW
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D004474:Ecthyma, Contagious / Q000235:genetics; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D007438:Introns / Q000235:genetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D004271:DNA, Fungal; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008670:Metals; D011134:Polysaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C008315:maltodextrin; D013213:Starch; D000516:alpha-Amylases