Gene cloning and characterization of a novel alpha-amylase from alkaliphilic Alkalimonas amylolytica.
A gene encoding an extracellular alpha-amylase (AmyA) was cloned from the alkaliphilic bacterium Alkalimonas amylolytica by enzymatic activity screening in Escherichia coli DH5alpha. The gene amyA consists of 1764 base pairs and was predicted to encode a 587-amino acid protein encompassing a 31-amino acid signal peptide. In addition, a 459-amino acid catalytic domain and a 97-amino acid starch-binding domain (SBD) were found. The SBD showed little similarity to other known SBDs; instead, it contains conserved amino acids typically belonging to the carbohydrate-binding module (CBM) family 20. AmyA could act on both granular and gelatinized starch. The catalytic domain of the enzyme showed little similarity to other known alpha-amylases. Rather, AmyA contains four characteristic conserved regions of glycoside hydrolase family 13. The recombinant enzyme was a liquefying enzyme with the highest activity at 50 degrees C and pH 9.5. The enzyme displayed a unique endo-product profile and action pattern on soluble starch to yield a series of malto-oligosaccharides ranging from maltose to maltoheptaose. The activity of the enzyme was enhanced by Co(2+), but not affected by 5 mM EDTA. Taken together, AmyA from A. amylolytica has potential to be used in paper, textile, detergent and other industries where starch needs to be degraded in an alkaline environment.
Wang
Nan
N
Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China.
Zhang
Yunhua
Y
Wang
Quanhui
Q
Liu
Jun
J
Wang
Honglei
H
Xue
Yanfen
Y
Ma
Yanhe
Y
D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000379:methods*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics*
D004492:Edetic Acid; D000516:alpha-Amylases