title: Production of Ca2+-Independent and Acidstable Recombinant α-Amylase of Bacillus acidicola Extracellularly and its Applicability in Generating Maltooligosaccharides.
abstract: The recombinant acidstable α-amylase (Ba-amy) of acidophilic bacterium Bacillus acidicola TSAS1 has been produced extracellularly using a combination of cloning (E. coli and P. pastoris) and physico-chemical treatment strategies. A total of 150,000 U/L of Ba-amy were attained under constitutive promoter in P. pastoris, which is 15-fold higher than that of the wild strain B. acidicola (10,000 U/L). The recombinant P. pastoris integrated two copies of Ba-amy under GAP promoter. The pure Ba-amy expressed in P. pastoris is a glycoprotein of 66 kDa, which is optimally active at pH 4.0 and 60 °C with a T1/2of 25 min at 70 °C. The Km, Vmaxand Kcatvalues of the recombinant Ba-amy are 1.66 mg/mL, 53.6 µmol/mg/min and 106.8/s, respectively. The enzyme generates maltose (30 %), maltotriose (20 %) and other higher maltooligosaccharides from starch, thus, useful in baking as an antistale. This is the first report on the optimization of extracellular production of recombinant acidic α-amylase of an acidophilic bacterium.
authors: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India.
authors: Deepak
authors: D
authors: Parashar
authors: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India. tsnarayana@gmail.com.
authors: T
authors: T
authors: Satyanarayana
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009844:Oligosaccharides / Q000096:biosynthesis*; D010843:Pichia / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C021705:maltooligosaccharides; D013213:Starch; D000516:alpha-Amylases; D002118:Calcium
keywords: Acidophile; Acidstable α-amylase; Bacillus acidicola; Escherichia coli; Maltooligosaccharides; Pichia pastoris