Purification and characterization of a novel α-amylase from a newly isolated Bacillus methylotrophicus strain P11-2

• This is the first to report on the enzyme production by Bacillus methylotrophicus.
• The α-amylase produced by strain P11-2 was purified to homogeneity.
• The end products of enzyme reaction toward soluble starch are different from others.
• The N-terminal sequence of the α-amylase showed no homology to other α-amylases.
• Based on its characters, α-amylase of strain P11-2 should be a novel α-amylase.

An aerobic bacterial strain P11-2 with high amylolytic activity was isolated from soil sample collected from wheat field of Jiyuan, China. The strain was identified as Bacillus methylotrophicus by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. The α-amylase was purified to homogeneity by a combination of 80% (NH4)2SO4 precipitation, DEAE FF anion exchange, and superdex 75 10/300 GL gel filtration chromatography. The purified α-amylase exhibited specific activity of 330.7 U/mg protein that corresponds to 13.1 fold purification. The relative molecular mass of the α-amylase was 44.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for enzyme activity were 7.0 and 70 °C, respectively. The α-amylase activity was stimulated by Mg2+, Ba2+, Al3+ and dl-dithiothreitol (DTT), however, Ca2+ almost had no activation or inhibition on the α-amylase. After 4 h of reaction toward soluble starch, the end products were glucose, maltose and maltotriose. The 10 residues of the N-terminal sequence of the purified α-amylase were SVKNGQILHA, which showed no homology to other reported α-amylases from Bacillus strain.