Biochemical characterization and structural insights into the high substrate affinity of a dimeric and Ca²⁺ independent Bacillus subtilis α‐amylase

Abstract

An extracellular amylase (AmyKS) produced by a newly isolated Bacillus subtilis strain US572 was purified and characterized. AmyKS showed maximal activity at pH 6 and 60°C with a half‐life of 10 min at 70°C. It is a Ca2+ independent enzyme and able to hydrolyze soluble starch into oligosaccharides consisting mainly of maltose and maltotriose. When compared to the studied α‐amylases, AmyKS presents a high affinity toward soluble starch with a Km value of 0.252 mg ml−1. Coupled with the size‐exclusion chromatography data, MALDI–TOF/MS analysis indicated that the purified amylase is a dimer with a molecular mass of 136,938.18 Da. It is an unusual feature of a non‐maltogenic α‐amylase. A 3D model and a dimeric model of AmyKS were generated showing the presence of an additional domain suspected to be involved in the dimerization process. This dimer arrangement could explain the high substrate affinity and catalytic efficiency of this enzyme.

Bacillus velezensis Identification and Recombinant Expression, Purification, and Characterization of Its Alpha-Amylase

“…BvAmylase exhibited the optimal activity at pH 6.0, and it was active at a narrow range of pH, displaying 86.63% and 69.05% activity at pH 5.0 and pH 7.0, respectively. These results were similar to AmyKS from Bacillus subtilis strain US572 with optimal pH 6.0 and showing 65.00% and 76.00% activity at pH 5.0 and pH 7.0, respectively [26]. Hence, this enzyme is suitable to be used in acidic conditions in industries.…”

“…In this study, SDS-PAGE result showed that the relative molecular weight of BvAmylase was approximately 72.0 kDa, which was similar to the α-amylase AmyJ33 (72.0 kDa) from Bacillus amyloliquefaciens JJC33M [22] and the α-amylase AmyBS-1 (72.3 kDa) in Bacillus subtilis AS01a [23], was higher than the α-amylase FMB1 (58.5 kDa) in Anoxybacillus ayderensis [5], the α-amylase (52.0 kDa) in Bacillus amyloliquefaciens [24], the α-amylase (56.0 kDa) in Bacillus sp. YX-1 [25] and the α-amylase AMY1 (47.0 kDa) in Massilia timonae [6], however, was lower than the α-amylase AmyKS (136.9 kDa) in Bacillus subtilis strain US572 [26] and the α-amylase (85.0 kDa) in Bacillus licheniformis AT70 [27].…”

“…Hence, this enzyme is suitable to be used in acidic conditions in industries. However, α-amylase from WangLB, B. subtilis JS-200449, and Bacillus subtilis showed the optimal activity at pH 9.0, pH 8.0, and pH 7.0, respectively [11,26,39]. In the literature, the optimal pH of α-amylase from Bacillus was even ranged from 4.5 to 10.5 [14,40].…”

Development of an active site titration reagent for α-amylases

“…Accordingly, a-amylases account for 25% of the world's multi-billion dollar enzyme market. 1,2 a-Amylases are endo-acting enzymes that cleave starch into malto-oligosaccharides, which are further degraded by exoacting a-glucosidases, glucoamylases, b-amylases and a-glucan phosphorylases and lyases. They are found in CAZy GH families 13, 57, 119 and 126, with the vast majority in the large GH13 family.…”

“…The active enzyme concentration can be quantied from such plots by extrapolating the steady-state portion back to the y-intercept (t ¼ 0) and tting the burst to eqn (1), as described previously. 11 Burst ¼ [E] 0 Â (k on /k on + k off ) 2 (1)…”