Heterologous Expression of a Thermostable α-Glucosidase from Geobacillus sp. Strain HTA-462 by Escherichia coli and Its Potential Application for Isomaltose–Oligosaccharide Synthesis

Abstract

Isomaltose–oligosaccharides (IMOs), as food ingredients with prebiotic functionality, can be prepared via enzymatic synthesis using α-glucosidase. In the present study, the α-glucosidase (GSJ) from Geobacillus sp. strain HTA-462 was cloned and expressed in Escherichia coli BL21 (DE3). Recombinant GSJ was purified and biochemically characterized. The optimum temperature condition of the recombinant enzyme was 65 °C, and the half-life was 84 h at 60 °C, whereas the enzyme was active over the range of pH 6.0–10.0 with maximal activity at pH 7.0. The α-glucosidase activity in shake flasks reached 107.9 U/mL and using 4-Nitrophenyl β-D-glucopyranoside (pNPG) as substrate, the Km and Vmax values were 2.321 mM and 306.3 U/mg, respectively. The divalent ions Mn2+ and Ca2+ could improve GSJ activity by 32.1% and 13.8%. Moreover, the hydrolysis ability of recombinant α-glucosidase was almost the same as that of the commercial α-glucosidase (Bacillus stearothermophilus). In terms of the transglycosylation reaction, with 30% maltose syrup under the condition of 60 °C and pH 7.0, IMOs were synthesized with a conversion rate of 37%. These studies lay the basis for the industrial application of recombinant α-glucosidase.

Biocatalytic synthesis of 2‐O‐α‐D‐glucopyranosyl‐L‐ascorbic acid using an extracellular expressed α‐glucosidase from Oryza sativa

“…that was expressed in E. coli , maintained 90% of activity after 30 h of incubation at 60°C. [ 46 ] It should be noted that the thermal stability of rAGL is an important factor related to the potential applications of enzymes in biotechnology.…”

Heterologous Expression of Thermotolerant α-Glucosidase in Bacillus subtilis 168 and Improving Its Thermal Stability by Constructing Cyclized Proteins

“…Most industrially applied α-glucosidases are derived from Aspergillus niger, generally more stable below 50 • C. It quickly becomes inactivated when the temperature is above 60 • C [13]. However, it is beneficial for reducing the viscosity of the reaction solution, improving the catalytic efficiency, and increasing the production of IMOs by adequately increasing the temperature of the transglycosylation reaction [14]. Poor thermal stability has become a limiting factor for the commercial application of α-glucosidase.…”

“…expressing α-glucosidase from an extreme environment [17]. Zhang et al heterologously expressed α-glucosidase (GSJ) from this source in Escherichia coli (E. coli) [14]. The optimal temperature of the recombinant α-glucosidase was 65 • C, and the t 1/2 (Half-life) was 84 h at 60 • C. Another method to improve the thermal stability of α-glucosidase is site-directed mutagenesis.…”

Molecular dynamics reveals insight into how N226P and H227Y mutations affect maltose binding in the active site of α-glucosidase II from European honeybee, Apis mellifera

“…Potential applications of HBGase II include production of isomalto-oligosaccharides (IMOs) [5]. Due to their functions in mammalian metabolic processes and its low calories, IMOs are widely used as prebiotics, food additives and feed ingredients [6,7]. Moreover, HBGase II could potentially be used in brewing industry, where α-glucosidase is used for malt hydrolysis [8,9].…”