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 K_m 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.

Keywords: heterologous expression; isomaltose-oligosaccharides; thermostability; transglycosylation; α-glucosidase.

Figure 1

Recombinant α-glucosidase (GSJ) production from E. coli BL21 (DE3). (A) Time profiles for batch cultivations of recombinant E. coli in shake flasks: ●, OD600 of the bacteria cells; ▲, the protein concentration of recombinant GSJ. (B) The intracellular enzyme activity of recombinant GSJ. (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of recombinant GSJ. Samples were run on a 10% SDS-PAGE gel. Proteins were visualized using Coomassie Brilliant Blue G-250. Lane M, molecular weight markers; Lane 1, E. coli BL21-pET28a; Lane 2-7, intracellular supernatant of recombinant E. coli after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction at 0 h, 6 h, 12 h, 21 h, 27 h and 33 h.

Figure 2

(A) The purification of recombinant GSJ from E. coli BL21 (DE3). Lane M, molecular weight markers; Lane 1, E. coli BL21-pET28a; Lane 2, crude enzyme; Lane 3, heat treatment; Lane 4, purified GSJ. (B) MALDI-TOF analyses of recombinant GSJ. The major signals are labelled as [M + 2H]²⁺ and [M + H]⁺, respectively.

Figure 3

Effect of temperature and pH on the activity and stability of recombinant GSJ. (A) Optimum temperature. Activity was measured between 30 °C and 90 °C at pH 7.0 for 10 min. The activity at optimum temperature was defined as 100%. (B) Thermostability. Assays were carried out in 50 mM phosphate buffer (pH 7.0) at 65 °C for 10 min after the incubation of the enzyme at 60 °C (●) or 65 °C (▲). The activity without heat treatment was defined as 100%. (C) Optimum pH. Assays were carried out at 65 °C for 10 min in buffers ranging in pH from 4.0 to 11.0. The activity at optimum pH was defined as 100%. (D) pH stability. Assays were carried out after the incubation of the enzyme in buffers ranging from pH 4.0–11.0 at 4 °C for 24–72 h.

Figure 4

Lineweaver–Burk plot of recombinant GSJ.

Figure 5

Effect of metal ions on recombinant GSJ. The activity was assayed after the incubation of the enzyme (30 ng) in 50 mM phosphate buffer (pH 7.0) containing 1 mM metal ion at 65 °C for 1 h.

Figure 6

Identification of the hydrolysis products by HPAEC-PAD-200. The hydrolysis products of soluble starch by commercial α-amylase and recombinant GSJ (A), commercial α-amylase and commercial α-glucosidase (B), commercial α-amylase (C). (D) Soluble starch. (E). Standard samples of glucose.

Figure 7

Identification of the transglycosylation products by HPAEC-PAD-200. (A) Standard samples of glucose (1), isomaltose (2), isomaltotriose (3), maltose (4) and panose (5). (B) maltose substrate (control group). (C) Transglycosylation products by recombinant GSJ.