Expression and Biochemical Characterization of a Thermostable Branching Enzyme from <b><i>Geobacillus thermoglucosidans</i></b>

Abstract

The branching enzyme (EC 2.4.1.18) catalyzes the formation of α-1,6 branch points in starch. In this study, the Geobacillus thermoglucosidans gene-encoding branching enzyme was expressed in Escherichia coli BL21 (DE3) and the protein was isolated and characterized. G. thermoglucosidans branching enzyme is a thermostable enzyme with an optimal reaction temperature of nearly 60°C and a half-life at 65°C of approximately 1.1 h. The activity of the recombinant enzyme is optimal at pH 7.5, with broad stability between pH 5.5 and 9.0. Its thermostability, relatively broad pH stability and optimal temperature near the temperature at which starch begins to gelatinize may make it easy to use in industrial production. Furthermore, the enzyme is activated by Mg²⁺, Ba²⁺, K⁺ and Na⁺ in a concentration-dependent manner and dramatically inhibited by Ni²⁺ and Co²⁺. Its substrate dependence, using amylopectin as the substrate, could be adequately fitted using the Michaelis-Menten equation, yielding a K_m of 0.99 mg/ml. High-performance anion exchange chromatography results showed that the chain length distribution of branching enzyme-treated waxy corn starch is indistinguishable from that of the branching enzyme-treated common corn starch. This enzyme may therefore be a promising tool for the enzymatic modification of starch.

Synthesis of highly branched α-glucans with different structures using GH13 and GH57 glycogen branching enzymes

“…However, many more bacterial GH13 GBEs have been characterized, among which the GBEs of Aquifex aeolicus (Hiroki Takata, Ohdan, Takaha, Kuriki, & Okada, 2003;M. Van Der Maarel, Vos, Sanders, & Dijkhuizen, 2003), Butyrivibrio fibrisolvens (Rumbak, Rawlings, Lindsey, & Woods, 1991), Deinococcus geothermalis and Deinococcus radiodurans (Palomo, Kralj, van der Maarel, & Dijkhuizen, 2009), Geobacillus stearothermophilus (Hiroki Takata et al, 1994), Geobacillus thermoglucosidans (Ban et al, 2016;Liu et al, 2017), Mycobacterium tuberculosis (Garg, Alam, Kishan, & Agrawal, 2007), Thermomonospora curvata (Fan, Xie, Zhan, Chen, & Tian, 2016), and Vibrio vulnificus (Jo, Park, Jeong, Kim, & Park, 2015). Today only three GH57 GBEs have been characterized; Pyrococcus horikoshii (Na et al, 2017), Thermococcus kodakarensis (Murakami, Kanai, Takata, Kuriki, & Imanaka, 2006), and Thermus thermophilus (Palomo et al, 2011).…”

Effects of Alcohol Extracts of Gluten on Oil Uptake of Fried Mahua

“…The molecular mass of gliadin‐like components was determined using size‐exclusion high‐performance liquid chromatography . The system was composed of a Waters 1525 Binary HPLC pump, a 200‐µL injection loop, a TSKgel 4000 SWXL 300 mm × 7.8 mm column for HPLC, a UV detector (165 Waters 2487 Dual λ Absorbance Detector) followed by an RI detector (Wyatt Optilab T‐rEX).…”

Evolutionary Stability of Salt Bridges Hints Its Contribution to Stability of Proteins

“…The reaction was initiated by adding an appropriate amount of wild type GBE and its mutants. The amount of newly synthesized α-1,6 linkages was determined by adding 1 mL sodium acetate (1 M, pH 3.5) and 1000 U of isoamylase to 2 mL sample, followed by incubating the mixture at 37 °C for 20 h. One unit is defined as producing 1 mmol of newly synthesized α-1,6 linkages formed per minute at 55 °C [3,13].…”

“…The resistance to thermal inactivation of GBE and its mutants was measured by incubating purified enzyme in 10 mM Tris-HCl buffer (pH 7.5) at 60 °C and 65 °C. Samples were taken at several time intervals and the residual activities were determined [3].…”