Characterization and solvent engineering of wheat β-amylase for enhancing its activity and stability

Abstract

The kinetic and thermodynamic parameters of wheat β-amylase (WBA) were characterized and various additives were evaluated for enhancing its activity and thermostability. WBA activity was examined by neocuproine method using soluble starch as substrate. The Michaelis constant (K(m)) and molecular activity (k(cat)) were determined to be 1.0±0.1% (w/v) and 94±3s(-1), respectively, at pH 5.4 and at 25°C. The optimum reaction temperature (T(opt)) for WBA activity was 55°C and the temperature (T(50)) at which it loses half of the activity after 30-min incubation was 50±1°C. Modifications of the solvent with 182mM glycine and 0.18% (w/v) gelatin have increased the T(50) by 5°C. Glycerol, ethylene glycol, dimethylformamide (DMF) and dimethyl sulfoxide have also slightly enhanced the thermostability plausibly through weakening the water structure and decreasing the water shell around the WBA protein. Ethanol and DMF activated WBA by up to 24% at 25°C probably by inducing favorable conformation for the active site or changing the substrate structure by weakening the hydrogen bonding. Its half-life in the inactivation at 55°C was improved from 23 to 48min by 182mM glycine. The thermodynamic parameters indicate that WBA is thermo-labile and sufficient stabilization was achieved through solvent modification with additives and that the heat inactivation of WBA is entropic-driven. It is suggested that WBA could be applied more widely in starch-saccharification industries with employing suitable additives.

Kinetic and thermodynamic analysis of the inhibitory effects of maltose, glucose, and related carbohydrates on wheat β-amylase

“…It is interesting to note that 182 mM glycine and 0.18% gelatin stabilize WBA by increasing its T 50 by 5 o C and that ethanol and dimethylformamide (DMF) increase WBA activity by 24%, although most additives have no effects on stability and have decreasing effect on the activity. Among the additives examined, arginine, 2-mercaptoethanol, glucose and maltose inhibited WBA strongly [12]. These lines of evidences suggest that the stability and activity of WBA could be changed by modification of the enzyme reaction system or solvent engineering.…”

“…Himaltosin GS (Lot 2S24A), a commercial preparation of WBA, was purchased from HBI Enzymes (Osaka, Japan). WBA was purified from the Himaltosin preparation according to the method described previously [12] to a homogeneous state as judged by polyacrylamide-gel electrophoresis (SDS-PAGE) with a molecular mass of 57.7 kDa. The preparation is composed of only WBA as a protein component [12].…”

“…Barley -amylase (BBA), SBA, and WBA are well characterized. The degrees of thermo-stability defined by T 50 , which is the temperature at which enzymes lose 50% of their activity after 30-min incubation, are 57 o C for BBA, 63 o C for SBA, and 50 o C for WBA [11,12].…”

“…Most recently, we have reported the effects of additives (carbohydrates, amino acids, organic solvents, proteins, detergents, etc.) on the activity and stability of WBA [12]. It is interesting to note that 182 mM glycine and 0.18% gelatin stabilize WBA by increasing its T 50 by 5 o C and that ethanol and dimethylformamide (DMF) increase WBA activity by 24%, although most additives have no effects on stability and have decreasing effect on the activity.…”

Treatment of ramie leaf β-amylase for preliminary purification

“…Plant-originating β-amylase is preferred due to its safety and high specificity compared with those of microbial sources (5,6). However, until now, the β-amylase source has been limited to several edible plants, such as barley, wheat, and soybean.…”