Research on the Determination Method of Enzymatic Activity for β-Glucan Synthase (ΒGS)

Abstract

Purpose To research out a suitable method for the determination of enzymatic activity for β-glucan synthase. Methods According to the methods for the determination of ΒGS in current reports, taking Grifola frondosa as material, a suitable method for the determination of enzymatic activity for ΒGS was researched out. ΒGS was isolated from the mycelium of Grifola frondosa. Taking glucose as substrate, ΒGS activity was reflected by the consumption of glucose. Consumption of glucose was measured by 3,5-dinitrosalicylic acid (DNS) method, the standard curve for glucose content measurement was plotted by determining absorbance values of different glucose concentrations at 540nm. One unit of ΒGS corresponds to the amount of enzyme which incorporated 1μg glucose into β-glucan in 1 minute. The optimum pH and temperature of the determination of enzymatic activity for ΒGS was determined respectively by carrying out the enzyme assay at different pH levels and temperatures. Results The method for the determination of enzymatic activity for ΒGS was a suitable, correct and usable method which can be popularized widely. The regression equation of the standard curve for glucose content measurement was y=0.0008x-0.0247, R2=0.9996. The optimum pH of the determination of enzymatic activity for ΒGS was pH=5.0 and the optimum temperature was 15°C. The suitable determination process for β-glucan synthase enzymatic activity is as follows: 1.0ml of enzyme was added in a test tube followed by 1.0 ml glucose (1.0mg/ml), and then the pH level was adjusted to 5.0. The test tube was incubated at 15°C for 10 minutes. Later, DNS reagent (1.5ml) was added to the test tube kept in boiling water for 5 minutes and cooled in water. A blank was prepared (2.0ml distilled water and 1.5ml DNS solution). Each solution was fixed the volume to 25ml, the color intensity was estimated at 540 nm using spectrophotometry. Conclusion A method for the determination of enzymatic activity for β-glucan synthase was researched out by taking Grifola frondosa as material, and it can be widely extended and applied by its characteristics of simple, rapid, high sensitivity and low cost.

The Purification of Grifolan Synthase and the Determination of its Molecular Weight

“…Taking glucose as substrate, enzyme reaction was carried out. Consumption of glucose was measured using DNS method [2]. Grifolan Synthase enzyme specific activity (Z) was calculated using Eq.…”

“…Grifolan, extracted from the fruiting bodies or mycelia of Grifola frondosa, has significant effects in anti-tumor, hypertension, diabetes, anti-hepatitis, anti-HIV and improving function of immune system proved by a large number of researches [1,3]. Recently Grifolan has been directly into clinical Phase II approved by the FDA as an anti-cancer agent of immunotherapy [1,2,3].…”

“…In these reports, the crude extract of β-glucan synthase was extracted and studied, but pure β-glucan synthase and its properties was not reported pesently. Grifolan Synthase has been isolated and its some enzymatic properties were studied [1,2], but the structure and function of Grifolan Synthase and its molecular weight have not been studied yet, these important researches is difficult to go through on if without the pure sample of Grifolan Synthase. In this paper, a purification method of Grifolan Synthase was researched out, and the molecular weight of grifolan synthase was studied.…”