Isolation of Grifolan Synthase <i>(GS)</i> and its Partly Enzymatic Properties

Abstract

Purpose To search for an isolation method of grifolan synthase, and research grifolan synthase partly enzymatic properties. Methods Taking Grifola frondosa as material, the mycelium was broken by ultrasonic, and then centrifugated, the supernatant was collected, GS was precipitated by different concentrations of ethanol, ammonium sulfate and acetone, the suitable isolation method of GS was researched out by taking enzyme activity and protein content as parameters. Reducing sugar, protein content and total sugar content were determined respectively by using 3,5-dinitrosalicylic acid (DNS) method, Coomassie brilliant blue method and phenol-sulfuric acid method. Taking glucose as substrate, GS activity was reflected by the consumption of glucose. Consumption of glucose was measured by DNS method. The optimal temperature and pH of GS enzyme reaction was determined by carrying out the enzyme assay at different temperatures and pH levels.The acid-base stability of GS was determined by subjecting GS to different pH levels for 60 minutes, and the heat stability of GS was determined by subjecting GS to different temperatures for 30 minutes. The direction of GS enzymatic reaction was determined by measuring the consumption of β-glucan in 1 minute. The possibility of GS existing extracellular was judged by determining GS activity in extracellular fermentation liquor. Results The proper isolation method of GS is that the mycelium was collected from fermentation liquor and broken by ultrasonic for 1min, then centrifugated at 5000rpm for 10 minutes and a supernatant was collected. Ammonium sulfate was added to the concentration of 60%, and then centrifugated at 12000rpm for 10 minutes at 4°C, the pellets was collected as GS crude enzyme. Using this method, 93.86mg of crude enzyme which enzyme activity was 5700U/mg was obtained from 100g mycelium with moisture content of 87.28%, extraction rate of crude enzyme was 0.7379%. The optimum pH of GS enzyme reaction was pH=5.0 and the optimum temperature was 15°C, GS was most stable at pH=5.0 and in the range of 30 °C to 50 °C. Just 0.6996μg β-glucan was hydrolyzed in 1 minute by GS which can actually consume 5700μg glucose per minute in the synthetic reaction of β-glucan, considering the error in actual measurement, it can be considered that GS is a one-way enzyme that it can only catalyze the synthesis of β-glucan. GS activity in extracellular Grifola frondosa fermentation liquor was -0.1875U/ml, indicating that GS is one kind of intracellular enzyme and without GS activity in fermentation liquor or extracellular. Conclusion An isolation method of grifolan synthase form the mycelium of Grifola frondosa was researched out, and grifolan synthase partly enzymatic properties were studied in this paper. It can lay the foundation for the further study on the structure and function of GS and grifolan production.

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

“…The mycelium was collected from fermentation liquor and broken by ultrasonic treatment for 1min, then centrifugated at 5000rpm for 10 minutes and supernatant was collected. Ammonium sulfate was added to the concentration of 60%, and then centrifugated at 12000rpm for 10 minutes at 4°C, Grifolan Synthase crude extract was collected from the supernatant [1].…”

“…Z= W/ (T×M) (1) where W is the consumption of glucose, (µg), T is the reaction time, (minute), M is the sample mass or volume, (mg or ml). Enzyme activity unit (U) was defined as: The amount of enzyme which incorporated 1µg glucose into β-glucan (grifolan) in 1 minute.…”

“…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.…”