菇類酒精去氫酶特性探討及對酒精產量的影響

Characterization of mushroom alcohol dehydrogenase and its influence on alcohol production

張瑋欣(Wei-Hsin Chang)

指導教授 ： 吳如雯

中臺科技大學/健康科學院/食品科技研究所/碩士(2009年)

摘要

酒類飲料主要使用製酒酵母菌 Saccharomyces cerevisiae來發酵製酒。有些食用菇類含有高活性的酒精去氫酶(alcohol dehydrogenase)並可發酵產生約 10％的酒精，也發現菇類含有預防癌症發生、提高免疫力的活性。本研究主要篩選含酒精去氫酶活性高的菇類及研究它們取代酵母菌種發酵製酒的可能性。從臺灣常栽培的食用菇類中發現四種菇類子實體(如秀珍菇、鴻喜菇、美白菇及金針菇)中含有高活性的酒精去氫酶。大部份菇類酒精去氫酶在 pH 7.0具有活性且分子量約為 100- 150 kDa，除了秀珍菇子實體萃取的酒精去氫酶可能有兩個其分子量分別約為 150 kDa及 80 kDa左右。菇類的酒精去氫酶活性在不同 pH時有明顯的差異，大部份菇類的酒精去氫酶最適範圍在 pH 6~8，此外，像是草菇、一般香菇、洋菇及蠔菇所含之酒精去氫酶的最適 pH分別是在酸性或鹼性中。
美白菇酒精去氫酶部份純化使用 50%~80%的硫酸銨沈澱、 Mono Q及 Blue Sepharose兩個管柱。經 Mono Q可得到酒精去氫酶約 16倍的純化倍率，但卻在 Blue Sepharose 時造成酒精去氫酶大量損失。從 SDS-PAGE上，發現部份純化的酒精去氫酶具有兩個明顯的 bands，其分子量約為 25 kDa及 22 kDa，推測美白菇酒精去氫酶可能是具有兩個次單元的四聚體蛋白質。
本研究另一方面主要探討美白菇菌絲在不同碳、氮源時酒精之產量的影響。發現美白菇菌絲生長最適 pH為 5.5，且葡萄糖與酵母萃取物是酒精發酵的關鍵成分，另外添加果糖也會提升酒精的產量。增加可發酵糖的含量可改善酒精產量，尤以 16° Brix（葡萄糖：果糖=1:1）為佳。而添加 2% yeast extract也可增加酒精的產量。 ADH活性增加可提高美白菇菌絲發酵的酒精產量。但在有氧、無氧的情況下以及其菌絲體生成量則對酒精產量並無直接相關性。

關鍵字

酒精發酵 ； 酒精去氫酶 ； 食用菇類 ； 美白菇

並列摘要

Wine drinks mainly use yeast Saccharomyces cerevisiae for ethanol fermentation. Some mushrooms have known to possess high alcohol dehydrogenase (ADH) and produce ethanol content higher than 10%. The potent mushroom-derived metabolites with antitumor and immunomodulating properties were also discovered. The objective of this research is to screen for mushrooms with high ADH activity and their potential use for ethanol fermentation. Four commercial mushrooms show highest and stable ADH activities, i.e. Pleurotus ostreatus, Hypsizygus marmoreus, Hypsizygus marmoreuas (white) and Flammulina velutipes. Most of mushrooms show ADH activity at pH 7.0 with a molecular weight of 100 to 150 kDa except Pleurotus ostreatus. It displays two distinct ADHs with a molecular weight of 80 and 150 kDa. The pH profile study of ADH activity differentiates among mushrooms studied. The highest activity of ADHs from the most of mushrooms is in the pH range of 6 to 8. However, some mushrooms, e.g. Volvariella volvacea, Lentinus edodes, Agaricus bisporus and Pleurotus cystidiosus display major ADH activities in the acidic or alkaline pH
Partial purification of ADH from Hypsizygus marmoreuas (white) was accomplished using 50-80% ammonium sulfate fractionation and two column chromatography, Mono Q and Blue Sepharose. Mono Q chromatography improves the purity fold to 16, but Blue sepharose chromatography causes a major lost of ADH activity. From a result of SDS-PAGE, the partial purified ADH demonstrates two major bands at 22 and 25 kDa. It hypothesizes that ADH from Hypsizygus marmoreuas (white) might be a tetrameric protein .
On the other hand, ethanol production using Hypsizygus marmoreuas (white) with different sources and concentration of carbon or nitrogen medium was studied. The optimum pH of growth for Hypsizygus marmoreuas (white) is 5.5. It showed that glucose and yeast extract are the key media components that influence ethanol production. Moreover, the addition of fructose arise the ethanol production. The increase of fermentable sugar content improves in the ethanol yield, especially at 16° Brix (glucose: Fructose =1: 1). The 2% of yeast extract also enhances the ethanol production. The increase of ADH activity in Hypsizygus marmoreuas (white) mycelia is in relation to higher ethanol formation. However, the fermentation in aerobic or anaerobic condition or the biomass of Hypsizygus marmoreuas (white) mycelia is irrelevant to the ethanol yield.

並列關鍵字

Hypsizygus marmoreuas (white) ； alcoholic fermentation ； alcohol dehydrogenase ； Edible mushroom

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