中國酒麴中澱粉糖化黴菌所分泌澱粉水解酵素之研究 第一報 Rhizopus tienchiuliensis所分泌澱粉水解酵素之分離純化及性質之研究

Studies on the Amylases of Amylomyces from Chinese Chiu-Chu (A Starter of Chinese Wine) I. Isolation, Purification and Properties of the Amylases from Rhizopus tienchiuliensis

劉伯文(Peh-Wen Liu) ； 陳勝和(Hsen-Ho Chen)

《化學》 51年3期 (1962/09) Pp. 111-126

https://doi.org/10.6623/chem.1962019

摘要

本研究係由甜酒釀麴分離之糖化黴菌Rhizopus tienchiuliensis中，抽提並精製澱粉水解酵素(amylase)。此澱粉水解酵素可分為液化(即α-amylase)及糖化(即β-amylase)兩部份。液化力價(liquefying activity)係用碘液呈色反應測驗；糖化力價(sacharifying activity)則用Bertrand法測驗其生產糖量。將Rhizopus tienchiuliensis之胞子接種於麥麩米糠=10:1之混合培養基上，保溫30-38℃；約29小時，即開始形成胞子時期，其液化及糖化力價達最高峰，胞子形成以後，則其力價漸次低減。粗製酵素液(即水浸抽液)分別用酒精精製法及硫酸銨沉澱精製法作比較試驗，結果以用硫酸銨者效率較優。依據Van Klinkenberg法用60%及80%濃度酒精分別沉澱α-,及β-amylase，結果其液化及糖化力價均集中於60%酒精沉澱部份；用80%酒精沉澱者，大部份為對澱粉呈不活性之物質。兩部份水解酵素最適pH值均在pH4.3-5.8之間；最適溫度均為55-57.5°C。兩者均能將麥芽糖分解為葡萄糖，但其力價不強。60%酒精沉澱部份澱粉水解酵素能抵抗高溫，以65℃處理5分鐘，只能破壞其液化力80%；至70°C，始完全被破壞。80%酒精沉澱部分澱粉水解酵素則對酸之抵抗力甚強，在pH值1.15時，尚有一部分糖化力，直至pH1.1時，其活力始完全喪失。60%酒精沉澱部份澱粉水解酵棄經高溫(60°C)將糖化力破壞後其活動pH範圍為pH1.6-11.7之間；pH1.6以下，或pH11.6以上，即完全失去活力，又60-80%酒精沉澱部分澱粉水解酵素用酸液(pH1.4)將液化力破壞後，其括動pH範圍為pH1.1-12.0之間;pH1.1以下，或pH12.2以上即完全失去活力。用60%及80%酒精分別沉澱之兩部分澱粉水解酵素作用於可溶性澱粉時，均可產生葡萄糖及麥芽糖，但前者之生產物在1小時以內尚有麥芽糖反應，1小時以後則麥芽糖之反應消失，後者之生產物則在5-12小時之間有麥芽糖之反應出現，12小時以後又漸次消失。又用60%及80%酒精沉澱之兩部分澱粉水解酵素分別作用直於麥芽糖時，均可生成葡萄糖，但在前者則麥芽糖之反應能在1小時內消失；然在後者（液化活力被破壞者）則雖作用24小時以上仍有麥芽糖之反應存留。酵素之本質為蛋白質，精製成結晶，向為至難之事，本研究室過去數年間在比方面之所作努力甚多，但迄未完全成功，此次用比較簡易之方法，將Rhizopus tienchiuliensis所分泌澱粉水解酵素精製後析出四方八面體結晶，殊為欣幸！其他詳細性質，仍在繼讀研究中。

並列摘要

In this experiment, extraction and purification of the Amylases from Rhizopus tienchiuliensis isolated from Chiu-Chu of Tien-chiu-niang (sweet wine) have been carrid out. The amylases were classified as a-Amylase (dextrinogenic) and 8-Amylase (Saccharogenic). The dextrinogenic activity was determined by iodine color reaction, while the saccharogenic activity by measurement of reducing sugar according to Bertrand's method. The spore of R. tienchiuliensis was inoculated into the medium consisting of wheat bran and rice bran (10:1) and incubated at 30-38°C. After 29 hours of incubation, it began to form spores and both dextrinogenic and saccharogenic activities of the mold reached maxima. The activities decreased, then, gradually after spore formation was completed. The precipitation efficiency of the enzyme from crude enzyme solution (aqueous extract) with ammonium sulfate was found to be better than that with alcohol. 60% and 80% of aqueous alcohols were used to separated a- and 8-amylases according to Van Klinkenberg's method. The results showed that most of the dextrinogenic and saccharogenic activities were found in the fraction precipitated with 60% of aq. alcohol, while that the fraction precipitated with 80% of aq. alcohol contained mainly of starch inactive substances. The optimum pH of 60% and 80% alcohol precipitated fraction were found to be at 4.3-5.8, and the optimum temperature at 55-57;5°C; Both the fractions can hydrolize maltose to glucose, but the activities were not strong. The 60% alcohol ppted. amylase was resistant to heat. Only 80% of the dextrinogenic activity was destroyed as it was treated at 65°C for 5 min. It retained some activities until 70°C. The 80% alcohol ppted, fraction was resistant to the action of acid. It retained some activities until pH. 1.1. The action range of 60% alcohol ppted, fraction was found to be between pH 1.6-11.7. Beyond this range, the activity was lost completely. Glucose and maltose were found in the hydrolysate of soluble starch with both fractions as hydrolyzing agents. Within an hour of action with the 60% alcohol ppted, fraction or between 5-12 hours of action with the 80% alcohol ppted. fraction, maltose reaction was found to be positive, however, it lost by further reaction. Both the fractions could hydrolyze maltose to glucose. With the 60% alcohol ppted, fraction, maltose was hydrolyzed completely within an hour, while with the another fraction, it existed for more than 24 hours. Enzyme is a protein in nature, so it is very difficult to crystallise out. Many studies on the crystallization of amylase have been carried out in our laboratory recently, but the results were not satisfactory. In this experiment, we have obtained the crystals of amylase. It seems to be octahedral crystals. Further studies will be proceeded to determine it's properties.