中國酒麴中糖化黴菌所分泌澱粉水解酵素之研究 第二報 黴菌（Rhizopus chungkuoensis）所分泌澱粉水解酵素之分離純化及其作用機構之探討

Studies on the Amylases from Amylomyces of Chinese Chiu-Chu. II. On the Isolation, Purification and Mechanism of Action of the Amylase from Rhizopus Chungkuoensis

劉伯文(Peh-Wen Liu) ； 陳勝和(Shen-Ho Chen) ； 邱德馨(TeH-Hsing Chiu)

《化學》 52年3期 (1963/09) Pp. 103-110

https://doi.org/10.6623/chem.1963016

摘要

將徽菌（Rhizopus chungkuoensis）培養於麩皮及米糠42小時後，抽出酵素液，經三次之硫酸銨鹽析及透析後，再用各種濃度之酒精分劃沉澱，測驗各部分沉澱物之液化及糖化力價，結果顯示其酵素活力，均集中於23-61%酒精沉澱部份，但各部份之液化及糖化力價比率大致相同；換而言之，卽不能用不同濃度之酒精分劃沉澱法，將澱粉液化（α-amylase）與澱粉糖化（β-amylase）兩酵素分開。這種酵素對澱粉液化及糖化之最適pH值均爲4.7-5.0；最適溫度均爲55-57.5°C，被破壞之pH值爲1.4以下及12.0以上，被破壞之溫度爲65°C 10分鐘，此等性質與前報告黴菌（Rhizopus teinchiulieusis）^1所分泌者大同小異，不分軒輊。Rhizopus chunkuoensis又可用Alumina Cr將其中之澱粉液化酵素（α-amylase）與澱粉糖化酵素（β-amylase）分開，因α-amylase被吸着於Alumina Cr，而β-amylase則遺留於溶液中，此結果與Waldschmidt-Leitz等^2對於麥芽中之澱粉水解酵素所得結果正相反，因彼等用Alumina Cr吸着麥芽amylase中之β-amylase，而α-amylase則遺留於溶液中。被Alumina Cr吸着之α-amylase用磷酸銨或鹽酸等溶液容易溶離（elute），且溶離之pH值範圍頗寬，在pH 1.8-8.4之範圍內，各pH之溶離效果無大差異。α-amylase作用於可溶性澱粉時，生產glucose, maltose, dextrin等；但對maltose無作用，β-amylase作用於可溶性澱粉時，只生產glucose，及殘餘之limit-dextrin，但對maltose亦可能分解，似爲一種a-amylo-glucosidase；亦可能爲β-amylase與maltase之混合物。用紙帶電氣泳動法測驗α-amylase時，未發現有不同方向或不同速度之分離spots，故可能爲單純之酵素，至於β-amylase中是否含有maltase？尚有待於進一步之研究。用Alumina Cr吸着α-amylase後之β-amylase溶液置於載玻璃片上風乾後，容易析出其四方八體（octahedron）結晶，但α-amylase之結晶迄未能成功。

並列摘要

In this experiments, the enzyme, Amylase, was purified from the aqueous extracts of Mold bran, which was prepared by culturing Rhizopus chungkuoensis on wheat bran-rice bran medium for about 42 hours, by repeated salting out with ammonium sulfate and dialyzing against tap and distilled water, then fractionated with various concentrations of alcohol. The active enzyme was found to exist mainly in the fraction precipitated with 23-61% of alcohol. The ratios of liquifying and saccharifying activities of each fractions were found to be nearly the same. It seems, therefore, impossible to separate the enzyme into α-amylase and S-amylase by simple alcohol fractionation. The optimum pH for both liquefication and saccharification of starch of this enzyme was found to be at 4.7-5.0, and the optimum temperatures 55-57.5°C. The enzyme was destroyed completely at the pH values lower than 1.4 or higher than 12.0, and at the temperature higher than 65°C in 10 min.. These properties were found to be similar to those of the enzyme isolated from Rhizopus tienchiuliensis reported recently. As the α-amylase of the enzyme could be adsorbed by Alumina Cr and the β-amylase remained unadsorbed, it was found to be successful to separate α-amylase from β-amylase by use of Alumina Cr adsorption. This results was just contrary to the results obtained by Waldschmidt Leitz et al. They found that β-amylase of malt amylase would be adsorbed by Alumina Cr, whereas α-amylase would not. The α-amylase adsorbed on Alumina Cr could be eluted easily with ammonium phosphate or HCI. The pH range of the eluting solution was very wide and the efficiencies were little affected by the acidity between pH 1.8 and 8.4. The α-amylase was found to be inactive to maltose and produce glucose, maltose and dextrin if acted upon soluble starch, while β-amylase be active to maltose and produce glucose and limit dextrin only. The latter one was considered as an α-amyloglucosidase or a mixture of β-amylase and maltase. In the paper electrophoresis of α-amylase, only one band was found in the paper strips, therefore, it was reasonable to cosider it as a single enzyme. As to β-amylase, there requires some further experiments to make sure whether it is a single enzyme or not. Octahedral crystals of β-amylase were obtained by putting the Alumina Cr treated enzyme solution on slide glass and letting it dry spontaneously, while the preparation of α-amylase crystals was still unsuccessful.