Purification and characterization of the secreted amylolytic enzyme from Alteromonas espejiana Bal 31 and its mutant.

• Main Authors: Meng-Ru Luo, 羅孟如
• Other Authors: Wu-Fu Tong
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/61218996193439070956

碩士 === 國立師範大學 === 生物學研究所 === 82 === A.espejiana Bal 31 野生型菌株及其突變株之澱粉水解酵素純化及物理， 化學性質探討為本研究之主題。利用硫酸銨沉澱，膠體過濾層析和親和性 管柱層析等方法將釋出於培養基的澱粉水解酵素加以純化分離。得到野生 型菌株澱粉水解酵素的純化倍率為 18 倍，突變株澱粉水解酵素則為 7 倍。兩者之分子量差異不大約為 80 KD。依酵素對可溶性澱粉之水解產物 分析結果推論，兩者皆為"liquefying".alpha. 澱粉水解酵素。 且其作 用最適溫度與最適 PH 值分別為攝氏 40 度及 7.0。所純化酵素對熱十分 敏感，突變株之澱粉水解酵素於攝氏 45 度作用 10 分鐘幾乎完全喪失活 性，野生型菌株澱粉水解酵素於攝氏45 度反應 10 分鐘，活性僅剩下約百 分之四十；若於 55 度作用 15 分鐘後即測不到活性。兩者作用的活化能 都有偏低的趨勢，二者分別為每莫耳 3.52 仟卡（ 野生型 ）及 4.01 仟 卡（ 突變株 ）。鈣離子可增加兩者對熱的耐受性，而鈉離子和氯離子對 突變株澱粉水解酵素之 耐熱性有提高的作用；若兩種離子同時添加則效果 更好，但野生型菌株澱粉水解酵素則否。然而兩者的活性均會受到 SDS、 尿素、papain 及 EDTA 的抑制，唯突變株澱粉水解酵素之耐受性均比野生 型菌株澱粉水解酵素高。Triton X 100及一般常見之金屬離子對酵素活性 並不具有明顯的抑制作用。兩者胺基酸的組成中，小分子厭水性胺基酸所 佔比例很高且均缺少 cysteine 和 arginine；這些特殊的胺基酸組成可能 可以增加蛋白質分子結構之彈性，使其得以適應溫度較低的海洋環境。 The properties of secreted amylolytic enzyme from A. espejiana Bal 31 and its mutant were investigated。The enzymes in caltured medium were purified by ammonium sulfate precipitation, gel filtration chromatography and affinity chromatography.The molecular weight of both enzymes were close and estimated to be about 80KD by SDS-PAGE. According to the substrate specificity and the hydrolytic productsanalysis, we interfer both enzymes are deduced to be the liquefyingtype of α-amylase. Both anzymes exhibit the same optimum pH andtemperature of 7.0 and 40℃ respectively. Relatived lower activationenergy and heatlabile of the enzymes were observed. Addition of calciumcould increase the thermostability of enzymes, but sodium and cholride ions showed more effective to the mutant's enzyme on the protection against heat. The tolerances of enzymes to the inactivators, such as urea,SDS,papain and EDTA were also different. However,triton X 100 and metal ions in our testing concentrations showed no obvious effects to enzyme activity. The amino acid composition of the enzymes displayed the unusual fraction of hydrophobic amino acids and the lacking of cysteine and arginine residues. This characteristic constitution might enhance the structural flexibility of enzyme protein to adapt to the marine enviroment.