Bacillus licheniformis B7 與Bacillus stearothermophilus Q8的細胞外耐熱性澱粉液化?之生產及性質之探討

Abstract

本實驗室前所分離兩株能生產耐熱性澱粉液化?的菌種，Bacillus licheniformis B7及Bacillus stearothermophilus Q8被選作為實驗菌株來進行本實驗。培養條件及養分需求情況經探討後得知兩菌株的最適生長環境為中性，而生產液化?需培養五天。花生粉萃取液當添加0.5％時對B. licheniformis B7的酵素產量可提高1.5倍；而B. stearothermophilus Q8則必須添加4％的花生粉萃取液，酵素產量提高1.4倍。 由兩株細菌所生產的澱粉液化?經六個步驟純化，可分別純化1467倍與1213倍，?收率為29％與27％。部分純化的酵素可添加牛血清蛋白，Ca2＋和Mg2＋以保護酵素活性。兩種酵素的最適作用酸鹼值為9.0，B. licheniformis B7的酵素經24小時不同酸鹼值處理後，在pH8和9很穩定，pH 7.0剩下91％的酵素活性，pH 10.0剩下48％的活性。經同樣的處理，B. stearothermophilus Q8的酵素在pH9.0十分穩定，而pH 8.0餘98％的酵素活性；pH 10.0則剩下41％的活性。兩酵素的最適作用溫度為90℃，當在100℃下作用時，酵素活性剩下86％和81％。添加10mM Ca2＋兩酵素在100℃下處理15分鐘，活性尚餘77％和67％。然而在不含基質的情況下，添加Ca2＋，可使得酵素經90℃處理2小時後，尚餘21％與10％的活性。0.1 mM和1 mM的Na＋，0.1mM的Mn2＋對酵素活性有促進作用。Fe3＋在0.1mM對B. licheniformis B7的酵素活性有促進作用。其他如Co2＋、Ag＋、Ni2＋、Cu2＋、Zn2＋、Cs＋、Ba2＋對酵素活性有抑制效果。OH-及N3-對酵素活性有促進作用，SO42-則僅對B. licheniformis B7的酵素活性有促進作用。Urea和KMnO4對酵素活性有抑制，2-Mercaptoethanel、SDS和Tween 80在低濃度時促進酵素活性。半乳糖、葡萄糖及麥芽糖對酵素活性沒有抑制作用。而果糖、甘露糖、木糖及乳糖對酵素活性具有抑制效果。對於多醣類的水解速率的順序為顆粒澱粉＞可溶性澱粉約等於玉米澱粉＞肝醣。

Two strains of thermostable alpha-amylase producing bacilli as Bacillus licheniformis B7 and Bacillus stearothermophilus Q8 were previously isolated in our laboratory. Cultural and nutritional requirements of those for the production of thermostable alpha-amylase were studied. The opitmum pH and incubation period for the enzyme production were 7.0 and 5 days, respectively. The effect of ground nut extract on enzyme production was also investigated. Enzyme production could increase 1.5 and 1.4 fold by adding a lower concentration of ground nut (0.5%) for B. licheniformis B7 and a higher concentration (4.0%) for B. stearothermophilus Q8. The enzymes of two bacilli were purified 1467-fold and 1213-fold with a 29% and 27% yield through a series of six steps. The partial purified enzymes could be protected by BSA, Ca2+, and Mg2+ and showed maxial activity at pH 9.0. Enzyme from B. licheniformis B7 showed 100% stability in the pH range 8 to 9; 91% stability at pH7.0 and 48% stability at pH 10.0 after 24 hrs treatment, but, enzyme from B. stearothermophilus Q8 only showed 100% stability at pH9.0; 98% stability at pH 8.0 and 41% stability at pH 10.0 after same treatment. Enzymes expressed optimal reaction temperature at 90℃ and 86% and 81% of the activities remained at 100℃ respectively. After 15 min incubation at 100℃ with the addition of 10 mM Ca2+, enzyme from B. licheniformis B7 retained 77% activity, on the contrary, enzyme from B. stearothermophilus Q8, with 10 mM Ca2+, only retained 67% activity. However, in the absence of substrate, after two hours incubation at 90℃, the enzymes retained 21% and 10% maximal activity respectively. Of the cations, Na at 0.1 mM and 1 mM, Mn2+ at 0.1 mM showed stimulatory effect on both enzymes, Fe3+ at 0.1 mM showed positive effect on the enzyme of B. licheniformis B7, whereas Co2+, Ag+, Ni2+, Cu2+, Zn2+, Cs+, Ba2+ were inhibitory. Of the anions, OH-, N3- showed an excitant effect, SO42- showed only positive effect on enzyme from B. licheniformis B7. Urea and KMnO4 addition resulted in the loss of enzyme activities; however, 2-Merc-aptoethnol, SDS and Tween 80 in low concn afforded protection of the enzyme activities. When the effects of certain organic compounds on enzyme activities were measured, galactose, glucose and maltose were non-inhibitory. Slight inhibition were cause by fructose, mannose, xylose and lactose. Hydrolysis of amylose, soluble starch, corn starch and glycogen, the relative hydrolysis sequence was about amylose, corn starch and soluble starch, glycogen.