β-淀粉酶的分离纯化及酶学性质研究
A Study of Isolation and Purification of β-Amylase and Its Enzymatic Properties

以“渝薯17”为实验材料，从淀粉生产废水中分离纯化β-淀粉酶.去皮、1:10(m/V)匀浆抽提，经过乙醇分级沉淀、DEAE-Sepharose离子交换层析和Superdex-200凝胶过滤层析获得电泳纯β-淀粉酶并对其酶学性质进行了研究.结果表明：该酶的比活力高达333.15 U/mg，显著高于市售枯草杆菌来源酶活(50 U/mg)；纯化倍数9.93倍，回收率66.60%；亚基分子量约为55.12 kD，全酶分子量约为223.54 kD；最适温度50 ℃，最适pH值6.2；50 ℃以下，pH值6~8具较好的稳定性.在最适条件下以可溶性淀粉为底物，测得K_m为0.001 36 g/mL，V_max为0.112 mg/mL·min；Mn²⁺，Pb²⁺，Li⁺，Zn²⁺，K⁺，Cu²⁺，草酸，SDS对该酶有不同程度的抑制作用，Co²⁺有激活作用，有机物作用不明显.
β-Amylase was extracted and purified from the starch production wastewater with the sweet potato variety 'Yushu 17' as the raw material. After peeling, 1:10 (m/V) homogenizing extraction, fractional precipitation with ethanol, DEAE-Sepharose ion exchange chromatography and Superdex-200 gel filtration chromatography, electrophoretically pure β-amylase was obtained. Then its enzymatic properties were studied. The results showed that the specific activity of β-amylase obtained in this study (333.15 U/mg) was significantly higher than that of commercially available sources of Bacillus subtilis (50 U/mg); its purification fold was 9.93 and its recovery rate was 66.6%; its subunit molecular weight was about 55.12 kD and its enzyme molecular weight was about 223.54 kD; and its optimum temperature was 50℃ and its optimum pH was 6.2. This β-amylase showed good stability with a temperature of < 50℃ and a pH of 6~8. Measured under the optimal conditions with soluble starch as the substrate, it had a Km of 0.001 36 g/mL and a Vmax of 0.112 mg/mL·min. In addition, Mn²⁺, Pb²⁺, Li⁺, Zn²⁺, K⁺, Cu²⁺, oxalic acid and SDS inhibited, in different extents, this β-amylase, Co²⁺ activated it, and organic matter had no obvious effect on it