Saha B C, Mathupala S P, Zeikus J G
Michigan Biotechnology Institute, Lansing 48909.
Biochem J. 1988 Jun 1;252(2):343-8. doi: 10.1042/bj2520343.
Clostridium thermohydrosulfuricum mutant Z 21-109 produced intracellular thermostable pullulanase and glucoamylase activities. The glucoamylase activity was inactivated by treating C. thermohydrosulfuricum cells with 10% (v/v) propan-1-ol at 85 degrees C in the presence of 5 mM-CaCl2. Pullulanase activity was selectively solubilized from cells by treatment with detergent and lipase. The solubilized pullulanase was purified by treatment with streptomycin sulphate and (NH4)2SO4 and by DEAE-Sephacel, octyl-Sepharose and pullulan-Sepharose chromatography. Pullulanase was purified 3511-fold and displayed homogeneity on SDS/polyacrylamide-gel electrophoresis. The pullulanase was a monomeric glycoprotein with an apparent Mr of about 136,500, and it displayed a pI of 5.9. The enzyme was enriched in both acidic and hydrophobic amino acids. The purified pullulanase was stable and optimally active at 90 degrees C. The optimum pH for activity and pH-stability ranges were 5.0-5.5 and 3.0-5.0 respectively. The enzyme was inhibited by cyclodextrins, EDTA and N-bromosuccinimide, but not by p-chloromercuribenzoate and acarbose. The pullulanase displayed a relative substrate specificity for hydrolysis of pullulan (100%) versus 75% for glycogen and 50% for soluble starch. The apparent Km, Vmax. and Kcat. values for enzyme activity on pullulan at 60 degrees C were 0.675 mg/ml, 122.5 mumol of reducing sugar formed/min per mg of protein and 16,240 min-1 respectively. The novel properties of this extremely thermostable pullulanase are discussed in relation to other purified starch-debranching enzymes.
嗜热栖热硫化叶菌突变体Z 21 - 109产生细胞内耐热性支链淀粉酶和糖化酶活性。在5 mM氯化钙存在下，于85℃用10%（v/v）丙醇处理嗜热栖热硫化叶菌细胞可使糖化酶活性失活。通过用去污剂和脂肪酶处理，支链淀粉酶活性可从细胞中选择性地溶解出来。溶解的支链淀粉酶经硫酸链霉素和硫酸铵处理以及通过DEAE - 琼脂糖凝胶、辛基 - 琼脂糖凝胶和支链淀粉 - 琼脂糖凝胶色谱法进行纯化。支链淀粉酶纯化了3511倍，在SDS /聚丙烯酰胺凝胶电泳上显示出均一性。该支链淀粉酶是一种单体糖蛋白，表观分子量约为136,500，其pI为5.9。该酶富含酸性和疏水性氨基酸。纯化的支链淀粉酶在90℃稳定且活性最佳。活性的最适pH值和pH稳定性范围分别为5.0 - 5.5和3.0 - 5.0。该酶受到环糊精、EDTA和N - 溴代琥珀酰亚胺的抑制，但不受对氯汞苯甲酸和阿卡波糖的抑制。该支链淀粉酶对支链淀粉水解表现出相对底物特异性（100%），对糖原的水解为75%，对可溶性淀粉的水解为50%。在60℃时，该酶对支链淀粉的酶活性的表观Km、Vmax和Kcat值分别为0.675 mg/ml、每毫克蛋白质每分钟形成122.5 μmol还原糖以及16,240 min-1。本文结合其他纯化的淀粉脱支酶讨论了这种极端耐热性支链淀粉酶的新特性。
