Kim C H, Nashiru O, Ko J H
Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dong-Guk University, Kyung-Ju City, Kyung-Pook, South Korea.
FEMS Microbiol Lett. 1996 May 1;138(2-3):147-52. doi: 10.1111/j.1574-6968.1996.tb08148.x.
A thermostable pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified to homogeneity from Thermus caldophilus GK-24 by chromatographic methods, including gel-filtration and ion-exchange chromatography. The specific activity of the enzyme was increased 431-fold with a recovery of 13.2%. The purified enzyme was a monomer, M(r) = 65 kDa as estimated by SDS-PAGE and gel filtration. The pI was 6.1. The enzyme was most active at pH 5.5. The activity was maximal at 75 degrees C and stable up to 95 degrees C for 30 min at pH 5.5. The enzyme was stable to incubation from pH 3.5 to pH 8.0 at 4 degrees C for 24 h. The activity of the enzyme was stimulated by Mn2+ and Mg2+ ions. Ni2+, Ca2+, Co2+ ions and EDTA did not inhibit the enzyme activity. The enzyme hydrolyzed the alpha-1,6 linkages of amylopectin, glycogens, alpha, beta-limited dextrin, and pullulan. The enzyme caused the complete hydrolysis of pullulan to maltotriose. The activity was inhibited by alpha-, beta-, or gamma-cyclodextrins. The N-terminal sequence [(AIa-Pro-Gln-(Asp or Tyr)- Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)] showed some similarity to those of bacterial pullulanases.
一种耐热性支链淀粉酶（支链淀粉6-葡聚糖水解酶，EC 3.2.1.41）已通过包括凝胶过滤和离子交换色谱在内的色谱方法从嗜热栖热菌GK-24中纯化至同质。该酶的比活性提高了431倍，回收率为13.2%。纯化后的酶为单体，通过SDS-PAGE和凝胶过滤估计其分子量（M(r)）为65 kDa。其等电点为6.1。该酶在pH 5.5时活性最高。活性在75℃时最大，在pH 5.5下高达95℃可稳定30分钟。该酶在4℃下于pH 3.5至pH 8.0孵育24小时仍稳定。该酶的活性受到Mn2+和Mg2+离子的刺激。Ni2+、Ca2+、Co2+离子和EDTA不抑制该酶的活性。该酶水解支链淀粉、糖原、α,β-极限糊精和支链淀粉的α-1,6键。该酶使支链淀粉完全水解为麦芽三糖。其活性受到α-、β-或γ-环糊精的抑制。N端序列[(AIa-Pro-Gln-(Asp或Tyr)-Asn-Leu-Leu-Xaa-ILe-Gly-Ala(Ser)]与细菌支链淀粉酶的序列有一些相似性。
