Purification and kinetic studies of wheat bran beta-amylase. Evaluation of subsite affinities.
Purification and kinetic studies of wheat bran beta-amylase. Evaluation of subsite affinities.
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麦麸β-淀粉酶的纯化和动力学研究。
DOI：
10.1093/oxfordjournals.jbchem.a130424
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发表时间：
1974
影响因子：
2.7
通讯作者：
Y. Morita
中科院分区：
文献类型：
--
作者：
M. Kato;K. Hiromi;Y. Morita
1.从小麦麸皮中纯化出β-淀粉酶[α-1，4-葡聚糖麦芽糖水解酶，EC 3.2.1.2]，经超离心分析和聚丙烯酰胺凝胶盘状电泳确定为单一蛋白组分。根据蓝值和还原值之间的关系，证明该酶制剂不含α-淀粉酶[EC 3.2.1.1]污染.在25°C和pH5.20条件下测定了β-淀粉酶催化水解G3、G7和G760的米氏常数K和分子活性k 0，其中Gn表示聚合度为n的线性底物。在底物浓度充分低于Km值的条件下，通过测量每种底物的初始速率，确定了一系列线性底物G3-G7、G12、5和G760的k 0/Km值。从k 0/Kmonn的依赖性，该酶的亚位点的数目估计为5。根据亚位点理论（30），使用k 0/Km和Km的值来评估每个亚位点的亚位点亲和力Ai，其中i是从底物的非还原性末端葡萄糖残基所处的末端亚位点开始计数的亚位点数目。计算了（A1+A2）、A3、A4和A5的值，并估算了A1的下限和A2的上限。A1和A4的大正值和A2和A3的大负值与麦芽四糖是最小的好底物和麦芽糖和苯基α-麦芽糖苷没有水解到任何可检测的程度的实验数据相一致。
1. β-Amylase [α-1,4-glucan maltohydrolase, EC 3.2.1.2] from wheat bran was purified to a single protein component as determined by ultracentrifugal analysis and polyacrylamide gel disc electrophoresis. The enzyme preparation was proved to be free from a-amylase [EC 3.2.1.1] contamination, as judged from the relationship between the blue value and the reducing value.2. The Michaelis constant Kmand the molecular activity k0were determined at 25°C and pH 5.20 for the β-amylase-catalyzed hydrolyses of G3, G7, and G760, where Gndenotes a linear substrate with degree of polymerizationn. The values of k0/Kmwere determined for a series of linear substrates, G3-G7, G12,5and G760from measurements of the initial rate for each substrate at a substrate concentration sufficiently lower than its Kmvalue.3. From the dependence of k0/Kmonn, the number of subsites of this enzyme was estimated to be five. According to the subsite theory(30), the values of k0/Kmand Kmwere used to evaluate the subsite affinityAiof each subsite, whereiis the subsite number counting from the terminal subsite at which the nonreducing end glucose residue of a substrate is to be situated. The values of (A1+A2), A3, A4and A5were evaluated, and the lower limit of A1and the upper limit of A2were also estimated. The large positive values of A1and A4, and the negative values of A2and A3are consistent with the experimental data that maltotetraose is the smallest good substrate and that maltose and phenyl α-maltoside are not hydrolyzed to any detectable extent.