Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology
Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology
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DOI：
10.1002/bit.10203
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发表时间：
2002-05-05
影响因子：
3.8
通讯作者：
Gupta, R
中科院分区：
文献类型：
--
作者：
Beg, QK;Saxena, RK;Gupta, R
使用中心复合外切设计（CCCD）测定来自莫哈韦芽孢杆菌的碱性蛋白酶的动力学常数，其中底物（酪蛋白）的浓度和测定温度围绕其中心点变化。测定了K-m、V-max、K-cat、活化能（E-a）和温度系数（q（10）），发现这些动力学常数的值与用常规方法得到的值相当。在45- 60 ℃范围内，随着反应温度的升高，酪蛋白的米氏常数（K-m）逐渐减小，最大反应速率（Vmax）逐渐增大。该蛋白酶在45、50、55和60 ℃下的Km分别为0.0357 mg/ml、0.0270 mg/ml、0.0259 mg/ml和0.0250 mg/ml，而在这些温度下的V-max值分别为74.07、99.01、116.28和120.48 μ g/ml/min，如通过响应面方法测定的。Arrhenius图表明，该酶在45 ℃以上经历热活化，直到60- 65 ℃，然后热失活。同样，活化能（E-a）在45- 55 ℃之间（9747 cal/mol）高于50- 60 ℃之间（4162 cal/mol）。(C)2002 Wiley Periodicals，Inc.
The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K-m,V-max, K-cat, activation energy (E-a) and temperature coefficient (q(10)) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michael-is-Menten constant (K-m) for casein decreased with corresponding increase in V-max, as reaction temperature was raised from 45-60degreesC. The protease exhibited K-m of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60degreesC, respectively, whereas V-max values at these temperatures were 74.07, 99.01, 116.28, and 120.48 mug/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45degreesC until 60-65degreesC followed by thermal inactivation. Likewise, the energy of activation (E-a) was more between 45-55degreesC (9747 cal/mol compared to E-a between 50-60degreesC (4162 cal/mol). (C) 2002 Wiley Periodicals, Inc.