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Calcium ion induced thermodynamic stability, bisubstrate specificity, and differential organic solvent tolerance of a predominantly β-sheet serine protease from Bacillus aquimaris VITP4.
Biotechnology and Applied Biochemistry ( IF 2.8 ) Pub Date : 2019-09-30 , DOI: 10.1002/bab.1811
Chittoor Jabeena Thaz 1 , Gurunathan Jayaraman 1
Affiliation  

The present study was aimed to get insights on the role of calcium ions on the thermodynamic stability, substrate specificity, and organic solvent compatibility of the extracellular protease produced by Bacillus aquimaris VITP4. Presence of Ca2+ enhanced the activity of the enzyme in the temperature range of 30-60 °C and increased the half-life from 164 to 234 Min. Circular dichroism experiments indicated that the temperature of half-denaturation (Tm ) of the protease increased from 76 to 86 °C. As judged by fluorescence emission profiles, the overall fold of the enzyme around the tryptophan residues could be similar. Further, thermal inactivation experiments revealed that the enzyme followed first order kinetics, with increase in energy for inactivation (Eai ) by 24.2 ± 1.2 kJ mol -1 in the presence of Ca2+ . Studies with synthetic peptides as well as with bovine serum albumin signified preferential hydrolysis of the peptide bonds at the C-terminus of alanine residues (with a kcat /KM of 141,400 M-1 Sec-1 ) and at the C-terminus of arginine residues with a lower specificity (72,400 M-1 Sec-1 ), indicating bisubstrate specificity of the enzyme. The enzyme was found to be compatible with organic solvents (50%, v/v) such as acetonitrile and butanol, indicating possible application under demanding nonaqueous conditions.

中文翻译:

钙离子诱导的水生芽孢杆菌VITP4的主要β-折叠丝氨酸蛋白酶的热力学稳定性,双底物特异性和不同的有机溶剂耐受性。

本研究旨在了解钙离子对水生芽孢杆菌VITP4产生的细胞外蛋白酶的热力学稳定性,底物特异性和有机溶剂相容性的作用。Ca2 +的存在增强了30-60°C温度范围内酶的活性,并将半衰期从164分钟增加到234分钟。圆二色性实验表明,蛋白酶的半变性(Tm)温度从76℃增加到86℃。通过荧光发射曲线判断,色氨酸残基周围酶的整体折叠可能相似。此外,热灭活实验表明,该酶遵循一级动力学,在存在Ca2 +的情况下,灭活能量(Eai)增加了24.2±1.2 kJ mol -1。用合成肽以及牛血清白蛋白进行的研究表明,丙氨酸残基的C末端(kcat / KM为141,400 M-1 Sec-1)和精氨酸残基的C末端的肽键优先水解。具有较低的特异性(72,400 M-1 Sec-1),表明该酶具有双底物特异性。发现该酶与有机溶剂(50%,v / v)(如乙腈和丁醇)相容,表明可能在要求的非水条件下使用。
更新日期:2020-01-09
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