Characterization of thermostable carboxypeptidase from high-altitude hot spring metagenome

被引:4
|
作者
Wani, Atif Khurshid [1 ]
Chopra, Chirag [1 ]
Ansari, Mushtaq Ahmad [2 ]
Dar, Mudasir A. [3 ]
Americo-Pinheiro, Juliana Heloisa Pine [4 ,5 ]
Singh, Reena [1 ]
机构
[1] Lovely Profess Univ, Sch Bioengn & Biosci, Phagwara 144411, Punjab, India
[2] King Saud Univ, Coll Pharm, Dept Pharmacol & Toxicol, Riyadh 11451, Saudi Arabia
[3] Jiangsu Univ, Biofuels Inst, Sch Environm & Safety Engn, Zhenjiang 212013, Peoples R China
[4] Sao Paulo State Univ UNESP, Sch Agr Sci, Dept Forest Sci Soils & Environm, Ave Univ 3780, BR-18610034 Botucatu, SP, Brazil
[5] Brazil Univ, Grad Program Environm Sci, St Carolina Fonseca 584, BR-08230030 Sa Paulo, SP, Brazil
关键词
Metagenomics; Hydrolases; Thermostable; Protease; Enzymes; BIOCHEMICAL-CHARACTERIZATION; PROTEASE; ALKALINE; SURFACTANTS; INHIBITION; REVEALS;
D O I
10.1016/j.ijbiomac.2024.133974
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
This study explored the metagenome of the Pir Panjal Hot Spring (PPHS) to identify thermostable hydrolases. The carboxypeptidase (CarP) gene was successfully amplified and cloned into Escherichia coli DH5-alpha cells, followed by expression in E. coli BL21-DE3 cells. The CarP enzyme was comprehensively characterized in vitro. Sequencing analysis revealed an open reading frame encoding a functional protein of 504 amino acids, with a molecular weight of 58.65 kDa and an isoelectric point of 4.81. The CarP protein was purified using Ni-His affinity chromatography, and the experimental molecular weight matched in silico predictions. The enzyme exhibited significant thermostability and alkaliphilic properties, with optimal activity at 70 degrees C and pH 10.0. Additionally, the presence of Zn+2 ions at concentrations of 5 and 10 mmol/L enhanced protease activity by 1.4 and 1.5-fold, respectively. This study reports the discovery of a novel, multifunctional, and thermostable CarP from hot-spring metagenomes. The enzyme's stability against high temperatures, metal ions, surfactants, and inhibitors, along with its specific substrate interactions, highlights its potential for various biotechnological applications.
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页数:12
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