Characterization of thermostable carboxypeptidase from high-altitude hot spring metagenome

• Published in: International journal of biological macromolecules 2024-09, Vol.276 (Pt 2), p.133974, Article 133974
• Main Authors: Wani, Atif Khurshid, Chopra, Chirag, Ansari, Mushtaq Ahmad, Dar, Mudasir A., Américo-Pinheiro, Juliana Heloisa Pinê, Singh, Reena
• Format: Article
• Language: English
• Subjects: Altitude; Amino Acid Sequence; Carboxypeptidases - chemistry; Carboxypeptidases - genetics; Carboxypeptidases - metabolism; Cloning, Molecular; Enzyme Stability; Enzymes; Escherichia coli - genetics; Hot Springs - microbiology; Hot Temperature; Hydrogen-Ion Concentration; Hydrolases; Metagenome; Metagenomics; Phylogeny; Protease; Substrate Specificity; Thermostable
• ISSN: 0141-8130; 1879-0003; 1879-0003
• DOI: 10.1016/j.ijbiomac.2024.133974

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-α 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 °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. [Display omitted] •Metagenomics allows access to the genetic material of unculturable microorganisms.•Carboxypeptidase (1515 bp) gene was successfully cloned and expressed in pJET1.2 and pET28a respectively.•His-tagged CarP protein (58.65 kDa) was purified using nickel column affinity chromatography.•CarP exhibits a pI and GRAVY of 4.81 and −0.447 respectively.•Zn2+ positively affects the activity of thermostable (70 °C) and alkalophilic (10.0) CarP.