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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. [ABSTRACT FROM AUTHOR]