# Serine Protease HtrA2 from Halophilic Archeon Haloarcula sp. TG1: Heterologous Expression, Characterization and Immobilization

## TL;DR

This paper studies a halophilic serine protease from Haloarcula sp. TG1, showing its potential for industrial use due to stability and effectiveness.

## Contribution

The study is the first to characterize and immobilize HtrA2 from Haloarcula sp. TG1 using PHB nanoparticles.

## Key findings

- Recombinant HtrA2 shows optimal activity at 50°C, pH 7.0, and 3.0 M NaCl.
- Immobilization on PHB nanoparticles improves HtrA2 reusability.
- HtrA2 effectively removes bloodstains from cotton fabric.

## Abstract

Halophilic proteases are valuable in industrial applications due to their resistance to harsh conditions. HtrA2 serine protease is widely distributed and conserved among eukaryotes and prokaryotes. However, HtrA2 proteases from archaea have been poorly characterized. In this study, htrA2 from haloarcheon Haloarcula sp. TG1 was cloned and corresponding nucleotide and amino acid sequences were analyzed. Recombinant HtrA2 was produced in Escherichia coli, and biochemical properties of purified HtrA2 were characterized. HtrA2 was immobilized for the first time using polyhydroxybutyrate (PHB) nanoparticles. Additionally, potential of HtrA2 as a detergent additive was evaluated by its bloodstain removal activity. Recombinant HtrA2 showed its optimum activity at 50 °C, pH 7.0, and 3.0 M NaCl. HtrA2 activity was highly retained over wide temperature (40 to 60 °C) and pH ranges (pH 5.0 to 11.0). Moreover, various organic solvents, inhibitors and metal ions were well tolerated by the enzyme. Acetone and Fe2+ significantly increased HtrA2 activity, while it was not inhibited by phenylmethylsulfonyl fluoride and sodium dodecyl sulfate. Also, immobilization of HtrA2 onto PHB nanoparticles improved its reusability. Furthermore, HtrA2 successfully removed the bloodstain from cotton fabric. This comprehensive characterization of HtrA2 demonstrates that recombinant HtrA2 obtained from Haloarcula sp. TG1 is promising for industrial applications.