An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K _cat). The enzyme was also calcium dependent and with 2 mM Ca⁺², the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca⁺², respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.

A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol, benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol; the enzyme was highly active at 60–80°C and displayed optimum activity at 70°C. The protease was significantly stable and catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis of the enzyme adversely. Crude enzyme preparation was more stable at 37°C in solvents as compared to partially purified and purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant protease from salt-tolerant alkaliphilic actinomycetes.

An alkaline protease gene of Bacillus lehensis JO-26 from saline desert, Little Rann of Kutch, was cloned and expressed in Escherichia coli BL21 (DE3). A 1,014-bp ORF encoded 337 amino acids. The recombinant protease (APrBL) with Asp 97, His 127, and Ser 280 forming catalytic triad belongs to the subtilase S8 protease family. The gene was optimally expressed in soluble fraction with 0.2 mM isopropyl β-D-thiogalactopyranoside (IPTG), 2% (w/v) NaCl at 28°C. APrBL, a monomer with a molecular mass of 34.6 kDa was active over pH 8–11 and 30°C−70°C, optimally at pH 10 and 50°C. The enzyme was highly thermostable and retained 73% of the residual activity at 80°C up to 3 h. It was significantly stimulated by sodium dodecyl sulfate (SDS), Ca²⁺, chloroform, toluene, n-butanol, and benzene while completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and Hg²⁺. The serine nature of the protease was confirmed by its strong inhibition by PMSF. The APrBL gene was phylogenetically close to alkaline elastase YaB (P20724) and was distinct from the well-known commercial proteases subtilisin Carlsberg (CAB56500) and subtilisin BPN′ (P00782). The structural elucidation revealed 31.75% α-helices, 22.55% β-strands, and 45.70% coils. Although high glycine and fewer proline residues are a characteristic feature of the cold-adapted enzymes, the similar observation in thermally active APrBL suggests that this feature cannot be solely responsible for thermo/cold adaptation. The APrBL protease was highly effective as a detergent additive and in whey protein hydrolysis.