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), Ca 2+ , chloroform, toluene, n-butanol, and benzene while completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and Hg 2+ . 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.

This report describes cultivation-dependent diversity, phylogeny and enzymatic potential of the haloalkaliphilic bacteria isolated from the unvegetated desert soil of yet unexplored, saline desert of Little Rann of Kutch (LRK), India. The LRK is a unique ecosystem displaying a combination of Dry Rann and Wet Rann. A total of 25 bacteria were isolated and characterized on the basis of colony morphology, biochemical profile, sugar utilization, secretion of the extracellular enzymes and antibiotic sensitivity. Further, the identification and phylogenetic relatedness of 23 bacteria were established by the analysis of 16S rRNA gene sequences. The phylogenetic analysis indicated that the isolates belong to the phylum , comprising low G + C, Gram-positive bacteria, with different genera: (~ 39%), (~ 30%), (~ 13%), (~ 13%), (~ 4%). Majority of the bacterial isolates produced proteases (30% isolates) followed by cellulases (24% isolates), CMCases (24% isolates) and amylases (20% isolates). , and predominantly produced hydrolases, while many produced multiple enzymes at high salinity and alkaline pH. Highest antibiotic resistance was observed against Ampicillin and Penicillin (32%) followed by Cefaclor (20%); Colistin, Cefoperazone and Cefotaxime (16%); Cefuroxime (12%); Gentamycin and Cefixime (8%); Erythromycin, Cefadroxil, Azithromycin, Co-trimoxazole, Amoxycillin, Norfloxacin, Cefpodoxime, Amikacin and Augmentin (4%). KJ1-10-99 and KJ1-10-93 representing< 97% of 16S rRNA gene sequence similarity belong to a novel lineage within the family . Comparison of the phenogram and phylogram revealed the contradiction of the phenogram pattern and the phylogenetic placement of the isolates. The isolates belonging to same species have shown considerable phenotypic variation. The study on the cultivable haloalkaliphilic bacteria of an unexplored enigmatic niche reflects ecological and biotechnological significance.

Two Gram-stain-positive, rod-shaped and endospore-forming bacteria that represent a single species, designated strains KJ1-10-99 and KJ1-10-93, were isolated from a saline desert of Little Rann of Kutch, Gujarat, India. Analysis of 16S rRNA gene sequences revealed that the isolates belonged to the family Bacillaceae and were closely related to each other with 16S rRNA gene sequence similarity of 99.9 %. However, these two isolates formed a novel phylogenetic branch within this family. Both strains were aerobic, catalase and oxidase positive, and could grow optimally at 37 °C and pH 9. Further, strains KJ1-10-99 and KJ1-10-93 grew optimally at a NaCl concentration of 7.5 and 15 % (w/v), respectively. Both strains shared highest sequence similarity with Fermentibacillus polygoni IEB3 (96.90 %) followed by Bacillus nanhaiisediminis NH3 (96.3 %) and Bacillus alkalinitrilicus ANL-iso4 (96.3 %). The major cellular fatty acids were anteiso-C15 : 0, anteiso-C17:0, C16 : 0, and iso-C15 : 0. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol in both strains. The predominant isoprenoid quinone was MK-7 in both the strains. The peptidoglycan contained meso-diaminopimelic acid (meso-DAP) as the diagnostic diamino acid. The DNA G+C content of strains KJ1-10-99 and KJ1-10-93 were 48.7 and 48.9 mol% respectively. Both strains could be distinguished from closest phylogenetic neighbours based on a number of phenotypic properties. On the basis of polyphasic taxonomic analysis and phylogenetic data, we conclude that the strains KJ1-10-99 (=LMG 29918=KCTC 33878) and KJ1-10-93 (=LMG 29919=KCTC 33877) represent a novel species of a new genus in the family Bacillaceae, order Bacillales, for which the name Desertibacillus haloalkaliphilus gen. nov., sp. nov. is proposed.

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