Genome, physicochemical properties and esterase gene cloning of novel lentibacillus and halobacillus species
Authors:   , Booncharoen Auttaporn 
Published: September 2023

A total of 831 moderately halophilic bacteria isolated from various samples in Thailand were screened for their lipolytic activity. Three hundred and forty-nine isolates showed activity on agar plate while 322 isolates exhibited in broth. The grouping and identification of 322 isolates based on DNA-fingerprinting using (GTG)5+ERIC2 primer and 16S rRNA gene sequence, 180 DNA patterns could be generated. The result indicated that the representative isolates belonged to genera Bacillus, Halobacillus, Lentibacillus, Marinobacter, Thallossobacillus, and Virgibacillus. On the basis of polyphasic taxonomic study and genome analysis, a selected moderately halophilic strain SSKP1-9T, Gram-staining-positive, aerobic, endospore-forming, rod-shaped was closely related to Lentibacillus juripiscarius TISTR 1535T and L. halophilus TISTR 1549T, 98.7 and 97.2%, respectively based on 16S rRNA gene sequence similarity. On the basis of these results, the strain represents a novel species of the genus Lentibacillus, for which the name Lentibacillus lipolyticus is proposed (SSKP1-9T=JCM 32625T=TISTR 2597T). A Gram-staining-positive, aerobic, endospore-forming, rod-shaped, strain SKP4-6T was closely related to Halobacillus salinus JCM 11546T, H. locisalis KCTC 3788T and H. yeomjeoni KCTC 3957T, with 98.6% respectively, based on 16S rRNA gene sequence similarity. On the basis of these results taxonomy and whole-genome analysis, strain SKP4-6T represents a novel species of the genus Halobacillus, for which the name Halobacillus fulvus is proposed (SKP4-6T =JCM 32624T=TISTR 2595T).

Based on whole-genome analysis, strain SSKP1-9T and SKP4-6T contained protein-encoding esterase for 8 and 5 genes, respectively. The genes were then cloned into the pSaltExSePR5 plasmid and transferred to Bacillus subtilis WB800. Two recombinant, >APNFEMBD_02578-C112-40 from strain SSKP1-9T and >NKILIEJB_01195-C9-35 from strain SKP4-6T showed the highest esterase activity and were therefore studied. These two genes identified major matches with closely related to a member of family acyl-CoA thioesterase and the alpha/beta hydrolase fold founded in genus Lentibacillus and Halobacillus, respectively. Of the two recombinant enzymes, the first one has a molecular weight of 16.2 kDa, and the other was 27.7 kDa, identical to the predicted protein size from the amino acid sequence. Two recombinant enzymes reached the maximum activity at 15-30% NaCl, and the highest activity was shown at 22 % NaCl. The recombinant >APNFEMBD_02578-C112-40 displayed the activity around 25-60 °C, and the maximum activity was observed at 40-55 °C while >NKILIEJB_01195-C9-35 had optimal activity at 55 °C and lost its activity rapidly above 65 °C of the remaining activity. They stable in NaCl concentration above 17% (w/v) after incubated at 55 °C for 1 hr. Two enzymes showed good stability and remained activity about 80% in a temperature range of 5-45 °C after 1 hr of incubation at 50 °C with or without NaCl. Theses enzymes reached high activity over a wide range of pH from 6.0 to 9.0, recording maximal activity at pH 7.0-8.0 and displayed excellent stable in with or without NaCl at various pH ranges (pH 6.0 to 9.0). Besides, the two recombinant enzymes were found to have higher esterase activity against p-nitrophenyl butyrate (C4:0) comparing to other esters substrate. Based on finding results, two recombinant enzymes were classified as halophilic and thermophilic esterase