Alkaline proteases are important enzymes in many industrial applications, especially as additives in laundry detergent industry. Though there are a number of Bacillus species which are reported to be producing proteases, the efficiency of a protease produced by a novel strain has to be studied in comparison to the others. Hence, in this study, an alkaline serine protease produced by a novel species Bacillus caseinilyticus was purified and characterized for its possible usage in detergent industry. Ammonium sulphate, dialysis and DEAE column chromatographic methods were used for purification of the isolated alkaline protease. The molecular weight of the protease was determined by SDS-PAGE and it was found to be 66 kDa. Peptide mass fingerprinting (PMF) was carried out using MALDI-TOF-TOF mass spectrometry and the peptides were found to be similar to that of subtilisin protease. Specific activity of purified protein was found to be 89.2 U/mg. Optimum pH and temperature for enzyme activity were at pH 8 and 60 °C, respectively, showing stability with 10 mM CaCl2. Phenyl methyl sulphonyl fluoride (PMSF) at both 5 and 10 mM concentrations completely inhibited the enzyme activity suggesting its serine nature. EDTA, metal ions Mg2+ and Ca2+ increased the enzyme activity. The one factor at a time optimisation of the protease production was carried to identify the important factors that affect its production. After optimisation, the protease was produced at lab scale, purified and characterised. This alkali, thermotolerant serine protease was found to be significantly stable in the presence of various surfactants and H2O2. Also, it was successfully able to remove blood stain when used as an additive along with commercial detergent suggesting its potential application in the laundry detergent industry.

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A Gram-stain positive, rod shaped aerobic bacterial strain designated Ka25(T) was isolated from a forest soil sample near Kasol, Himachal Pradesh, India. Based on 16S rRNA gene sequence analysis it was identified that strain Ka25(T) belongs to the class Actinobacteria and is most closely related to Nocardioides daejeonensis KCTC 19772(T) (97.2 %), Nocardioides dubius JCM 13008(T) (96.9 %), Nocardioides psychrotolerans CGMCC 1.11156(T) (96.8 %), Nocardioides pacificus DSM 27278(T)(96.7 %) and Nocardioides daphniae JCM 16608(T)(96.4 %) sequence similarity respectively. The chemotaxonomic properties of strain Ka25(T) are consistent with those of the members of the genus Nocardioides: MK-8(H4) is the predominant menaquinone, iso-C16 : 0, iso-C17 : 0 and C18 : 1ω9c were identified as the predominant cellular fatty acids and the cell-wall peptidoglycan is based on LL-2,6-diaminopimelic acid. The DNA G+C content of strain Ka25(T) was determined to be 71.6 mol %. The results of the polyphasic taxonomic analysis allowed a clear differentiation of strain Ka25(T) from all other members of the genus Nocardioides. Strain Ka25(T) is thus considered to represent a novel member of the genus Nocardioides, for which the name Nocardioides solisilvae sp. nov. is proposed. The type strain is Ka25(T) (=KCTC39528(T) =MCC2762(T)).

Two novel (18C and 6C) Gram-stain-positive, rod shaped, motile and endospore-forming bacterial strains were isolated from Lonar soda lake, India. Based on 16S rRNA gene sequence analysis, strains 18C and 6C were identified as belonging to the class Firmibacteria, and were most closely related to Bacillus cohnii KCTC 3572 (99.3 and 99.9%, respectively), Bacillus zhanjiangensis KCTC 13713 (97.4 and 98.0%, respectively), Bacillus halmapalus LMG 17950 (97.0 and 97.6%, respectively) and other members in the genus Bacillus (<97.0%). However, the DNA-DNA relatedness between 18C and 6C and B. cohnii KCTC 3572 (49.6 ± 0.9 and 51.6 ± 0.7, respectively), B. zhanjiangensis KCTC 13713 (42.9 ± 0.8 and 47.1 ± 0.3, respectively) and B. halmapalus LMG 17950 (39.9 ± 0.8 and 40.8 ± 0.3, respectively) indicated that the novel strains were distantly related to these strains. Further, the high 16S rRNA gene sequence similarity (100%) and DNA-DNA relatedness (90 ± 5%) suggested that strains 18C and 6C were members of a genomospecies. The strains grew optimally at a pH of 7.5 with 2-3% (w/v) NaCl and temperature of 37 °C. Strains 18C and 6C were catalase and oxidase negative. The cell wall of strain 18C contained meso-diaminopimelic acid as the diagnostic diamino acid, which was in contrast with its nearest neighbour B. cohnii KCTC 3572, which contained ornithine and aspartic acid. Polar lipids include diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), an unknown phospholipid (PL) and three unknown lipids (L1-3). The predominant isoprenoid quinone was MK-7. iso-C (32.5%) was the predominant fatty acid and significant proportions of anteiso-C (19.5%), C (11.5%), iso-C (9.5%) and anteiso-C (6.3%) were also detected. The DNA G + C content of strains 18C and 6C were 39.3 and 39.2 mol%, respectively. The results of molecular, biochemical and chemotaxonomic tests showed a clear differentiation of strains 18C and 6C from all other members of the genus Bacillus, for which the name Bacillus catenulatus sp. nov. is proposed. The type strain is 18C (=KCTC 33781 = CGMCC 1.15475).

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