• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1054-1059
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• 2005

An extracellular protease was identified from Bacillus subtilis KCCM 10257 by N-terminal sequencing and mass spectral analysis. The molecular mass of the extracellular protease was estimated to be 28 kDa by SDS-PAGE. Sequencing of the N-terminal of the protease revealed the sequence of A(G,S,R)QXVPYG(A)V(P,L)SQ. The N-terminal sequence exhibited close similarity to the sequence of other proteases from Bacillus sp. A mass list of the monoisotopic peaks in the MALDI-TOF spectrum was searched after peptide fragmentation of the protease. Six peptide sequences exhibiting monoisotopic masses of 1,276.61, 1,513.67, 1,652.81, 1,661.83, 1,252.61, and 1,033.46 were observed from the fragmented protease. These monisotopic masses corresponded to the lytic enzyme L27 from Bacillus subtilis 168, and the Mowse score was found to be 75. A doubly charged Top product (MS) at a m/z of 517.3 exhibiting a molecular mass of 1034.6 was further analyzed by de novo sequencing using a PE Sciex QSTAR Hybrid Quadropole-TOF (MS/MS) mass spectrometer. MS/MS spectra of the Top product (MS) at a m/z of 517.3 obtained from the fragmented peptide mixture of protease with Q-star contained the b-ion series of 114.2, 171.2, 286.2, 357.2, 504.2, 667.4, 830.1, and 887.1 and y-ion series of 147.5, 204.2, 367.2, 530.3, 677.4, 748.4, 863.4, and 920.5. The sequence of analyzed peptide ion was identified as LGDAFYYG from the b- and y-ion series by de novo sequencing and corresponded to the results from the MALDI-TOF spectrum. From these results the extracellular protease from Bacillus subtilis KCCM 10257 was successfully identified with the lytic enzyme L27 from Bacillus subtilis 168.

• Journal of Aquaculture
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• v.19 no.1
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• pp.40-46
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• 2006

Bacillus subtilis strain with highly active chitosanase was isolated from the intestine of Sebastiscus marmoratus (scorpion fish). It was named as Bacillus subtilis CH1 by morphological, biochemical and 165 rRNA gene analysis. The optimal conditions for chitosanase production were investigated. The optimum carbon and nitrogen sources for Bacillus stibtilis CH1 were 2% starch and 1% yeast extract respectively. Unlike other chitosanases, the expression of this chitosanase was not induced or slightly induced with chitosan. The chitosanase secreted into the medium were concentrated with ammonium sulfate precipitation and purified by gel permeation chromatography. The molecular weight of purified chitosanase was 30 kDa. The optimum pH and temperature of purified chitosanase were 5.5 and $60^{\circ}C$ respectively. The purified chitosanase was continuously thermostable at $40^{\circ}C$ and showed stable activity between pH 6.0 and 8.0. Chitosanase activity of Bacillus subtilis CH1 under optimum condition was 4.1 units/ml.

• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.580-587
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• 1995

In order to produce the bacteriolytic enzyme, bacterial strains capable of excreting a large amount of the enzyme were screened from the coastal sea water samples in Pusan. The bacterial strain SH-1, which showed the highest activity among 43 bacteriolytic enzyme producing bacteria, was finally selected for further studies. The strain SH-1 was an endospore-forming grampositive rod, and the position of spore was paracentral. These morphological characteristics assigned the isolated strain to the morphological group I classified by Gordon. The fatty acid composition of the bacterial stain was analyzed to be consisted of branched chains of iso-Cn and anteiso-Cn. Based on the percent content of the branched chain (93.85%), the isolates could be identified as a species of Bacillus. According to the experimental results of the API system (API 50CHB & API 20E) the strain was identified as Bacillus subtilis. Numerical texonomy, in which 82 major characters were examined using several species of Bacillus as the standard bacteria, indicated that the strain SH-1 showed 90% similarity to Bacillus subtilis. Thus, the isolated strain SH-1 could be identified as Bacillus subtilis.

• Microbiology and Biotechnology Letters
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• v.24 no.3
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• pp.299-303
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• 1996

Isolation, identification, and culture conditions of a lytic enzyme producing microorganism against Lacto- bacillus plantarum were investigated. The selected strain was gram-positive, rod (0.7 $\times$ 2.7 $\mu$m in size), and non-motile. The strain did not have any flagella and spores. According to its cultural and physiological characteristics, the strain was identified as Bacillus sp. The optimal pH and temperature for the production of lytic enzyme were 8.0 and 30$\circ$C, respectively. The maximum enzyme activity showed 1.5 units/ml in the medium composed of 1% peptone and 0.1% NaCl.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.599-606
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• 2004

Gram-positive antagonistic bacilli were isolated from agricultural soils for possible use in biocontrol of plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and/or Pythium ultimum. Among the 65 antagonistic Gram-positive soil isolates, 22 strains were identified as Bacillus species by 16S rDNA sequence analyses. Four strains, including DF14, especially exhibited multiple antagonistic properties against the three damping-off fungi. Genotypic properties of the Bacillus isolates were characterized by rapid molecular fingerprinting methods using repetitive extragenic palindromic-PCR (REP-PCR), ribosomal intergenic spacer-length polymorphisms (RIS-LP), 16S rDNA PCR-restriction fragment length polymorphisms (PCR-RFLP), and strain-specific PCR assays. The results indicated that the REP-PCR method was more valuable than the RIS-LP and 16S rDNA PCR-RFLP analyses as a rapid and reliable approach for bacilli typing and identification. The use of strain-specific primers designed based on 16S rDNA sequence comparisons enabled it to be possible to selectively detect a strain, DF14, which is being used as a biocontrol agent against damping-off fungi.

• Korean Journal of Veterinary Service
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• v.39 no.1
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• pp.1-6
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• 2016

In this study, Bacillus licheniformis which has been used as probiotics was isolated from Korean traditional fermented soybean. A total of 69 strains were presumptively identified as B. licheniformis by phenotypic methods. Based on PCR amplification and 16S rRNA gene sequencing, the multilocus sequence typing of gyrA and rpoB, followed by phylogenetic analysis was performed. The isolates were distinctly differentiated and found to be closely related to B. amyloliquefaciens, B. subtilis, and B. aerius. The partial 16S rRNA gene sequences of those strains matched those of B. sonorensis (97%) and B. aerius (98%) in the phylogenetic tree. In contrast, multilocus phylogenetic analysis (MLPA) showed that only 61 (86.9%) out of 69 strains were B. licheniformis. The rest of those strains were found to be B. subtilis (5.8%), B. amyloliquefaciens (2.9%), and B. sonorensis (2.9%), respectively. Therefore, our results suggested that since the 16S rRNA gene sequencing alone was not sufficient to compare and discriminate closely related lineages of Bacillus spp., it was required to analyze the MLPA simultaneously to avoid any misleading phenotype-based grouping of these closely related species.

• Journal of the Korean Applied Science and Technology
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• v.37 no.6
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• pp.1535-1544
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• 2020

The primary objective of this study was to isolate and identify amine oxidase-producing probiotic Bacillus strains from traditional fermented soybean paste. Biogenic amines (BA)-forming bacteria isolated from the samples were identified as Bacillus sp. TS09, Bacillus licheniformis TS17, Bacillus subtilis TS19, Bacillus cereus TS23, Bacillus sp. TS30, Bacillus megaterium TS31, B. subtilis TS44, Bacillus coagulans TS46 and Bacillus amyloliquefaciens TS59. Meanwhile, B. subtilis TS04 and TS50 isolated from the same samples exhibited good probiotic properties, including the tolerance to artificial gastric juice and bile salts, the adhesion to intestinal epithelial cells, and the production of bacteriocin(s) active against BA-forming bacteria (Bacillus sp. TS30 and B. subtilis TS44). In addition, the amine oxidase produced by B. subtilis TS04 and TS50 significantly decreased the formation of BA, especially cadaverine, putrescine, and tyramine, therefore, these strains could be considered good potential probiotic candidates to prevent or reduce BA accumulation in food products.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.543-547
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• 1991

An alkalophilic microorganism, SH-8, was isolated from soil samples. It was a Grampositive, catalase-positive, spore-forming and motile rod which was capable of growth in aerobic condition at the initial pH 9.0 or above up to 11.0 and between 15 and $42^{\circ}C$ The characteristics of this strain resembled those of the Bacillus group of bacteria. The mutant, Bacillus sp. SH- 8M, was selected from Bacillus sp. SH-8 by U.V. mutagenesis and was able to grow at pH 6.9 up to 11.0. These two strains will be suitable for the comparative study on growth and enzyme production at various pH conditions.

• Fisheries and Aquatic Sciences
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• v.22 no.8
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• pp.17.1-17.9
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• 2019

Bacillus is a diverse genus consisting of more than 200 species with extensive genetic diversity. Their beneficial effects in industrial shrimp farming have been well documented. However, little is known about the biodiversity of the Bacillus spp. in this aquaculture system. Taxonomic analysis by 16S rRNA sequencing does not always allow species-level identification of Bacillus spp. In this study, 26 Bacillus isolates from two industrial Litopenaeus vannamei shrimp ponds in Bac Lieu Province, Vietnam, were analyzed for their genetic diversity by multi-locus sequence typing (MLST). A total of 22 sequence types were identified and segregated into four distinct clusters, corresponding to B. subtilis, B. velezensis, B. siamensis, and B. licheniformis. Bacillus subtilis and B. velezensis accounted for more than 73% of the Bacillus isolates. Notably, the MLST scheme exhibited high discriminatory power and might be further simplified to be a convenient method to identify species of the genus Bacillus.

• Food Science and Biotechnology
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• v.17 no.3
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• pp.519-526
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• 2008

To improve the quality of traditional kochujang, strains with high protease and amylase activity were isolated and identified from Sunchang traditional kochujang. Twenty-three strains strongly producing protease and 16 strains strongly producing $\alpha$- and $\beta$-amylase were isolated by using 1% isolated soy protein agar medium and 2% starch agar medium, respectively. Protease activities of the IA7, I5, and IA2 strain were 22.5, 21.2, and 20.6 unit/mL, respectively, and were higher than those of the other strains. Stains with high $\alpha$-amylase activity included K9 (967.8 unit/mL), K14 (828.3 unit/mL), K13 (662.5 unit/mL), K8 (601.5 unit/mL), and K11 (405.9 unit/mL). The $\beta$-amylase activity of the K11 strain was the highest, 34.3 unit/mL, among the isolated strains. Based on morphological, physiological properties, and API 50CHB-kit test for assimilation of 49 carbohydrates, 8 strains selected according to protease, $\alpha$-amylase, and $\beta$-amylase activities were tentatively identified as Bacillus megaterium (IA2), Bacillus subtilis (IA7, 15), Bacillus amyloliquefaciens (K8, K9, K11, and K13), and Bacillus stearothermophillus (K14). The IA7, 15, and K11 strains were finally identified as B. subtilis (99% ID) based on 16S rDNA sequencing.