• Journal of Microbiology and Biotechnology
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• v.33 no.2
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• pp.195-202
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• 2023

Protease is a widely used enzyme particularly in the detergent industry. In this research, we aimed to isolate alkaline protease-producing bacteria for characterization as a laundry detergent additive. The screening of alkaline protease production was investigated on basal medium agar plus 1% skim milk at pH 11, with incubation at 30℃. The highest alkaline protease-producing bacterium was 6BS15-4 strain, identified as Bacillus gibsonii by 16S rRNA gene sequencing. While the optimum pH was 12.0, the strain was stable at pH range 7.0-12.0 when incubated at 45℃ for 60 min. The alkaline protease produced by B. gibsonii 6BS15-4 using dairy effluent was characterized. The optimum temperature was 60℃ and the enzyme was stable at 55℃ when incubated at pH 11.0 for 60 min. Metal ions K⁺, Mg²⁺, Cu²⁺, Na⁺, and Zn²⁺ exhibited a slightly stimulatory effect on enzyme activity. The enzyme retained over 80% of its activity in the presence of Ca²⁺, Ba²⁺, and Mn²⁺. Thiol reagent and ethylenediaminetetraacetic acid did not inhibit the enzyme activity, whereas phenylmethylsulfonyl fluoride significantly inhibited the protease activity. The alkaline protease from B. gibsonii 6BS15-4 demonstrated efficiency in blood stain removal and could therefore be used as a detergent additive, with potential for various other industrial applications.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.197-208
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• 2014

A novel protease gene from Bacillus gibsonii, aprBG, was cloned, expressed in B. subtilis, and characterized. High-level expression of aprBG was achieved in the recombinant strain when a junction was present between the promoter and the target gene. The purified recombinant enzyme exhibited similar N-terminal sequences and catalytic properties to the native enzyme, including high affinity and hydrolytic efficiency toward various substrates and a superior performance when exposed to various metal ions, surfactants, oxidants, and commercial detergents. AprBG was remarkably stable in 50% organic solvents and retained 100% activity and stability in 0-4 M NaCl, which is better than the characteristics of previously reported proteases. AprBG was most closely related to the high-alkaline proteases of the subtilisin family with a 57-68% identity. The secretion and maturation mechanism of AprBG was dependent on the enzyme activity, as analyzed by site-directed mutagenesis. Thus, when taken together, the results revealed that the halo-solvent-tolerant protease AprBG displays significant activity and stability under various extreme conditions, indicating its potential for use in many biotechnology applications.

• Journal of Species Research
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• v.7 no.2
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• pp.114-122
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• 2018

As part of a larger study of indigenous prokaryotic species diversity in South Korea, various samples from Namhangang were subjected to analyses. Fresh water, underwater sediment, and moss-inhabiting aerobic and anaerobic bacteria were isolated. 22 of the isolates were identified as unrecorded bacterial species in Korea that had ${\geq}98.7%$ 16S rRNA gene sequence similarity with published species. The aerobic strains isolated were Kurthia gibsonii and Massilia plicata. Also identified were four facultative anaerobic strains: Bacillus hisashii, Enterococcus rotai, Paenibacillus vini, and Pediococcus pentosaceus. 16 strictly anaerobic strains were identified as Bacteroides xylanolyticus, Carnobacterium maltaromaticum, Clostridium argentinense, Clostridium beijerinckii, Clostridium butyricum, Clostridium cavendishii, Clostridium diolis, Clostridium frigidicarnis, Clostridium perfringens, Clostridium saccharoperbutylacetonicum, Clostridium sphenoides, Clostridium subterminale, Cutibacterium acnes, Paraclostridium bifermentans, Prevotella paludivivens, and Romboutsia lituseburensis. Based on the examination of morphological, cultural, physiological, and biochemical properties of the isolates, descriptive information of these previously unrecorded species is provided here.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.9
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• pp.1304-1312
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• 2013

The effects of storage period and aerobic deterioration on the bacterial community were examined in Italian ryegrass (IR), guinea grass (GG), and whole-crop maize (WM) silages. Direct-cut forages were stored in a laboratory silo for 3, 7, 14, 28, 56, and 120 d without any additives; live counts, content of fermentation products, and characteristics of the bacterial community were determined. 2,3-Butanediol, acetic acid, and lactic acid were the dominant fermentation products in the IR, GG, and WM silages, respectively. The acetic acid content increased as a result of prolonged ensiling, regardless of the type of silage crop, and the changes were distinctively visible from the beginning of GG ensiling. Pantoea agglomerans, Rahnella aquatilis, and Enterobacter sp. were the major bacteria in the IR silage, indicating that alcoholic fermentation may be due to the activity of enterobacteria. Staphylococcus sciuri and Bacillus pumilus were detected when IR silage was spoiled, whereas between aerobically stable and unstable silages, no differences were seen in the bacterial community at silo opening. Lactococcus lactis was a representative bacterium, although acetic acid was the major fermentation product in the GG silage. Lactobacillus plantarum, Lactobacillus brevis, and Morganella morganii were suggested to be associated with the increase in acetic acid due to prolonged storage. Enterobacter cloacae appeared when the GG silage was spoiled. In the WM silage, no distinctive changes due to prolonged ensiling were seen in the bacterial community. Throughout the ensiling, Weissella paramesenteroides, Weissella confusa, and Klebsiella pneumoniae were present in addition to L. plantarum, L. brevis, and L. lactis. Upon deterioration, Acetobacter pasteurianus, Klebsiella variicola, Enterobacter hormaechei, and Bacillus gibsonii were detected. These results demonstrate the diverse bacterial community that evolves during ensiling and aerobic spoilage of IR, GG, and WM silages.

• Journal of Dairy Science and Biotechnology
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• v.28 no.2
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• pp.7-11
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• 2010

In recent times, antimicrobial resistance has been a concern because of its relation to national health and food safety. In this study, we reviewed the management of antibiotics and antimicrobial-resistant microorganisms in domestic and foreign countries and analyzed microorganisms and antimicrobial resistance in dairy farms and raw milk. The isolates from dairy farms were Micrococcus spp., Staphylococcus chromogens, Escherichia coli, Bacillus licheniformis, Escherichia coli O157:H7, Pseudomonas plecoglossicida, Enterobacter spp., and Kluyvera intermedia. Rummeliibacillus stabekisii, Paenibacillus badius, Pantoea agglomerans, B. oleronius, B. fusiformis, and B. badius were isolated from feed E. coli and Kurthia gibsonii, from feces and S. pasteuri, S. aureus, S. chromogenes, and Salmonella spp., from raw milk. Pathogens isolated from dairy farms and raw milk were tested for susceptibility to 20 types of antibiotics. E. coli (EAEC) and E. coli O157:H7 (EHEC) isolated from dairy farms, E. coli (EAEC) isolated from feces, and Salmonella spp. isolated from raw milk showed resistance to multiple antibiotics. These results show that antimicrobial-resistant microorganisms should be more effectively managed to improve the safety of dairy farms.