• Journal of Microbiology and Biotechnology
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• v.13 no.1
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• pp.35-42
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• 2003

A 1,3 kb cellulase gene encoding novel bifunctional cellulase-chitosanase activity was cloned from biopolymer-producing alkali-tolerant B. lichenformis NBL420 in E. coli. A recombinant cellulase-chitosanase, named CelA, was expressed and purified to homogeneity. The activity staining and the enzymatic characterization of the purified CeIA revealed bifunctional activities on carboxymethyl cellulose (CMC) and glycol-chitosan. The similar characteristics of the enzymatic activities at the optimum pH, optimum temperature, and thermostability Indicated that CelA used a common catalytic domain with relaxed substrate specificity. A comparison of the deduced amino acids in the N-terminal region revealed that the mature CelA had a high homology with the previously identified bifunctional cellulase-chitosanase of Myxobacter sp. AL- 1.

• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.7-14
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• 2013

In the Bacillus amyloliquefaciens ${\alpha}$-amylase (BAA), the loop (residues 176-185; region I) that is the part of the calcium-binding site (CaI, II) has two more amino acid residues than the ${\alpha}$-amylase from Bacillus licheniformis (BLA). Arg176 in this region makes an ionic interaction with Glu126 from region II (residues 118-130), but this interaction is lost in BLA owing to substitution of R176Q and E126V. The goal of the present work was to quantitatively estimate the effect of ionic interaction on the overall stability of the enzyme. To clarify the functional and structural significance of the corresponding salt bridge, Glu126 was deleted (${\Delta}$E126) and converted to Val (E126V), Asp (E126D), and Lys (E126K) by site-directed mutagenesis. Kinetic constants, thermodynamic parameters, and structural changes were examined for the wild-type and mutated forms using UV-visible, atomic absoption, and fluorescence emission spectroscopy. Wild-type exhibited higher $k_{cat}$ and $K_m$ but lower catalytic efficiency than the mutant enzymes. A decreased thermostability and an increased flexibility were also found in all of the mutant enzymes when compared with the wild-type. Additionally, the calcium content of the wild-type was more than ${\Delta}E126$. Thus, it may be suggested that ionic interaction could decrease the mobility of the discussed region, prevent the diffusion of cations, and improve the thermostability of the whole enzyme. Based on these observations, the contribution of loop destabilization may be compensated by the formation of a salt bridge that has been used as an evolutionary mechanism or structural adaptation by the mesophilic enzyme.

• Microbiology and Biotechnology Letters
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• v.4 no.4
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• pp.166-178
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• 1976

Taxonomic study of 11 strains of Bacillus coagulans and 14 strains of 13 spccies of Bacillus by deoxyribonucleic acid (DNA)-DNA hybridization were conducted. Among the 11 strains of B. coagulans, 6 were isolated from soil and the rest were the authentic strains obtained from American Type culture collection (ATCC) or the Institute for Fermentation, Osaka (IFO). All strains were examined to confirm as they are expected species of B. coagulans by the methods of Cordon et al. according to Bergey's Manual (8th ed.). The intraspecific DNA homology indexes among the 11 strains of B. coagulans using strain ATCC 7050 as the standard ($^3$H labeled input DNA) showed 76％ or, more, respectively. These findings accorded well with the results of the conventional taxonomic study according to the Bergey's Manual. The interspecific DNA homology indexes between B. coagulant strain ATCC 7050 and the type cultures of B. subtilis (168), B. licheniformis (IFO 12107), B. pumilus (IFO 12110), B. firmus (ATCC 14575), B. lentus (ATCC 10840), B. circulans (ATCC 4513), B. macelans (ATCC 8244), B. polymyxa (ATCC 842), B. sphaericus (ATCC 14577), B. brevis (ATCC 8246, IFO 12334), B. laterosporus (ATCC 64), and B. pantothenticus (ATCC 14576) respectively, showed 2 to 4％, while that of between B. coagulans ATCC 7050 and Escherichia coli K-12 was less than 1 ％.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.167-172
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• 2000

Twenty-five halotolerant gram-positlve bacteria were isolated from the coastal seawater 01 Cheju Island and Incheon J&yakdo Chemosystematic and phenotypic characteristics were used to iuvestigate the taxonomic position of these bacteria. According to their chemosystematic characteristics, the twenty-tive isolates were divided into 4 groups. Group 1 bacteria possesed 40.1 to 49.9 inol% m DNA G+C content, menaquinone-7 as a major quinone, and meso-Alpm as a diamino acid of peptidoglycan. Group 1 tam were identified as Bacilluspumilis, Bacillus lichenifbrrizis, Bacillus megaterium, Bncill~rsubtilis. Group 2 bacteria possessed 63.9 to 66.4 mol% and MK-8. They were all in the genus Arth~obaclm-. Group 3 bacteria possessed 31.0 to 37.6 mol% and MK-7. They were identified as Staphylococcus haeniol.viicvs, Siaph~~lococc~is sapropl~j~ticns, and Siaphylococcus intermediru.. Group 4 bacterium possessed 72.0mol% and MK-8 and was identified as ~Lficrococcus ltdtezm. Bacillus species accounted for 68% of h e total isolates.

• Journal of the Korean Society of Food Science and Nutrition
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• v.35 no.8
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• pp.973-978
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• 2006

This study was conducted to investigate the solubility, antioxidative and antimicrobial activity of the freeze dried chitosan-ascorbate (CAs) and chitosan-acetate (CAc). In the results of solubility, CAs was soluble over 0.5% in distilled water, vinegar, green tea, soju (distilled liquor), beer and red wine, while it was not soluble in soy sauce, soy milk, milk, orange juice, coffee, sesame oil, soy milk and soybean oil. The solubility of CAc in the liquid foods was similar to those of CAs, but it was soluble less than 0.1% in beer, and formed curd in red wine. Electron donating activity, antioxidative activity and SOD activity of CAs were 48.2, 90.6 and 67.5%, respectively, while the activities of the CAc were 0, 40.0 and 10.0%, respectively. The minimal inhibitory concentrations of CAs and CAc were $200\;{\mu}g/disc$ against Bacillus circulans, Bacillus brevis, Bacillus licheniformis, Bacillus arabitane and Bacillus sterothermophillus, $400\;{\mu}g/disc$ against Escherichia coli O157, Listeria monocytogenous, Bacillus cereus and Bacillus subtilis. There was no significant difference in Hunter's L* value between CAs and CAc $(81.95{\sim}82.97)$, but Hunter's a* and b* values of the CAs was higher than those of CAc. While sour and bitter tastes of CAs were lower than those of CAc, there was no significant difference in astringent taste. From these results, it suggested that CAs has more extensive utility in liquid foods with antimicrobial and antioxidant activity as well as sensory quality compared to CAc.

• Proceedings of the Microbiological Society of Korea Conference
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• 1991.04a
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• pp.225-236
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• 1991

The genes encoding the thermostable $\alpha$-amylase and maltogenic amylase from Bacillus lichenciformis were cloned and expressed in E. coli. The recombinant plasmid pTA322 was found to contain a 3.1kb EcoRI genomic DNA fragment of the thermostable $\alpha$-amylase. The cloned $\alpha$-amylase was compared with the B. licheniformis native $\alpha$-amylase. Both $\alpha$-amylase have the same optimal temperature of $70^{\circ}C$ and are stable in the pH range of 6 and 9. The complete nucleotide sequences of the thermostable $\alpha$-amylase gene were determined. It was composed of one open reading rame of 1,536 bp. Start and stop codons are ATG and TAG. From the amino acid sequence deduced from the nucleotide sequence, the cloned thermostable $\alpha$-amylase is composed of 483 amino acid residues and its molecular weight is 55,200 daltons. The content of guanine and cytosine is $47.46mol\%$ and that of third base codon was $53_41mol\%$. The recombinant plasmid, pIJ322 encoding the maltogenic amylase contains a 3.5kb EcoRI-BamHI genomic DNA fragment. The optimal reaction temperature and pH of the maltogenci amylase were $50^{\circ}C$ and 7, respectively. The maltogenic amylase was capable of hydrolysing pullulan, starch and cyclodextrin to produce maltose from starch and panose from pullulan. The maltogenic amylase also showed the transferring activity. The maltogenic amylase gene is composed of one open reading frame of 1,734bp. Start and stop codons are ATG and ATG. At 2bp upstream from start codon, the nucleotide sequence AAAGGGGGAA seems to be the ribosome-binding site(RBS, Shine-Dalgarno sequence). A putative promoter(-35 and-10 regions) was found to be GTTAACA and TGATAAT. From deduced amino acid sequence from the nucleotide srquence, this enzyme was comosed of 578 amino acid residues and its molecular weight was 77,233 daltons. The content of guanine and cytosine was $48.1mol\%$. The new recombinant plasmid, pTMA322 constructed by inserting the thermostable $\alpha$-amylase gene in the EcoRI site of pIJ322 to produce both the thermostable $\alpha$-amylase and the maltogenic amylase were expressed in the E. coli. The two enzymes expressed from E. coli containing pTMA322 was reacted with the $15\%$ starch slurry at $40^{\circ}C$ for 24hours. The distribution of the branched oligosaccharides produced by the single-step process was of the ratio 50 : 50 between small oligosaccharide up DP3 and large oligosaccharide above DP3.

• KSBB Journal
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• v.23 no.2
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• pp.125-130
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• 2008

We investigated the biological activities of ethanol extract from the seawater algae, Chlorella elliposidea C020 such as antibacterial activity, anti-oxidant activity, tyrosinase inhibitory activity and hemolytic activity against human erythrocytes. Extract was obtained from various solvent, methanol, ethanol, acetone and ethanol + acetone (1:1, v/v%), 95% ethanol proved to be best extraction solvents. The contents of ethanol extract were higher in freeze-dried sample than that in frozen-thawing. Antibacterial activities of ethanol extract showed strong inhibitory effect against Bacillus subtilis PM125, Bacillus licheniformis and fish pathogenic bacteria, Vibrio parahaemolyticus KCTC2471 and Edward tarda NUF251. However, this extract didn't worked against antifungal activity against Candida albicans KCTC1940. And, ethanol extract was without hemolytic activity against human erythorocytes. The ethanol extract showed 75% of free radical scavanging effect on 2.0 mg/mL using DPPH method. In tyrosinase inhibition assay of ethanol extract, $IC_{50}$ (Inhibition Concentration) was measured as 10.87 mg/mL. Conclusionally, ethanol extract of Chlorella elliposidea C020 has good candidate for bioactive materials.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.433-473
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• 1988

Two isolates of C-7 and S-34, which were identified as Bacillus licheniformis and Enterobacter cloacae, were shown the highest phytase productivities among the 23 and 44 strains isolated from the fermenting corn and soybean meals, respectively. The phytase productivity with B. lichenifrmis was maximized at pH 6.0, 3$0^{\circ}C$ after 5 days of incubation and E. cloacae was maximized at pH 1.0, 35$^{\circ}C$ after 5 days of incubation. The bacterial phytase productivity with each bacterium was significantly increased or decreased by the addition of various concentrations of 6 carbon and 7 nitrogen sources including glucose, sucrose, KNO$_3$, and NH$_4$Cl.

• Journal of Animal Science and Technology
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• v.63 no.6
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• pp.1314-1327
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• 2021

Bacillus is characterized by the formation of spores in harsh environments, which makes it suitable for use as a probiotic for feed because of thermostability and high survival rate, even under long-term storage. This study was conducted to investigate the effects of Bacillus-based probiotics on growth performance, nutrient digestibility, intestinal morphology, immune response, and intestinal microbiota of weaned pigs. A total of 40 weaned pigs (7.01 ± 0.86 kg body weight [BW]; 28 d old) were randomly assigned to two treatments (4 pigs/pen; 5 replicates/treatment) in a randomized complete block design (block = BW and sex). The dietary treatment was either a typical nursery diet based on corn and soybean meal (CON) or CON supplemented with 0.01% probiotics containing a mixture of Bacillus subtilis and Bacillus licheniformis (PRO). Fecal samples were collected daily by rectal palpation for the last 3 days after a 4-day adaptation. Blood, ileal digesta, and intestinal tissue samples were collected from one pig in each pen at the respective time points. The PRO group did not affect the feed efficiency, but the average daily gain was significantly improved (p < 0.05). The PRO group showed a trend of improved crude protein digestibility (p < 0.10). The serum transforming growth factor-β1 level tended to be higher (p < 0.10) in the PRO group on days 7 and 14. There was no difference in phylum level of the intestinal microbiota, but there were differences in genus composition and proportions. However, β-diversity analysis showed no statistical differences between the CON and the PRO groups. Taken together, Bacillus-based probiotics had beneficial effects on the growth performance, immune system, and intestinal microbiota of weaned pigs, suggesting that Bacillus can be utilized as a functional probiotic for weaned pigs.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.230-233
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• 2002

Superoxide is involved in causing inflammation, cancer, and arteriosclerosis in many cases. Taking antioxidant material can be helpful in preventing the diseases. Natural food such as barley, wormwood, sea tangle, and soybean contain antioxidant ingredients. Antioxidant activity increase was determined by fermenting them with microorganism. To determine the activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution was used. When barley, wormwood, sea tangle, and soybean were fermented with Bacillus lichenifomis Bl, antioxidant activities of each fermented product increased 2.6, 1.6, 2.7, and 1.7 folds, respectively. Also, absorbance of fermented soybean was higher than that of soybean at the range of 250~290nm, which might be involved in differences of antioxidant activity of the two. Paraquat suppressed Esherichia coli DH5$\alpha$ growth by making superoxide inside the strain. However, when ethanol extract from fermented soybean was added into the GM (glucose-mineral) media containing the strain, its growth was recovered, suggesting that ethanol extract can move across E. coli, and can function as anti-oxidant material in vivo. Thus, it will be possible to develope antioxidant material from fermented soybean which can be taken orally.