• Microbiology and Biotechnology Letters
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• v.17 no.6
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• pp.587-592
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• 1989

A strain of alkalophilic Bacillus sp. YS-309 capable of producing large amount of $\beta$-galactosidase has been isolated from soil sample. Intracellular $\beta$-galactosidase was purified 6.9 folds by procedures including ammonium sulfate precipitation, DEAE-cellulose chromatography, gel-filtration, DEAE-Sephadex A-50 chromatography with over-all yield of 17.8%. The molecular weight of native enzyme was 205, 000 by HPLC, and SDS-polyacrylamide gel electrophoresis showed that the enzyme consisted of 4 identical subunits with a molecular weight of 56, 000.

• Journal of Microbiology
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• v.34 no.4
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• pp.305-310
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• 1996

A microorganism producing carboxymethyl-cellulase (CMCase) was isolated from 300 soil and compost samples. The isolate was identified as Bacillus sp. by $Biolog^{TM}$ test and fatty acid analysis, and named as Bacillus sp. KD1014. The isolate could degrade, in addition to CMC, various kinds of polysaccharides such as levan, xylan, starch, and filter paper but hardly degrade microcrystalline Avicel. The optimum growth and CMCase production of the isolate was observed between 16-and 25 hr-culture at 45$^{\circ}C$ and pH 5.0. The maximum CMCase activity was observed at pH 4.5 and 6$0^{\circ}C$. The CMCase was found to bind to Avicel. The CMCase was internally cleaved as growth continued. When crude supernatant was used for activity staining, three major bands were detected on a native gel, however, only one major band was detected on a denaturating gel after removal of the detergent.

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.62-69
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• 1999

In order to investigate the critical amino acid residues involved in the catalytic activities of $\beta$-cyclodextrin glucanotransferase ($\beta$-CGTase) excreted by Bacillus firmus var. alkalophilus, the amino acid residues in $\beta$-CGTase were modified by various site-specific amino acid modifying reagents. The cyclizing and amylolytic activities of $\beta$-CGTase were all seriously reduced after treatment with Woodward's reagent K (WRK) modifying aspartic/glutamic acid, N-bromosuccinimde (NBS) modifying tryptophan, and diethylpyrocarbonate (DEPC) modifying histidine residues. The roles of tryptophan and histidine residues in $\beta$-CGTase were further investigated by measuring the protection effect of various substrates during chemical modification, comparing protein mobility in native and affinity polyacrylamide gel electrophoresis containing soluble starch, and comparing the $K_m$ and $V_{max}$ values of native and modified enzymes. Tryptophan residues were identified as affecting substrate-binding ability rather than influencing catalytic activities. On the other hand, histidine residues influenced catalytic ability rather than substrate-binding ability, plus histidine modification had an effect on shifting the optimum pH and pH stability.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.244-250
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• 2000

An ${\alpha}-glucosidase$ gene (aglA) from thermophilic Bacillus sp. DG0303 was cloned, sequenced, and expressed in Escherichia coli. The aglA was localized to the 2.1-kb PvuI-XmnI region within the 5.9-kb DNA insert of the gybrid plasmid pAG1. The gene consisted of an open reading frame of 1,686 bp with an unusual GTG initiation codon and TGA termination codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 562 amino acid residues with a M, of 66,551 dalton. A comparative amino acid sequence analysis revealed that DG0303 ${\alpha}-glucosidase$ is related to bacillary oligo-1, 6-glucosidases. The Bacillus sp. DG0303 ${\alpha}-glucosidase$ showed a high sequence identity (36-59%) to the B. flavocaldarius, B. cereus, and B. thermoglucosidasius oligo-1, 6-glucosidases. The number of prolines in theses four ${\alpha}-glucosidases. was observed to increase with increasing thermostability of these enzymes. The cloned ${\alpha}-glucosidase was purified from E. coli $DH5{\alpha}$ bearing pAG1 and characterized. The recombinant enzyme was identical with the native enzyme in its optimum pH and in its molecular mass, estimated by sodium dodecy1 sulfate-polyacrylamide gel electrophoresis. The temperature optimum of the cloned ${\alpha}-glucosidase$ was lower than that of the native enzyme.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.410-418
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• 2023

Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains of Bacillus subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on the bacABCDEF operon. Examination of the sequence surrounding the upstream of the bac operon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of the bac operon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of the bac operon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.632-637
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• 2000

This research was undertaken to restore the biological activity of cyclodextrin glucanotransferase (CGTase) of Bacillus macerans origin expressed as inclusion bodies in recombinant Escherichia coli. The optimum concentration of urea used as a denaturant was 8 M. The supplementation of 0.5 M urea into a dialysis buffer increased the refolding efficiency by preventing any protein aggregation. The influence of the protein concentration, temperature, and pH were also investigated. The protein concentration was found to be the most important factor in the refolding efficiency. The optimum temperature was 15-$25^{\circ}C$ and the optimum pH was 6.0. The maximum specific activity of the CGTase refolded under the optimum conditions was 92.2 U/mg, corresponding to 72% of the native CGTase. A comparison of the secondary structure between the native and the refolded CGTase showed that the relative ratio of the $\alpha$-helix content in the native to the refolded CGTase was 1:0.82.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.274-282
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• 2019

Mercury-resistant ($Hg^R$) bacteria were isolated from heavy metal polluted wastewater and soil collected near to tanneries of district Kasur, Pakistan. Bacterial isolates AZ-1, AZ-2 and AZ-3 showed resistance up to $40{\mu}g/ml$ against mercuric chloride ($HgCl_2$). 16S rDNA ribotyping and phylogenetic analysis were performed for the characterization of selected isolates as Bacillus sp. AZ-1 (KT270477), Bacillus cereus AZ-2 (KT270478) and Bacillus cereus AZ-3 (KT270479). Phylogenetic relationship on the basis of merA nucleotide sequence confirmed 51-100% homology with the corresponding region of the merA gene of already reported mercury-resistant Gram-positive bacteria. The merE gene involved in the transportation of elemental mercury ($Hg^0$) via cell membrane was cloned for the first time into pHLV vector and transformed in overexpressed C43(DE3) E. coli cells. The recombinant plasmid (pHLMerE) was expressed and the native MerE protein was obtained after thrombin cleavage by size exclusion chromatography (SEC). The purification of fusion/recombinant and native protein MerE by Ni-NTA column, dialysis and fast protein liquid chromatography (FPLC/SEC) involved unfolding/refolding techniques. A small-scale reservoir of wastewater containing $30{\mu}g/ml$ of $HgCl_2$ was designed to check the detoxification ability of selected strains. It resulted in 83% detoxification of mercury by B. cereus AZ-2 and B. cereus AZ-3, and 76% detoxification by Bacillus sp. AZ-1 respectively (p < 0.05).

• Journal of Life Science
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• v.12 no.1
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• pp.77-86
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• 2002

Chitin, a $\beta$-1,4 polymer of N-acetyl-D-glucosamine, is one of the most abundant organic compounds in nature. Chitinase (EC 3.2.1.14) is an enzyme that degrades chitin to chito-oligosaccharides, diacetyl rhitobiose and N-acetyl-D-glucosamine. An extracellular chitinase-producing bacterial strain was isolated from soil and named to as Bacillus subtilis JK-56. Optimum culture condition of B. subtilis JK-56 for the production of chitinase was 1% chitin, 0.5% polypepton, 0.1% KCl, 0.05% MnS $O_4$.4$H_2O$, 37$^{\circ}C$, initial pH 7.0 and 40 hour culture time. When B. subtilis JK-56 was grown in the optimum medium, one major active band and two minor active bands were detected by native-PAGE and active staining of the gel. Among them, the major band was purified from the culture supernatant by 70% ammonium sulfate precipitation and native-PAGE with BIO-RAD Model 491 Prep-Cell and named as Chi-56A. Its molecular weight was estimated to be 53kDa monomer and the isoelectric point (pI) was pH 4.3. The pH and temperature for the optimum activity of Chi-56A were pH 6.0 and $65^{\circ}C$, respectively. Chi-56A was stable up to $65^{\circ}C$ and in alkaline region. Its $K_{m}$ value for colloidal chitin was 17.33g/L. HPLC analysis of the reaction products confirmed that Chi-56A was an exo type chitinase.e.

• Applied Biological Chemistry
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• v.14 no.2
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• pp.137-148
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• 1971

Studies were carried out to investigate the main fermentation microorganisms and their flora changes during Korean native soy-sauce fermentation. Korean native Maeju loaves collected from 5 Do's were separated into surface and inner parts. Four different soy-sauces-the surface part Maeju fermented soy-sauce, the inner part, the surface and inner part combined Maeju fermented soy-sauce, and the semi-Japanese type soy-sauce were fermented and the changes of fermentation microorganism flora and the various chemical components during the period of their fermentations were studied. Besides, 14 home-made soy-sauces collected from 14 different places all over Korea were examined in comparison with the laboratory soy-sauces and to determine the characteristics of Korean native soy-sauce. The results were as follows: 1. The main microorganisms in Korean native soy-sauce fermentation were determined as; Aerobic bacteria: Bacillus subtilis, Bacillus pumilus Lactic acid bacteria: Pediococcus halophilus, Leuconostoc mesenteroides Yeasts: Torulopsis datila, Saccharomyces rouxii 2. Microflora changes during Korean native soy-sauce fermentation were as follows; Aerobic bacteria increased until the 2nd week of fermentation and then gradually decreased. The lactic acid bacteria increased until the 3rd week, after which decreased. When the lactic acid fermentation lowered the pH value to below the 5.4, yeasts were able to grow and participate the fermentation. As the production of organic acids amounted, to a certain height, the growth of all microorganisms lead to the period of decline or death at about the 2nd month of fermentation. After boiling of soy-sauce most microorganisms except a few of Bacillus sp. disappeared. Occosionally yeasts and lactic acid bacteria survived depending upon the composition of soy-sauce. 3. Changes of general chemical components influencing the microflora were investigated for the period of Korean native soy-sauce fermentation. Tetal acidity, salt concentration and total nitrogen were increasing steadily over the entire period of fermentation. pH values were dropping to a certain degree of about 4.5. Salt concentration and pH value seemed to be the important factors influencing the microflora. 4. The microflora were influenced by chemical components of soy-sauce. Aerobic bacteria were able to survive in all soy-sauce as they made spores. Growth of lactic acid bacteria was inhited at 23-26% of salt concentration and pH 4.8. Soy-sauce yeasts started to grow only at pH below 5.4 and seemed to be inhibited at around 26% of salt concentration under pH 4.5-4.7. 5. The open kettle boiling of soy-sauce, the characteristic process of Korean native soy-sauce manufacturing, was effective to sterilize microorganisms, increase the salt concentration, and coagulate proteins. 6. The average viable cell counts of microorganism found in collected samples of home-made Korean native soy-sauces were; Aerobic bacteria: $53{\times}10^2\;cell/ml$ Lactic acid bacteria: 34 cell/ml Yeasts: 14 cell/ml The average values of chemical compositions of samples of home-made Korean native soy-sauce were; Salt concentration: 28.9% pH value: 4.79 Total acidity(lactic acid): 0.91g/100ml Total nitrogen: 1.09g/100ml

• The Korean Journal of Food And Nutrition
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• v.9 no.4
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• pp.447-451
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• 1996

Lytic activities of the egg white lysozyme from Korea-native Ogol fowl against the alkalophilic and thermophilic Bacillus sp. TA-11 were investigated and compared. Lytic activity of the Ogol fowl lysozyme for Bacillus sp. TA-11 was the highest for the cell of post-logarithm phase and optimum concentration of the lysozyme was 0.25%, Optimum reaction pH and temperature were 4.5 and 35$^{\circ}C$, respectively. Lytic activity of egg white lysozyme from fowl for Bacillus sp. TA-11 was the highest for the cell of stationary phase and optimum concentration of the lysozyme was 0.5%. Optimum reaction pH and temperature were 5.5 and 4$0^{\circ}C$, respectively.