• Korean Journal of Microbiology
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• v.44 no.2
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• pp.116-121
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• 2008

In my search of microbial source of novel enzymes, a marine actinomycetes, A1460, producing haloperoxidase was isolated from macroalgae from south sea, Korea and studied for physiological and biochemical properties. The haloperoxidation reaction was followed by the bromination of phenol red in the presence of hydrogen peroxide and potassium bromide. The haloperoxidase was partially purified from the cell extract with $35\sim75%$ ammonium sulfate precipitation, High-Q anion exchange chromatography, gel filtration chromatography, hydroxyapetite chromatography and hydrophobic interaction chromatography to a yield of 42% and purification fold of 70. This enzyme showed relatively high heat stability without losing 50% of activity after 1 hr incubation at $60^{\circ}C$. The highest activity was found at $45^{\circ}C$, and the optimal pH was about pH 7, but higher stability was observed at pH 8. Azide and cyanide ion showed strong inhibition at less than 1 $\mu M$ level suggesting that the enzyme was Fe ion dependent haloperoxidase.

• Microbiology and Biotechnology Letters
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• v.10 no.4
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• pp.275-281
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• 1982

One strain of Streptomyces sp. (AS-707) isolated from soil was found to produce a biologically active substance that showed a strong inhibitory activity against proteolytic enzymes viz. trypsin, papain, $\alpha$-chymotrypsin, Azotobacter protease, and Bacillus pretense. The substance was separated from culture filtrate by ion exchange column chromatography using Amberlite IRC-50 and CM-cellulose column chromatography. It was found that the recovery yield was 26% as activity basis. The substance was stable in wide pH range from 2.0 to 12.0 at 37$^{\circ}C$, but it was unstable in alkaline pH values at 6$0^{\circ}C$. The activity was thermostable to give 90% activity compared to the intact sample when it was treated at pH5.6 at 10$0^{\circ}C$ for 2 hours.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1724-1730
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• 2012

An ${\alpha}$-L-arabinofuranosidase (TmAFase) from Thermotoga maritima MSB8 is a highly thermostable exo-acting hemicellulase that exhibits a relatively higher activity towards arabinan and arabinoxylan, compared with other glycoside hydrolase 51 family enzymes. In the present study, we carried out the enzymatic characterization and structural analysis of TmAFase. Tight domain associations found in TmAFase, such as an inter-domain disulfide bond (Cys306 and Cys476) in each monomer, a novel extended arm (amino acids 374-385) at the dimer interface, and total 12 salt bridges in the hexamer, may account for the thermostability of the enzyme. One of the xylan binding determinants (Trp96) was identified in the active site, and a region of amino acids (374-385) protrudes out forming an obvious wall at the substrate-binding groove to generate a cavity. The altered cavity shape with a strong negative electrostatic distribution is likely related to the unique substrate preference of TmAFase towards branched polymeric substrates.

• Journal of Animal Science and Technology
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• v.59 no.9
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• pp.20.1-20.9
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• 2017

Background: Ribonuclease (RNase) is one of the few toxic proteins that are present constantly in snake venoms of all types. However, to date this RNase is still poorly studied in comparison not only with other toxic proteins of snake venom, but also with the enzymes of RNase group. The objective of this paper was to investigate some properties of RNase from venom of Vietnam cobra Naja atra. Methods: Kinetic methods and gel filtration chromatography were used to investigate RNase from venom of Vietnam cobra. Results: RNase from venom of Vietnam cobra Naja atra has some characteristic properties. This RNase is a thermostable enzyme and has high conformational stability. This is the only acidic enzyme of the RNase A superfamily exhibiting a high catalytic activity in the pH range of 1-4, with $pH_{opt}=2.58{\pm}0.35$. Its activity is considerably reduced with increasing ionic strength of reaction mixture. Venom proteins are separated by gel filtration into four peaks with ribonucleolytic activity, which is abnormally distributed among the isoforms: only a small part of the RNase activity is present in fractions of proteins with molecular weights of 12-15 kDa and more than 30 kDa, but most of the enzyme activity is detected in fractions of polypeptides, having molecular weights of less than 9 kDa, that is unexpected. Conclusions: RNase from the venom of Vietnam cobra is a unique member of RNase A superfamily according to its acidic optimum pH ($pH_{opt}=2.58{\pm}0.35$) and extremely low molecular weights of its major isoforms (approximately 8.95 kDa for RNase III and 5.93 kDa for RNase IV).

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.349-354
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• 1985

A 4.7 kb Hind III fragment containing $\alpha$-amylase gene of Bacillus stearothermophilus IAM 11062 was cloned in Escherichia coil HB101, using plasmid pBR322 and runaway plasmid pSY343 as a vector. The cloned gene was stably maintained and expressed In E.coli. The constructed strain of E. coli have at least 3 times higher amylase activity than the donor strain, of B. stearothermophilus. About 75％ of the $\alpha$-amylase produced by the constructed strain of E. coli was localized in the periplasm and it was found that the enzymes can be released by an osmotic shock using EDTA. The enzymatic properties of L-amylase produced in E. coli were very similar to those produced by B. stearothermophilus in terms of optimum temperature, heat stability and molecular weight.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.244-255
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• 2019

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at $60^{\circ}C$. We solved the crystal structure of PbXI at $1.94-{\AA}$ resolution to investigate the origin of its thermostability. The PbXI structure shows a $({\beta}/{\alpha})_8$-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the ${\beta}4-{\alpha}6$ loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the ${\beta}1-{\alpha}2$ loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible ${\beta}1-{\alpha}2$ loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate low-temperature purpose-specific XI enzymes.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.123-129
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• 2007

The trehalose $({\alpha}-D-glucopyranosyl-[1,1]-{\alpha}-D-glucopyranose)$ biosynthesis genes MhMTS and MhMTH, encoding a maltooligosyltrehalose synthase (MhMTS) and a maltooligosyltrehalose trehalohydrolase (MhMTH), respectively, have been cloned from the hyperthermophilic archaebacterium Metallosphaera hakonesis. The ORF of MhMTS is 2,142 bp long, and encodes 713 amino acid residues constituting a 83.8 kDa protein. MhMTH is 1,677 bp long, and encodes 558 amino acid residues constituting a 63.7 kDa protein. The deduced amino acid sequences of MhMTS and MhMTH contain four regions highly conserved for MTSs and three for MTHs that are known to constitute substrate-binding sites of starch-hydrolyzing enzymes. Recombinant proteins obtained by expressing the MhMTS and MhMTH genes in E. coli catalyzed a sequential reaction converting maltooligosaccharides to produce trehalose. Optimum pH of the MhMTS/MhMTH enzyme reaction was around 5.0 and optimum temperature was around 70 C. Trehalose-producing activity of the MhMTS/ MhMTH was notably stable, retaining 80% of the activity after preincubation of the enzyme mixture at $70^{\circ}C$ for 48 h, but was gradually abolished by incubating at above $85^{\circ}C$. Addition of thermostable $4-{\alpha}-glucanotransferase$ increased the yield of trehalose production from maltopentaose by 10%. The substrate specificity of the MhMTS/MhMTH-catalyzed reaction was extended to soluble starch, the most abundant maltodextrin in nature.

• Microbiology and Biotechnology Letters
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• v.4 no.3
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• pp.91-97
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• 1976

During the course of studies on the production and utilization of thermostable ${\alpha}$-amylase from a thormophilic actinomycete species isolated from soil, partial characterization of the ${\alpha}$-amylase has been (arried out. The optimum pH for the dextrinogenic activity of the enzyme was found to be 6.5 and the maximum reaction rate was achieved at a temperature range of 55$^{\circ}$ to 65$^{\circ}C$. Calcium ion was recognized to have a slight effect in activating the enzyme, while heavy metal salts especially ferrous and cupric ions showed a remarkable inhibition effect. The enzyme was best protected iron thermal denaturation at pH 8.0 with tris-HCI buffer；inactivation was rapid at higher or lower pH values. Furthermore, its thermal stability was greatly increased by calcium ion, particulary at the final concentration of 1${\times}$10$\^$－2/ mole in the reaction mixture. The Km value for the ${\alpha}$-amylase was calculated to be 2.17${\times}$10$\^$－4/g per $m\ell$ and the energy of activation for the dextrinogenic reaction to be 12,000${\pm}$580 ㎈ per mole.

• Journal of Life Science
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• v.25 no.2
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• pp.180-188
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• 2015

The bacterial strain isolated from Kimchi showed antibacterial activity against Micrococcus luteus IAM 1056. The selected strain was identified as Lactococcus lactis by 16S rRNA nucleotide sequence analysis and named as Lactococcus sp. KD 28. The treatment of culture supernatant with proteinase K removed antibacterial activity, indicating its proteinaceous nature, a bacteriocin. This bacteriocin was sensitive to hydrolytic enzymes such as ${\alpha}$-chymotrypsion, trypsin, proteinase K, lipase, ${\alpha}$-amylase and subtilisin A. The bacteriocin was highly thermostable and resistant to heating at $80^{\circ}C$ for up to an hour but 50 % of the total activity was remained at $100^{\circ}C$ for 30 min. The pH range from 2.0 to 8.0 had no effect on bacteriocin activity and it was not affected by solvents such as acetonitrile, isopropanol, methanol, chloroform and acetone up to 50% concentration. The bacteriocin showed antibacterial activity against M. luteus IAM 1056, Lactobacillus delbrueckii subsp. lactis KCTC 1058, Enterococcus faecium KCTC 3095, Bacillus cereus KCTC 1013, B. subtilis KCTC 1023, Listeria ivanovii subsp. ivanovii KCTC 3444, Staphylococcus aureus subsp. aureus KCTC 1916, B. megaterium KCTC 1098 and B. sphaericus KCTC 1184. The bacteriocin was purified through ammonium sulfate concentration, SP-Sepharose chromatography and RP-HPLC. The molecular weight was estimated to be about 3.4 kDa by tricine-SDS-PAGE analysis.

• Microbiology and Biotechnology Letters
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• v.45 no.1
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• pp.43-50
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• 2017

A xylan-degrading bacterium (strain WJ-1) was isolated from soil collected from Jeju Island, Republic of Korea. Strain WJ-1 was characterized as a gram-positive, aerobic, and spore-forming bacterium. The predominant fatty acid in this bacterium was anteiso-$C_{15:0}$ (42.99%). A similarity search based on 16S rRNA gene sequences suggested that the strain belonged to the genus Streptomyces. Further, strain WJ-1 shared the highest sequence similarity with the type strains Streptomyces spinoveruucosus NBRC 14228, S. minutiscleroticus NBRC 13000, and S. glaucescens NBRC 12774. Together, they formed a coherent cluster in a phylogenetic tree based on the neighbor-joining algorithm. The DNA G+C content of strain WJ-1 was 74.7 mol%. The level of DNA-DNA relatedness between strain WJ-1 and the closest related species S. glaucescens NBRC 12774 was 85.7%. DNA-DNA hybridization, 16S rRNA gene sequence similarity, and the phenotypic and chemotaxonomic characteristics suggest that strain WJ-1 constitutes a novel subspecies of S. glaucescens. Thus, the strain was designated as S. glaucescens subsp. WJ-1 (Korean Agricultural Culture Collection [KACC] accession number 92086). Additionally, strain WJ-1 secreted thermostable endo-type xylanases that converted xylan to xylooligosaccharides such as xylotriose and xylotetraose. The enzymes exhibited optimal activity at pH 7.0 and $55^{\circ}C$.