• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.271-278
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• 2020

Glycerol dehydrogenase (GlyDH) catalyzes the oxidation of glycerol to dihydroxyacetone (DHA), which is the first step in the glycerol metabolism pathway. GlyDH has attracted great interest for its potential industrial applications, since DHA is a precursor for the synthesis of many commercially valuable chemicals and various drugs. In this study, GlyDH from Klebsiella pneumoniae (KpGlyDH) was overexpressed in E. coli and purified to homogeneity for biochemical and molecular characterization. KpGlyDH exhibits an exclusive preference for NAD⁺ over NADP⁺. The enzymatic activity of KpGlyDH is maximal at pH 8.6 and pH 10.0. Of the three common polyol substrates, KpGlyDH showed the highest k_cat/K_m value for glycerol, which is three times higher than for racemic 2,3-butanediol and 32 times higher than for ethylene glycol. The k_cat value for glycerol oxidation is notably high at 87.1 ± 11.3 sec^-1. KpGlyDH was shown to exist in an equilibrium between two different oligomeric states, octamer and hexadecamer, by size-exclusion chromatography analysis. KpGlyDH is structurally thermostable, with a T_m of 83.4℃, in thermal denaturation experiment using circular dichroism spectroscopy. The biochemical and biophysical characteristics of KpGlyDH revealed in this study should provide the basis for future research on its glycerol metabolism and possible use in industrial applications.

• Journal of Microbiology and Biotechnology
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• v.31 no.3
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• pp.464-474
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• 2021

Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3-1) and Streptomyces sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners' putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37℃. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H2O2) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.

• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.1032-1040
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• 2006

Some Pseudomonas species can grow on styrene as a sole carbon and energy source. From the new isolate Pseudomonas putida SN1, the genes for styrene catabolism were cloned and sequenced. They were composed of four structural genes for styrene monooxygenase (styA and styB), styrene oxide isomerase (styC), and phenylacetaldehyde dehydrogenase (styD), along with two genes for the regulatory system (styS and styR). All the genes showed high DNA sequence (91% to 99%) and amino acid sequence (94% to 100%) similarities with the corresponding genes of the previously reported styrene-degrading Pseudomonas strains. A recombinant Escherichia coli to contain the styrene monooxygenase from the SN1 was constructed under the control of the T7 promoter for the production of enantiopure (S)-styrene oxide, which is an important chiral building block in organic synthesis. The recombinant E. coli could convert styrene into an enantiopure (S)-styrene oxide (ee >99%) when induced by IPTG The maximum activity was observed as 140 U/g cell, when induced with 1 mM IPTG at $15^{\circ}C$.

• Plant Resources
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• v.8 no.1
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• pp.60-66
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• 2005

Lysine is the first essential amino acid for optimal nutrient quality in rice grain. For the narrow genetic diversities of lysine contents in rice, somaclonal variation was the source of mutation in our breeding program. Biochemical selection was conducted using 1 mM S-(2-aminoethyl) cysteine followed by two passages of 5 mM lysine plus threonine in the callus subculture medium. The lysine contents in endosperm of all progenies recovered from the biochemical selection were higher than those of their donor cultivar 'Hwayeongbyeo'. These elevated lysine levels of mutants were successfully transmitted to $M_4$ generation. The lysine contents in endosperm varied 3.85 to $4.80\%$ compare to their donor cultivar 'Hwayeongbyeo' was $3.85\%$. Three of high-lysine germplasms, Lys-l, Lys-2 and Lys-7 were selected by biochemical selection and rapid screening methods. DNA analysis showed that a new insertion of Tos 17 which mapped to rice chromosome 11 on the high-lysine mutant, Lys-2.

• Advances in environmental research
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• v.9 no.3
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• pp.215-232
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• 2020

Crude oils are essential source of energy. It is majorly found in geographical locations beneath the earth's surface and crude oil is the main factor for the economic developments in the world. Natural crude oil contains unrefined petroleum composed of hydrocarbons of various molecular weights and it contains other organic materials like aromatic compounds, sulphur compounds, and many other organic compounds. These hydrocarbons are rapidly getting degraded by biosurfactant producing microorganisms. The present study deals with the isolation, purification, and characterization of biosurfactant producing microorganism from oil-contaminated soil. The ability of the microorganism producing biosurfactant was investigated by well diffusion method, drop collapse test, emulsification test, oil displacement activity, and blue agar plate method. The isolate obtained from the oil contaminated soil was identified as Bacillus subtilis. The identification was done by microscopic examinations and further characterization was done by Biochemical tests and 16SrRNA gene sequencing. Purification of the biosurfactant was performed by simple liquid-liquid extraction, and characterization of extracted biosurfactants was done using Fourier transform infrared spectroscopy (FTIR). The degradation of crude oil upon treatment with the partially purified biosurfactant was analyzed by FTIR spectroscopy and Gas-chromatography mass spectroscopy (GC-MS).

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.4
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• pp.318-322
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• 2004

Lactic acid is an environmentally benign organic acid that could be used as a raw material for biodegradable plastics if it can be inexpensively produced by fermentation. Two genes (ldhL and ldhD) encoding the L-(+) and D-(-) lactate dehydrogenases (L-LDH and D-LDH) were cloned from Lactobacillus sp., RKY2, which is a lactic acid hyper-producing bacterium isolated from Kimchi. Open reading frames of ldhL for and ldhD for the L and D-LDH genes were 962 and 998 bp, respectively. Both the L(+)- and D(-)-LDH proteins showed the highest degree of homology with the L- and D-lactate dehydrogenase genes of Lactobacillus plantarum. The conserved residues in the catalytic activity and substrate binding of both LDHs were identified in both enzymes.

• Journal of Animal Science and Technology
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• v.58 no.3
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• pp.10.1-10.10
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• 2016

Background: Airag, alcoholic sour-tasting beverage, has been traditionally prepared by Mongolian nomads who naturally ferment fresh mares' milk. Biochemical and microbiological compositions of airag samples collected in Ulaanbaatar, Mongolia and physiological characteristics of isolated lactic acid bacteria were investigated. Methods: Protein composition and biochemical composition were determined using sodium dodecyl sulfate-gel electrophoresis and high performance liquid chromatography, respectively. Lactic acid bacteria were identified based on nucleotide sequence of 16S rRNA gene. Carbohydrate fermentation, acid survival, bile resistance and acid production in skim milk culture were determined. Results: Equine whey proteins were present in airag samples more than caseins. The airag samples contained 0.10-3.36 % lactose, 1.44-2.33 % ethyl alcohol, 1.08-1.62 % lactic acid and 0.12-0.22 % acetic acid. Lactobacillus (L.) helveticus were major lactic acid bacteria consisting of 9 isolates among total 18 isolates of lactic acid bacteria. L. helveticus survived strongly in PBS, pH 3.0 but did not grow in MRS broth containing 0.1 % oxgall. A couple of L. helveticus isolates lowered pH of skim milk culture to less than 4.0 and produced acid up to more than 1.0 %. Conclusion: Highly variable biochemical compositions of the airag samples indicated inconsistent quality due to natural fermentation. Airag with low lactose content should be favorable for nutrition, considering that mares' milk with high lactose content has strong laxative effect. The isolates of L. helveticus which produced acid actively in skim milk culture might have a major role in production of airag.

• Microbiology and Biotechnology Letters
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• v.48 no.3
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• pp.358-365
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• 2020

An organic solvent- and bleach-stable protease-producing strain was isolated from a polluted river water sample and identified as Aeromonas veronii OB3 on the basis of biochemical properties (API 20E) and 16S rRNA sequence analysis. The strain was found to hyper-produce alkaline protease when cultivated on fish waste powder-based medium (HVSP, 4080 U/ml). The biochemical properties and compatibility of OB3 with several detergents and additives were studied. Maximum activity was observed at pH 9.0 and 60℃. The crude protease displayed outstanding stability to the investigated surfactants and oxidants, such as Tween 80, Triton X-100, and H₂O₂, and almost 36% residual activity when incubated with 1% SDS. Remarkably, the enzyme demonstrated considerable compatibility with commercial detergents, retaining more than 100% of its activity with Ariel and Tide (1 h, 40℃). Moreover, washing performance of Tide significantly improved by the supplementation of small amounts of OB3 crude protease. These properties suggest the potential use of this alkaline protease as a bio-additive in the detergent industry and other biotechnological processes such as peptide synthesis.

• KSBB Journal
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• v.21 no.6 s.101
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• pp.414-417
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• 2006

For developing functional biomaterials, chemical analysis of Lespedeza cuneata including minerals, amino acids and vitamin were investigated. Minerals of L. cuneata were found to be calcium, potassium, magnesium, phosphorus, sodium, iron, manganese, zinc and copper. Among the free amino acid, proline was 33.77 mg% that were 67.2% of free amino acids and essential amino acids were 7.49 mg%. Total amino acids were analyzed as 2,817 mg% and the content of glutamic acid (496.00 mg%) was highest. In case of vitamin, the highest components was vitamin E with 33.03 mg%.

• Korean Journal of Clinical Laboratory Science
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• v.46 no.4
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• pp.136-139
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• 2014

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the severe nosocomial infectious agents. The traditional diagnostic methods including biochemical test, antibiotic susceptibility test and PCR amplification are time consuming and require much work. The Surface enhanced Raman spectroscopy (SERS) biosensor is a rapid and powerful tool for analyzing the chemical composition within a single living cell. To identify the biochemical and genetic characterization of clinical MRSA, all isolates from patients were performed with VITEK2 gram positive (GP) bacterial identification and Antibiotic Susceptibility Testing (AST). Virulence genes of MRSA also were identified by DNA based PCR using specific primers. All isolates, which were placed on a gold coated nanochip, were analyzed by a confocal Raman microscopy system. All isolates were identified as S. aureus by biochemical tests. MRSA, which exhibited antibiotic resistance, demonstrated to be positive gene expression of both femA and mecA. Furthermore, Raman shift of S. aureus and MRSA (n=20) was perfectly distinguished by a confocal Raman microscopy system. This novel technique explained that a SERS based confocal Raman microscopy system can selectively isolate MRSA from non-MRSA. The study recommends the SERS technique as a rapid and sensitive method to detect antibiotic resistant S. aureus in a single cell level.