• Korean Journal of Food Science and Technology
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• v.34 no.4
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• pp.552-558
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• 2002

Polyphenol oxidase from Flammulina velutipes was purified and characterized. Purification of polyphenol oxidase was achieved by ammonium sulfate precipitation, Superdex G-200 gel filtration chromatography, Phenyl superose affinity chromatography, Mono-Q anion exchange chromatography and Superdex S-200 gel filtration chromatography on FPLC. After these purification steps specific activity of purified polyphenol oxidase increased to 199.1 units/mg. Polyphenol oxidase from F. velutipes was composed of a single polypeptide with molecular weight of about 40 kDa. Optimum pH and temperature for the enzyme reaction were found to be 6.0 and $25^{\circ}C$, respectively. The activity of the enzyme gradually decreased at acidic pH between 3 and 5, and the enzyme lost its activity at alkaline pH between 8 and 10. This enzyme exhibited high substrate specificity to o-diphenols. Km-values for L-DOPA and caffeic acid were found to be 3.97 mM and 1.78 mM, respectively. 2-mercaptoethanol, L-ascorbic acid, sodium bisulfite, EDTA and $Mg^{2+}$ inhibited the activity of pholyphenol oxidase and $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$ and $Ni^{2+}$ increased enzyme activity. The activity of enzyme was well maintained at $-70^{\circ}C$ for over 4 months, and at $-20^{\circ}C$ for 1 months.

• Journal of Life Science
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• v.19 no.8
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• pp.1039-1046
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• 2009

The cytochrome P450s (P450s) metabolizing natural products are among the most versatile biological catalysts known in plants, but knowledge of the structural basis for their broad substrate specificity has been limited. The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants however, all attempts to express and purify the protein corresponding C3H gene have failed. As a result, no conditions suitable for the unambiguous assay of the enzyme are known. The detailed understanding of the mechanism and substrate-specificity of C3Hdemands a method for the production of active protein on the milligram scale. We have developed a bacterial expression and purification system for the plant C3H, which allows for the quick expression and purification of active wild-type C3H via introduction of combinational mutagenesis. The modified cytochrome P450 C3H ($C3H_{mod}$) could be purified in the absence of detergent using immobilized metal affinity chromatography and size exclusion chromatography following extraction from isolated membranes in a high salt buffer and catalytically activated. This method makes the use of isotopic labeling of C3H for NMRstudies and X-ray crystallography practical, and is also applicable to other plant cytochrome P450 proteins.

• Journal of Life Science
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• v.22 no.2
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• pp.209-219
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• 2012

The properties of lactate dehydrogenase (LDH, EC 1.1.1.27) eye-specific $C_4$ isozyme were studied by polyacrylamide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme kinetics. Furthermore, we proposed the optimal conditions for measuring the activity of LDH eye-specific $C_4$ isozyme. The isozymes were detected in the cytosol of eye tissues from Lepomis macrochirus and Micropterus salmoides and were more similar to the $A_4$ than the $B_4$ isozyme. LDH/CS in the eye tissue of L. macrochirus was increased in September, so the ratio of anaerobic metabolism was high. The electrophoretic patterns of mitochondrial LDH were similar to those of cytosolic LDH in the eye tissues of L. macrochirus and Micropterus salmoides. LDH eye-specific $C_4$ isozyme from eye tissue was purified by preparative native-PAGE. The activities of LDH eye-specific $C_4$ isozymes in L. macrochirus and M. salmoides were reduced at concentrations greater than 0.2 mM and 0.1 mM of pyruvate, respectively. These concentrations remained at 5.2% and 15.8% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The LDH activities of eye tissues were reduced at concentrations greater than 22 mM and 24 mM of lactate, respectively, in L. macrochirus and M. salmoides. The ${K_m}^{PYR}$ of eye-specific $C_4$ was 0.088 mM in L. macrochirus and it was 0.033 mM in M. salmoides. The activities of cytosolic and mitochondrial eye-specific $C_4$ isozymes were high in ${\alpha}$-ketobutyric acid. Furthermore, the activities of eye tissue and eye-specific $C_4$ isozyme had to be measured with 0.5 mM of pyruvate and a buffer solution of pH 7.5. As a conclusion, the eye-specific $C_4$ isozyme in M. salmoides has a high affinity for pyruvate and exhibits maximum activity at a lower concentration of pyruvate and at higher concentration of lactate than that in L. macrochirus. Therefore, it seems that the energy produced by the LDH eye-specific $C_4$ isozyme in M. salmoides was used at the first stage of predatory behavior.

• Journal of Life Science
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• v.29 no.4
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• pp.492-498
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• 2019

Previously, three kinds of lipases, lipAD1, lipAD2, and lipAD3, and lipase chaperone, lipBD, of Acinetobacter schindleri DYL129 isolated from soil sample were reported. In this report, three lipase and lipase chaperone were cloned into the pET32a(+) or pGEX-6P-1 vectors for protein expression in Escherichia coli, and named as pETLAD1, pETLAD2, pETLAD3 and pETLBD or pGEXLAD1, pGEXLAD 2, pGEXLAD3 and pGEXLBD, respectively. Protein expression rate was higher in pET system than in pGEX system. Although LipAD1 and LipAD2 were produced as inclusion bodies, their expression levels were high. So LipAD1 and LipAD2 could be solubilized in 8 M urea buffer and purified. LipAD3 and LipBD were overexpressed in soluble form and purified. Those proteins were purified by His-tag affinity chromatography connected in AKTA prime system. The activities of the purified lipases were demonstrated with 1% tributyrin agar plate. After purification, molecular mass was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LipAD1 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl butyrate, LipAD2 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl myristate, and LipAD3 showed high activity toward ${\rho}$-nitrophenyl acetate, ${\rho}$-nitrophenyl butyrate, and ${\rho}$-nitrophenyl miristate, respectively. Three lipases, LipAD1, LipAD2, and LipAD3, showed optimal reaction at $50^{\circ}C$ using ${\rho}$-nitrophenyl butyrate, as substrate.

• Korean Journal of Microbiology
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• v.51 no.4
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• pp.355-363
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• 2015

The present work quantified the growth of young biofilm in a model distribution system that was fed with chlorinated drinking water at a hydraulic retention time of 2 h. Bacterial biofilms grew on the surface of polyvinyl chloride (PVC) slides at a specific growth rate of $0.14{\pm}0.09day^{-1}$ for total bacteria and $0.16{\pm}0.08day^{-1}$ for heterotrophic bacteria, reaching $3.1{\times}10^4cells/cm^2$ and $6.6{\times}10^3CFU/cm^2$ after 10 days, respectively. The specific growth rates of biofilm-forming bacteria were found to be much higher than those of bulk-phase bacteria, suggesting that biofilm bacteria account for a major part of the bacterial production in this model system. Biofilm isolates exhibited characteristic kinetic properties, as determined by ${\mu}_{max}$ and $K_S$ values using the Monod model, in a defined growth medium containing various amounts of acetate. The lowest ${\mu}_{max}$ value was observed in bacterial species belonging to the genus Methylobacterium, and their slow growth seemed to confer high resistance to chlorine treatment (0.5 mg/L for 10 min). $K_S$ values (inversely related to substrate affinity) of Sphingomonas were two orders of magnitude lower for acetate carbon than those of other isolates. The Sphingomonas isolates may have obligate-oligotrophic characteristics, since the lower $K_S$ values allow them to thrive under nutrient-deficient conditions. These results provide a better understanding and control of multi-species bacterial biofilms that develop within days in a drinking water distribution system.