• Journal of Microbiology and Biotechnology
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• v.32 no.5
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• pp.551-563
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• 2022

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50℃ and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30℃, 40℃, and 50℃, respectively. The enzyme reserved its activity at 30℃ and 37℃ up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC₅₀ of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant Lasparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC₅₀ values of 1.53 and 18 IU, respectively.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.19-22
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• 2007

Acetohydroxyacid synthase (E.C.2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by $Ni^{2+}-charged$ HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, $MgCl_{2}$ and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at $37^{\circ}C$. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained.

• Preventive Nutrition and Food Science
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• v.11 no.1
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• pp.1-5
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• 2006

Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid that have been used to reduce the incidence, growth and metastasis of breast, colon, prostate and gastric cancer in animals. CLA could reduce tumor growth by altering angiogenesis; a process requiring associated angiogenic factors such as vascular endothelial growth factor (VEGF). In this study, we determined whether CLA could modulate the expression of VEGF in murine mammary tumor cells and adipocytes. The c9, t11-CLA isomer reduced VEGF transcripts and protein when mammary tumor cells were stimulated with PMA. That isomer also reduced VEGF expression in un stimulated mouse 3T3-L1 adipocytes. Since VEGF can be regulated by cyclooxygenase-2 (COX-2), we determined whether CLA could alter COX-2 enzyme expression and $PGE_2$ production. The c9, t11-CLA isomer reduced not only COX-2 enzyme expression but also $PGE_2$ production. Thus, c9, t11-CLA could modulate neovascularization by alteration of VEGF expression from mammary tumor cells and adipocytes by reducing COX-2 metabolites.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.157-167
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• 2000

A thermostable chitosanase gene from the isolated strain, Bacillus sp. CK4, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30 kDa enzyme in size. The deduced amino acid sequence of the chitosanase from Bacillus sp. CK4 exhibits 76.6%, 15.3%, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. CK4 belongs to the Cluster III group with Bacillus subtilis. The size of the gene was similar to that of a mesophile, Bacillus subtilis showing a higher preference for codons ending in G or C. The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues were changed to E50D/Q, E62D/Q, and D66N/E by site-directed mutagenesis. The D66N/E mutants enzymes had remarkably decreased kinetic parameters such as $V_{max}$ and k$\sub$cat/, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three cysteine residues at position 49, 72, and 211. Titration of the Cys residues with DTNB showed that none of them were involved in disulfide bond. The C49S and C72S mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However the half-life of the C211S mutant enzyme was less than 60 min at 80$^{\circ}C$, while that of the wild type enzyme was about 90 min. Moreover, the residual activity of C211S was substantially decreased by 8 M urea, and fully lost catalytic activity by 40% ethanol. These results show that the substitution of Cys with Ser at position 211 seems to affect the conformational stability of the chitosanase.

• Natural Product Sciences
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• v.16 no.1
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• pp.1-5
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• 2010

$\beta$-Glucuronidases (E.C. 3.2.1.31) from Escherichia coli, Helix pomatia, and bovine liver activity have been investigated on 7-O-glucuronides (baicalin, wogonoside, and luteolin-7-O-glucuronide) and 3-O-glucuronides (quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide). Bovine liver enzyme was not active on any of these substrates. E. coli and H. pomatia enzymes were active on 7-O-glucuronides, however, 3-O-glucuronides were resistant to $\beta$-glucuronidase hydrolysis. These results suggest that glucuronic acid at 7-position is more susceptible to E. coli and H. pomatia $\beta$-glucuronidases than that at 3-position. In addition, the subtle difference of aglycone structure on 7-O-glucuronides affected the preference of enzyme. E. coli enzyme was favorable for the hydrolysis of baicalin, however, H. pomatia enzyme was found to be efficient for the hydrolysis of wogonoside. Both enzymes showed the similar hydrolytic activity towards luteolin-7-O-glucuronide. When the Scutellaria baicalensis crude extract was subjected to enzymatic hydrolysis, baicalin and wogonoside were successfully converted to their aglycone counterparts with H. pomatia at 50 mM sodium bicarbonate buffer pH 4.0. Accordingly, the enzymatic transformation of glycosides may be quite useful in preparing aglycones under mild conditions.

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.341-346
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• 2011

To study the effects of salinity-sodicity on bacterial population and enzyme activities, soil samples were collected from the Bay of Yellow Sea, Incheon, South Korea. In the soils nearest to the coastline, pH, electrical conductivity ($EC_e$), sodium adsorption ratio (SAR), and exchangeable sodium percentage (ESP) were greater than the criteria of saline-sodic soil, and soils collected from sites 1.5-2 km away from the coastline were not substantially affected by the intrusion and spray of seawater. Halotolerant bacteria showed similar trends, whereas non-tolerant bacteria and enzymatic activities had opposite trends. Significant positive correlations were found between EC, exchangeable $Na^+$, and pH with SAR and ESP. In contrast, $EC_e$, SAR, ESP, and exchangeable $Na^+$ exhibited significant negative correlations with bacterial populations and enzyme activities. The results of this study indicate that the soil chemical variables related with salinity-sodicity are significantly related with the sampling distance from the coastline and are the key stress factors, which greatly affect microbial and biochemical properties.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1979.04a
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• pp.115.2-115
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• 1979

By employing a two-stage continuous culture system, some of important physiological parameters involved in cellulase bicsynthesis have been evalua-ted with an ultimate objective of detigning an op-timally controlled tellulase process. Volumetric and specific cellulase productivities obtained were 90 IU/liter/hr and 8IU/g biomass/hr respectively. The maximum specific enzyme productivity observed was 14.8 IU/g hiomass/hr. The optimal dilution rate in the second stage which corresponded to the maximum enzyme productivity was 0.026-0.028 hr$^{-1}$ , and the specific growth rate in the second stage ihat suported maximum specific enzyme productivity was equal to orslightly less than zero. The maintenance coefficients deter-mined for oxygen and for carbon source are M$_{o}$=0.85mmmole/g biomass/hr and M$_{c}$=0.14 mmole hexose/g bio mass/hr respectively. The yield constants determined are; Y(x/o) =32.3g biomass/mole oxygen, Y (x/c) =1.1g bio-mass/g carbon or 0.44g biomass/g hexose, Y(x/n) = 19.6g biomass/g nitrogen for the enzyme produc-tion stage and 12.5g biomass/g nitrogen for the cell growth stage.e.e.

• Journal of Nutrition and Health
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• v.36 no.3
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• pp.245-254
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• 2003

The aim of this study was to investigate the effects of P/S ratio of fatty acid and antioxidant (vitamin E, selenium) supplements on the serum lipid levels and hepatic antioxidant enzyme activity in rats. Female 16-week-old Sprague-Dawley rats were fed 6 different experimental diets for 4 weeks. While the peroxidizability index (PI) levels of fatty acids in the experimental diets were fixed at 81.22, the levels of P/S ratio of fatty acids were formulated at 0.38, 1.00, 4.81 (LP, MP, HP). These diets were supplemented with vitamin E (1,000 mg/kg diet) and selenium (2.5 mg/kg diet) (LP-S, MP-S, HP-S). This study showed that the serum concentrations of total-cholesterol and HDL-C increased with the increasing of the P/S ratio in the diet (p <0.05). Antioxidant supplementation significantly lowered the concentrations of triglyceride (TG) and VLDL-C of serum (p<0.05). Levels of thiobarbituric acid reactive substance (TBARS) in the liver tended to decrease with the increasing of the P/S ratio in the diet (p<0.001), but antioxidant enzyme activity in the liver was not significantly different. In addition, antioxidant supplementation significantly lowered TBARS level in the liver (p<0.05), but had no effect on antioxidant enzyme activity except for glutathione reductase (p<0.05). In conclusion, it is necessary to consider the properties of dietary fatty acids and antioxidants supplementation for the prevention of cardiovascular diseases.

• The Korean Journal of Pesticide Science
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• v.2 no.2
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• pp.10-15
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• 1998

The bimolecular inhibition rate constants of carbofuran and N-dimethylphosphinothioyl carbofuran(PSC) to acetylcholinesterase(AChE) were $7.7{\times}10^{5}\;M^{-1}{\cdot}min^{-1}$ and $1.2{\times}10^{3}\;M^{-1}{\cdot}min^{-1}$, respectively. These results showed that PSC required a bioactivation process for its toxic action because it didn't inhibit the target enzyme effectively. The potency of PSC as an inhibitor of AChE increased when PSC and AChE were incubated with microsomes fortified with NADPH compared with microsome alone. Piperonyl butoxide(PBO) addition to these coupled systems greatly reduced the inhibition of the target enzyme by blocking the bioactivation process. In vivo inhibition study of mouse brain AChE, $I_{50}$ value for AChE was 28 mg/kg for PSC and the value increased to 57 mg/kg when PBO was pretreated. This result showed that cytochrome $P_{450}$ would also play a role in the bioactivation process of PSC in vivo. And conversioin of carbofuran from PSC was 55 % in a chemical oxidation system using meta-chloroperoxybenzoic acid. The oxidative activation of PSC to carbofuran was shown to be essential for showing its toxicological action and cytochrome $P_{450}$ was identified as an important enzyme which participated in this process.

• Food Science and Preservation
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• v.30 no.4
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• pp.654-662
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• 2023

The Aster glehni extract has many therapeutic and medicinal values. Therefore, it is essential to set appropriate conditions for enzyme treatment to efficiently extract A. glehni. In this study, changes in the quality of A. glehni extract depending on the concentration and time of enzyme treatment was investigated to increase its effective utilization. Compared to the control, the pH of the extract of A. glehni its soluble solid content increased with the enzyme treatment. The color of the A. glehni extract changed from green-yellow to reddish-yellow with the increase in treatment duration. The fructose and sucrose contents of the extract were the highest at 7.73% and 6.78%, respectively, in the control group without the enzyme treatment. Glucose and maltose contents were 6.91% and 4.44% in the C group (3.2% enzyme concentration and 60 min for enzyme treatment), respectively. Total polyphenol content, which shows antioxidant activity, was the highest at 7.38 mg GAE/g in the E group (1.6% of enzyme concentration and 120 min for enzyme treatment). 2,2-diphenylpicrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) showed the highest radical scavenging activity in the C group (3.2% of enzyme concentration and 60 min for enzyme treatment). These results enable setting appropriate conditions of enzyme treatment in terms of enzyme concentration and time for the production of dry powders using A. glehni extract.