• Journal of Microbiology
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• v.40 no.1
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• pp.33-37
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• 2002

An enzymatic reaction for the production of D-alanine from D-aspartic acid and pyruvate as substrates by a thermostable D-amino acid aminotransferase (D-AAT) was investigated at various conditions In the temperature range of 40-70$\^{C}$ and pH range of 6.0-9.5. The D-AAT was produced with recombinant E. coli BL21, which hosted the chimeric plasmid pTLK2 harboring the D-AAT from the novel thermophilic Bacillus sp. LK-2. The enzyme reaction was shown to follow the Ping Pong Bi Bi mechanism. The K$\_$m/ values for D-aspartic acid and pyruvate were 4.38 mar and 0.72 mM, respectively. It was observed that competitive inhibition by D-alanine, the product of this reaction, was evident with the inhibition constant K$\_$i/ value of 0.1 mM. A unique feature of this reaction scheme is that the decorboxylation of oxaloacetic acid, one of the products, spontaneously produces pyruvate. Therefore, only a catalytic amount of pyruvate is necessary for the enzyme conversion reaction to proceed. A typical time-course kinetic study skewed that D-alanine up to 88 mM could be produced from 100 mM of D-aspartic acid with a molar yield of 1.0.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.116-122
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• 2007

An efficient enzyme system for the synthesis of L-tyrosine was developed using a fed-batch reactor with continuous feeding of phenol, pyruvate, and ammonia. A thermo- and chemostable tyrosine phenol-lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM phenol at pH 8.5 and $40^{\circ}C$. The fed-batch system (working volume, 0.51) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4M ammonium chloride (pH 8.5). The system produced 130g/I of L-tyrosine within 30h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-tyrosine was 94% on the basis of the supplied phenol.

• BMB Reports
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• v.40 no.6
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• pp.1002-1008
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• 2007

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli $DH5\alpha$ cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-$41^{\circ}C$) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at $37^{\circ}C$ for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at $-80^{\circ}C$.

• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.43-48
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• 2013

Oreorchis coreana Finet is threatened globally by over-collection from its natural habitats for horticultural purposes. Its rarity in nature makes this plant one of the most endangered species in Korea. In this study, we investigated the effects of sodium hypochlorite (NaOCl) on orchid seed viability and seed germination. An in vitro bioassay swelling test using immature seeds was compared with a standard chemical procedure using triphenyl tetrazolium chloride (TTC) to test seed viability. In general, the bioassay was more appropriate for estimating embryo viability after a prolonged pre-treatment (more than 1 h) in 1% NaOCl, a surface sterilant often used to enhance germination of seeds of terrestrial plants. Therefore, an efficient method for investigating in vitro swelling of immature seeds is urgently needed. We established a method for determining the viability and swelling of O. coreana seeds via in vitro examination of immature seeds. Treatment of immature seeds with 1% NaOCl for 10 min greatly enhanced the extent of swelling of immature zygote embryos when compared to untreated seeds. These data obtained here appear to be comparable to viability and swelling that occurs in O. coreana seeds via asymbiotic germination.

• Biomedical Science Letters
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• v.23 no.2
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• pp.80-88
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• 2017

Ginkgo biloba extract (EGb 761) is a standardized extract of Ginkgo biloba leaves and has anti- atherosclerosis properties. Many patients with atherosclerosis disorders take Ginkgo biloba extracts to supplement current therapy. In addition, normal healthy individuals also take Ginkgo biloba extracts for prophylactic purposes. However, it is unknown whether supplementation of Gingko biloba extracts in healthy individuals offer a benefit. In this study, we assessed whether EGb 761 could provide beneficial effects on serum cholesterol levels in normal mice. Wild-type C56Bl/6 mice were orally administered EGb 761 at 25 mg/kg (Group 3) or 50 mg/kg (Group 4) every other day for 40 days. We found that the serum levels of HDL-cholesterol (HDL-C) were significantly increased in EGb 761 and lovastatin treated groups. Treatment with EGb 761 and lovastatin resulted in reduced serum total cholesterol and LDL-cholesterol (LDL-C) compared to control group. Serum lecithin cholesterol acyltransferase (LCAT) levels were higher in EGb 761 and lovastatin treated group compared to the control group. However, no difference was observed in serum APO A-I levels between the control group and treatment group. These results suggest that EGb 761 can increase HDL-C resulting in increased serum LCAT levels.

• Journal of Microbiology and Biotechnology
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• v.30 no.3
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• pp.391-397
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• 2020

In this study, we used a novel α-L-arabinopyranosidase (AbpBs) obtained from ginsenoside-converting Blastococcus saxobsidens that was cloned and expressed in Escherichia coli BL21 (DE3), and then applied it in the biotransformation of ginsenoside Rb₂ into Rd. The gene, termed AbpBs, consisting of 2,406 nucleotides (801 amino acid residues), and with a predicted translated protein molecular mass of 86.4 kDa, was cloned into a pGEX4T-1 vector. A BLAST search using the AbpBs amino acid sequence revealed significant homology with a family 2 glycoside hydrolase (GH2). The over-expressed recombinant AbpBs in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinopyranose moiety attached to the C-20 position of ginsenoside Rb₂ under optimal conditions (pH 7.0 and 40℃). Kinetic parameters for α-L-arabinopyranosidase showed apparent K_m and V_max values of 0.078 ± 0.0002 μM and 1.4 ± 0.1 μmol/min/mg of protein against p-nitrophenyl-α-L-arabinopyranoside. Using a purified AbpBs (1 ㎍/ml), 0.1% of ginsenoside Rb₂ was completely converted to ginsenoside Rd within 1 h. The recombinant AbpBs could be useful for high-yield, rapid, and low-cost preparation of ginsenoside Rd from Rb₂.

• Journal of Applied Biological Chemistry
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• v.52 no.3
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• pp.109-115
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• 2009

The gene encoding a putative aspartate aminotransferase in Xanthomonas oryzae pv. oryzae (Xoo) was cloned using PCR technique. The gene was ligated with pET-21(a) vector containing His6 tag and expressed in E. coli BL21(DE3). Affinity purification of the recombinant aspartate aminotransferase with Ni-NTA resin resulted in one band by SDS-PAGE analysis. The purified enzyme showed a molecular weight of 43 kDa, as expected. The enzyme was the most active toward L-aspartate as an amino donor, indicating that the purified enzyme is one of aspartate aminotrans-ferases exist in Xoo. Optimal activity of the enzyme was observed at around pH 7.5 and stability was much higher at alkaline pH rather than acidic pH values. The enzyme was considerably activated by the presence of manganese ion, showing about 157% of control activity at 1.0 mM.

• Radiation Oncology Journal
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• v.13 no.1
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• pp.33-40
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• 1995

Purpose : To assess the efficacy and toxicity of the preoperative radiotherapy with or without chemotherapy in locally advanced rectal cancer Methods : Forty three patients (clinically diagnosed stages above or equal to Astler-Coiler stage B2 without distant metastasis) were assigned to preoperative radiotherapy alone arm (n=16) or combined preoperative radiotherapy and chemotherapy arm (n=27). Preoperative radiotherapy of 4500 cGy to whole pel-vis +/-540 cGy boost to primary site and concurrent chemotherapy of 2 cycles of 5-FU (500 mg/$m^2$) and leucovorin (20 mg/$m^2$) were used. Fifteen patients of preoperative radiotherapy alone arm and 19 of combined arm received surgical resection after preoperative treatment. Results : During the preoperative treatment, no significant complication was developed in both groups. Pathologic results were as follows; complete remission 1, Bl 1, B2 6, C1 2, C2, 5 in preoperative radiotherapy alone arm and complete remission 2, Bl 8, B2 4, C2 3, D 2 in combined arm. Postoperative complications were delayed perineal wound healing in three patients, intestinal obstruction in three patients (one managed by conservative medical treatment, two by surgical treatment). Conclusion : The combined preoperative radiotherapy and chemotherapy arm was more effective in pathological response and lymph node negativity rate than the preoperative radiotherapy alone arm. Both the preoperative radiotherapy alone arm and the combined arm were generally well tolerated and did not result in an increased postoperative morbidity.

• Journal of Korean Medicine for Obesity Research
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• v.11 no.1
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• pp.47-60
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• 2011

Objectives The objective of this study was to contribute to the development of pharmacupuncture for obesity treatment by reviewing the studies of pharmacupuncture experimentations and clinical trials. Methods We searched the papers with keywords of 'obesity' and 'pharmacupuncture' in the search site, RISS, Oriental medicine advanced searching integrated system(OASIS), Korean traditional knowledge portal, the society of Korean medicine for obesity research, Korean pharmacupuncture institute, the Korean academy of oriental rehabitation medicine and Korean acupuncture & moxibustion society. Results 1. We reviewed 37 articles searched. 27 articles(73.0%) were animal experimentations, 5 articles(13.5%) were cell experimentations, 4 articles(10.8%) were clinical trials and 1 article(2.7%) was study analysis. 2. The herbs, using for animal experimentations, were atratylodes japonica, coix lachrymajobi, ephedra sinica, crataegus pinnatifida, wild ginseng and etc. Acupucture points were joksamni(ST36), zhongwan(CV12), gansoo(BL18), pungnyung(ST40), umnungchon(SP9), bisu(BL20), gokji (LI11), cheun-chu(ST25) and etc. 3. For cell experimentations, preadipocytes and adipocytes performed on cell cultures with using rats, 3T3-L1 preadipocytes and porcine skin including fat tissue were treated with fel ursi, bovis calculus, ephedrae herba, spirodelae herba, wild ginseng. 4. For clinical trials, Sangsik no.1, Bigiheo, ephedra, green tea and sweet bee venom were injected at the region where a lot of fat like zhongwan(CV12), xiawan(CV10), kwanwon(CV4), cheun-chu(ST25) and thigh. Conclusion Through animal and cell experimentations and clinical trials, the treatment of obesity using local acupuncture therapy was effective. For clinical use, however, it is considered that animal and cell experimentation and clinical trial's connection using one kind of herb and studies about more clinical trials and associated side effects are needed.

• Journal of Dairy Science and Biotechnology
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• v.29 no.1
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• pp.49-54
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• 2011

Bile salt hydrolase (BSH, EC 3.5.1.24) activity, which cleaves amide bond between carboxyl group (bile acid) and amino group (glycine or taurine), is commonly detected in gut-associated species of human and animal. During the screening of BSH active strains from the fecal samples of elderly human volunteers, strain CU30-2 was isolated on the basis of the highly active BSH producing activity. A bsh gene of the isolate was cloned into the pET22b expression vector and overexpressed in Escherichia coli BL21 (DE3) Gold by induction with 1mM IPTG. The overexpressed BSH enzyme with 6x His-tag was purified with apparent homogeneity using a $Ni^+$-NTA agarose column and characterized. The BSH enzyme of E. faecalis CU30-2 exhibited approximately 50 times higher activity against glycol-conjugated bile salts than tauro-conjugated bile salts having the highest activity against glycocholic acid. Considering the prevalence of E. faecalis strains in the human GI tract and glycol-conjugates dominated bile acid composition of human bile, further study is needed to investigate the impact of the BSH activity exerted by E. faecalis strains to the host as well as to the BSH producing strains.