• 한국미생물·생명공학회지
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• 제27권3호
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• pp.184-190
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• 1999

To acquire an industrially useful biocatalyst for the enzymatic synthesis and production of various D-amino acid aminotransferase (D-AAT) activity. The enzyme activity was found from 110 strains of isolated thermophiles revealing its wide occurrence in thermophiles. Enzyme activity and thermal stability of the D-AAT producers were compared. Finally we have selected four thermophiles as producers of potent biocatalysts for the D-amino acid production; two thermophiles, Bacillus sp. Lk-1 and LK-2, having higher specific activity and two thermophiles, B. stearothermophilus KL-01 and Bacillus sp. KLS-01, having higher thermal stability than the D-AAT producers. Taxonomic and physiological characteristics of the four isolated thermophiles were described herein.

• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.48-54
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• 2008

A putative ${\omega}$-aminotransferase gene, cc3143 (aptA), from Caulobacter crescentus was screened by bioinformatical tools and overexpressed in E. coli, and the substrate specificity of the ${\omega}$-aminotransferase was investigated. AptA showed high activity for short-chain ${\beta}$-amino acids. It showed the highest activity for 3-amino-n-butyric acid. It showed higher activity toward aromatic amines than aliphatic amines. The 3D model of the ${\omega}$-aminotransferase was constructed by homology modeling using a dialkylglycine decarboxylase (PDB ID: 1DGE) as a template. Then, the ${\omega}$-aminotransferase was rationally redesigned to increase the activity for 3-amino-3-phenylpropionic acid. The mutants N285A and V227G increased the relative activity for 3-amino-3-phenylpropionic acid to 3-amino-n-butyric acid by 11-fold and 3-fold, respectively, over that of wild type.

• BMB Reports
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• 제35권3호
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• pp.306-312
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• 2002

The effects of salts on the biochemical properties of D-amino acid aminotransferase from Bacillus sp. YM-1 have been studied to elucidate both the inhibitory effects of salts on the activity and the protective effects of salts on the substrate-induced inactivation. The results from UV-visible spectroscopy studies on the reaction of the enzyme with D-serine revealed that salt significantly reduced the rate of the formation of the quinonoid intermediate and its accumulation. The kinetic and spectroscopy studies of the reaction with $\alpha$-[$^2H$]-DL-serine in different concentrations of NaCl provided evidence that the rate-limiting step was changed from the deprotonation of the external aldimine to another step(s), presumably to the hydrolysis of the ketimine. Gel filtration chromatography data in the presence of NaCl showed that the enzyme volume was reduced sharply with the increasing NaCl concentration, up to 100 mM. An additional increase of the NaCl concentration did not affect the elution volume, which suggests that the enzyme has a limited number of salt-binding groups. These results provide detailed mechanistic evidence for the way salts inhibit the catalytic activity of D-amino acid aminotransferase.

• Journal of Microbiology
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• 제40권1호
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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.

• 한국식품위생안전성학회지
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• 제15권1호
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• pp.51-54
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• 2000

1. E.coli에서 별도의 expression vector를 사용하지 않고도 Bacillus 균주 유래의 유용효소(D-Amino acid aminotransferase;D-AAT, Aspartate aminotransferase;AspAT, Alanine dehydrogenase;AlaDH 등)의 유전자의 5'-up stream부위의 발현 기구를 검토한 결과 각 유용 효소의 유전자들의 5'-upstream부위에 존재하는 프로모터들이 연속적으로 존재한다는 사실이 유추되었고, E.coli의 SD sequence와 매우 상동성이 높은 서열 또한 존재함을 확인하였다. 2. 유용 효소 유전자의 번역과 관련된 5'-upstream 부위의 분석을 통하여 유용 효소의 유전자들은 E.coli의 ribosomal RNA와 매우 안정한 SD pairing을 형성(D-AAT의 경우: -13.0kcal/mol, AspAT의 경우; -9.5kcal/mol, AlaDH의 경우 -15.8kca1/mol)할 수 있음을 확인하여, 이러한 높은 자유에너지 변화는 E.coli내에서 유용 효소의 번역에 기여함을 예상할 수 있다.

• Journal of Applied Biological Chemistry
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• 제56권2호
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• pp.89-93
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• 2013

Gamma-aminobutyric acid (GABA) aminotransferase (gabT) and succinic semialdehyde dehydrogenase (gabD) genes from Pseudomonas fluorescens KCCM 12537 were cloned into a single pETDuet-1 vector and co-expressed in Escherichia coli BL21(DE3) simultaneously. The mixture of both enzymes, called GABase, is the key enzyme for the enzymatic analysis of GABA. The molecular mass of the GABA aminotransferase and succinic semialdehyde dehydrogenase were determined to be 52.8 and 46.7 kDa following computations performed with the pI/Mw program, respectively. The GABase activity between pH 6.0 and 9.0 for 24 h at $4^{\circ}C$ remained over 75%, but under pH 6.0 decreased rapidly. The GABase activity between 25 and $35^{\circ}C$ by the treatment at pH 8.6 for 30 min remained over 80%, but over $35^{\circ}C$ decreased rapidly. When the activity against GABA was defined as 100%, the purified GABase activity against 5-aminovaleric acid having a similar structure to GABA showed 47.7% and GABase activity against ${\beta}$-alanine, ${\varepsilon}$-amino-n-caproic acid, $_L$-ornithine, $_L$-lysine, and $_L$-aspartic acid showed between 0.3 to 2.3%. The GABA content was analyzed with this co-expressed GABase, compared with the other GABase which was available commercially. As a result, the content of GABA extracted from brown rice, dark brown rice, and black rice were $26.4{\pm}3.5$, $40.5{\pm}4.7$ and $94.7{\pm}9.3{\mu}g/g$, which were similar data of other GABase in the error ranges.

• Asian-Australasian Journal of Animal Sciences
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• 제30권3호
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• pp.370-378
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• 2017

Objective: An experiment was conducted to investigate the effects of a specific mixture of essential oils (MEO), containing thyme, clove and cinnamon EO, on rumen microbial fermentation, nutrient apparent digestibility and blood metabolites in fistulated sheep. Methods: Six sheep fitted with ruminal fistulas were used in a repeated measurement design with two 24-d periods to investigate the effect of adding MEO at 0 (control), 0.8, and 1.6 mL/d on apparent nutrient digestibility, rumen fermentation characteristics, rumen microbial population and blood chemical metabolites. Animals were fed with a 50:50 alfalfa hay:concentrate diet. Results: Ruminal pH, total volatile fatty acids (VFA) concentration, molar proportion of individual VFA, acetate: propionate ratio and methane production were not affected with MEO. Relative to the control, Small peptides plus amino acid nitrogen and large peptides nitrogen concentration in rumen fluid were not affected with MEO supplementation; while, rumen fluid ammonia nitrogen concentration at 0 and 6 h after morning feeding in sheep fed with 1.6 mL/d of MEO was lower (p<0.05) compared to the control and 0.8 mL/d of MEO. At 0 h after morning feeding, ammonia nitrogen concentration was higher (p<0.05) in sheep fed 0.8 mL/d of MEO relative to 1.6 mL/d and control diet. Ruminal protozoa and hyper ammonia producing (HAP) bacteria counts were not affected by addition of MEO in the diet. Relative to the control, no changes were observed in the red and white blood cells, hemoglobin, hematocrit, glucose, beta-hydroxybutyric acid, cholesterol, total protein, albumin, blood urea nitrogen and aspartate aminotransferase and alanine aminotransferase concentration. Apparent total tract digestibility of dry matter, crude proten, organic matter, and neutral detergent fiber were not influenced by MEO supplementation. Conclusion:The results of the present study suggested that supplementation of MEO may have limited effects on apparent nutrient digestibility, ruminal fermentation and protozoa and HAP bacteria count, blood cells and metabolites.

• 운동영양학회지
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• 제24권4호
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• pp.28-33
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• 2020

[Purpose] This study aimed to investigate the effects of branched-chain amino acid (BCAA) supplement on delayed onset muscle soreness (DOMS) by analyzing the maximum muscle strength and indicators of muscle damage. [Methods] Twelve men with majors in physical education were assigned to the BCAA group and placebo group in a double-blinded design, and repeated measurements were conducted. DOMS was induced with an isokinetic exercise. Following BCAA administration, the changes in the knee extension peak torque, flexion peak torque, aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) concentrations were analyzed. The maximum knee muscle strength was measured at the baseline (pre-D0) following BCAA administration for 5 days before exercise (-D5, -4D, -3D, -2D, -1D). In contrast, the post-treatment measurements (D3) were recorded after BCAA administration for 3 days (post-D0, D1, D2). Blood samples were obtained before (pre-D0), immediately after (post-D0), 24 h (D1), 48 h (D2), and 72 h (D3) after the exercise to analyze the indicators of muscle strength. BCAA was administered twice daily for 8 days (5 days and 3 days before inducing DOMS and during the experimental period, respectively). [Results] There was no difference in the flexion peak torque between the groups. However, the BCAA group showed a significantly higher extension peak torque at D3 (second isokinetic exercise), compared to the placebo group (p<.05). There was no difference in AST changes between the groups. Nonetheless, the CK and LDH were significantly reduced in the BCAA group, compared to the placebo group. There was no correlation between the extension peak torque and flexion peak torque. However, the CK and LDH increased proportionately in DOMS. Moreover, their concentrations significantly increased with a decreasing peak torque (p<.01). [Conclusion] An exercise-induced DOMS results in a decrease in the peak torque and a proportional increase in the CK and LDH concentrations. Moreover, the administration of BCAA inhibits the reduction of the extension peak torque and elevation of CK and LDH concentrations. Therefore, BCAA might be administered as a supplement to maintain the muscle strength and prevent muscle damage during vigorous exercises that may induce DOMS in sports settings.

• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.710-716
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• 2016

Gamma-aminobutyric acid (GABA) is a non-protein amino acid, which is an important inhibitor of neurotransmission in the human brain. GABA is also used as the precursor of biopolymer Nylon-4 production. In this study, the carbon flux from the tricarboxylic acid cycle was directed to the GABA shunt pathway for the production of GABA from glucose. The GABA shunt enzymes succinate-semialdehyde dehydrogenase (GabD) and GABA aminotransferase (GabT) were co-localized along with the GABA transporter (GadC) by using a synthetic scaffold complex. The co-localized enzyme scaffold complex produced 0.71 g/l of GABA from 10 g/l of glucose. Inactivation of competing metabolic pathways in mutant E. coli strains XBM1 and XBM6 increased GABA production 13% to reach 0.80 g/l GABA by the enzymes co-localized and expressed in the mutant strains. The recombinant E. coli system developed in this study demonstrated the possibility of the pathway of the GABA shunt as a novel GABA production pathway.

• 한국미생물·생명공학회지
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• 제35권1호
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• pp.11-16
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• 2007

Pseudoalteromonas carrageenovora 유래의 arylsulfatase 유전자는 PCR로 증폭한 후 Geobacillus toebii의 D-amino acid aminotransferase(D-ATT) 유전자 유래의 구성적 발현 promoter를 함유하는 pHCE-IA vector로 subcloning 하였다. 4-Methylumbelliferyl sulfate가 포함된 LB 평판배지 상에서 자란 형질전환체 Escherichia coli BL2l (DE3)/pHCE-AST는 360 nm상에서 4-methylumbellifrrone에 의한 강한 형광을 보였고, 이는 대장균에서 arylsulfatase가 활성형으로 생산되었음을 의미하였다. E. coli BL21 (DE3)/pHCE-AST를 0.4% glycerol 또는 0.4% glucose가 포함된 LB 배지로 배양했을 때 arylsulfatase활성은 glycerol이 포함된 배지에서 활성이 더 높게 나타났다. 2% glycerol이 포함된 LB배지에서 arylsulfatase 활성은 약 15.0 unit/ml에 달했으며, 이는 1% glycerol을 첨가해서 배양했을 때보다 2.6배 이상의 높은 발현 수준이였다. 재조합 arylsulfatase 효소로 제조된 agarose와 시판용 agarose를 DNA markers를 이용해서 전기영동 성능을 비교했을 때 우수한 이동성과 분리능을 보였다. 본 연구의 결과, E. coli에서 과발현 생산된 arylsulfatase 효소를 이용하여 전기영동용 고순도 agarose생산 공정에 적용 가능함을 확인하였다.