• Journal of Microbiology and Biotechnology
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• 제7권6호
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• pp.435-437
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• 1997

The role of $Ca^{2+}$ in making functional malate synthase in Corynebacterium glutamicum was investigated using the cloned DNA coding for the enzyme. Introduction of cloned aceB into C. glutamicum overexpressed malate synthase as judged by SDS-PAGE. However, the increase in enzyme activity of the expressed malate synthase did not match the level of overexpression observed in SDS-PAGE. Addition of $Ca^{2+}$ to the growth medium specifically increased the activity. The malate synthase could be stained with ruthenium red in a $Ca^{2+}$-specific manner. This agrees with the previous observation which reported a potential $Ca^{2+}$-binding domain in the N-terminal region of the protein.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.547-552
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• 2004

Streptomyces thermovulgans malate synthase (stMS) is known to be more thermostable and thermoactive than S. coelicolor malate synthase (scMS). Therefore, based on the amino acid sequence of stMS, 3 scMS mutants, namely P186R, T8PL9P, and T8PL9PP186R, were created by site-directed mutagenesis in an attempt to engineer a more thermoactive and thermostable enzyme. An enzymatic analysis of the wild-type and mutant MS revealed that P186R and T8PL9PP186R were more thermoactive than the wild-type scMS and T8PL9P. Furthermore, all 3 mutants exhibited a greater thermo stability than scMS, thereby suggesting that both R186 and P8P9 can cause increased thermo stability in scMS.

• Journal of Microbiology and Biotechnology
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• 제4권4호
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• pp.256-263
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• 1994

The aceB gene, encoding for malate synthase, one of the key enzymes of glyoxylate bypass, was isolated from a pMT1-based Corynebacterium glutamicum gene library via complementation of an Escherichia coli aceB mutant on an acetate minimal medium. The aceB gene was closely linked to aceA, separated by 598 base pairs, and transcribed in divergent direction. The aceB expressed a protein product of Mr 83, 000 in Corynebacterium glutamicum which was unusually large compared with those of other malate synthases. A DNA-sequence analysis of the cloned DNA identified an open-reading frame of 2, 217 base pairs which encodes a protein with the molecular weight of 82, 311 comprising 739 aminoo acids. The putative protein product showed only limited amino acid-sequence homology to its counteliparts in other organisms. The N-terminal region of the protein, which shows no apparent homology with the known sequences of other malate synthases, appeared to be responsible for the protein s unusually large size. A potential calciumbinding domain of EF-hand structure found among eukaryotes was detected in the N-terminal region of the deduced protein.

• Journal of Microbiology and Biotechnology
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• 제20권6호
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• pp.1006-1010
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• 2010

Pseudomonas putida JM37 metabolized glyoxylate at a specific rate of 55 g/g dry biomass/day. In order to investigate their role, three genes encoding enzymes that are potentially involved in the conversion of glyoxylate were disrupted; namely, tartronate semialdehyde synthase (gcl), malate synthase (glcB), and isocitrate lyase (aceA). Strains with transposon insertion in either of these genes were isolated from a 50,000 clone library employing a PCR-guided enrichment strategy. In addition, all three double mutants were constructed via targeted insertion of a knock-out plasmid. Neither mutation of gcl, glcB, and aceA nor any of the respective double mutations influenced glyoxylic acid conversion, indicating that P. putida JM37 may possess other enzymes and pathways for glyoxylate metabolism.

• 미생물학회지
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• 제23권3호
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• pp.230-234
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• 1985

Malonate를 유일한 탄소원으로 활용할 수 있는 세균을 토양으로부터 분리하였다. 이 세균은 행태, 배양, 생리 그리고 생화학적 연구를 통하여 Acinetobacter calcoaceticus임이 확인되었다. 이 미생물을 malonate를 유일한 탄소원으로 하는 배지에서 배양하였을 갱우, malonyl CoA synthetase, isocitrate lyase 및 malate, synthase가 유도 되었다. 따라서 이 미생물에서도 Pseudomonas fluorescens에서 제안되었던 대사경로 즉 $malonate{\rightarrow}malonyl-CoA{\rightarrow}acetyl-CoA{\rightarrow}glyoxylate\;cycle$을 통하여 malonate를 이용하는 것으로 판단된다.

• International Journal of Oral Biology
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• 제42권2호
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• pp.55-61
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• 2017

Recent studies indicate that mitochondria are an important source of reactive oxygen species (ROS) in the spinal dorsal horn. In our previous study, application of malate, a mitochondrial electron transport complex I substrate, induced a membrane depolarization, which was inhibited by pretreatment with ROS scavengers. In the present study, we used patch clamp recording in the substantia geletinosa (SG) neurons of spinal slices, to investigate the cellular mechanism of mitochondrial ROS on neuronal excitability. DNQX (an AMPA receptor antagonist) and AP5 (an NMDA receptor antagonist) decreased the malate-induced depolarization. In an external calcium free solution and addition of tetrodotoxin (TTX) for blockade of synaptic transmission, the malate-induced depolarization remained unchanged. In the presence of DNQX, AP5 and AP3 (a group I metabotropic glutamate receptor (mGluR) antagonist), glutamate depolarized the membrane potential, which was suppressed by PBN. However, oligomycin (a mitochondrial ATP synthase inhibitor) or PPADS (a P2 receptor inhibitor) did not affect the substrates-induced depolarization. These results suggest that mitochondrial substrate-induced ROS in SG neuron directly acts on the postsynaptic neuron, therefore increasing the ion influx via glutamate receptors.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.542-549
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• 2010

In the present study, overexpression, purification, and characterization of Aeropyrum pernix K1 chaperonin B in E. coli were investigated. The chaperonin $\beta$-subunit gene (ApCpnB, 1,665 bp ORF) from the hyperthermophilic archaeon A. pernix K1 was amplified by PCR and subcloned into vector pET21a. The constructed pET21a-ApCpnB (6.9 kb) was transformed into E. coli BL21 Codonplus (DE3). The transformant cell successfully expressed ApCpnB, and the expression of ApCpnB (61.2 kDa) was identified through analysis of the fractions by SDS-PAGE (14% gel). The recombinant ApCpnB was purified to higher than 94% by using heat-shock treatment at $90^{\circ}C$ for 20 min and fast protein liquid chromatography on a HiTrap Q column step. The purified ApCpnB showed ATPase activity and its activity was dependent on temperature. In the presence of ATP, ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}$ and $50^{\circ}$, respectively. Specifically, the activity of malate dehydrogenase (MDH) at $85^{\circ}$ was greatly stabilized by the addition of ApCpnB and ATP. Coexpression of pro-carboxypeptidase B (pro-CPB) and ApCpnB in E. coli BL21 Codonplus (DE3) had a marked effect on the yield of pro-CPB as a soluble and active form, speculating that ApCpnB facilitates the correct folding of pro-CPB. These results suggest that ApCpnB has both foldase and holdase activities and can be used as a powerful molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.

• Journal of Applied Biological Chemistry
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• 제42권1호
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• pp.1-6
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• 1999

Poly-${\beta}$-hydroxybutyrate (PHB) and enzymes related PHB metabolism have been measured in nitrogen-fixing symbiosis of chickpea and cowpea plants. Bacteroids from chickpea and cowpea contained PHB to 0.8% and 43% of their dry weight, respectively, whereas the free-living cells CC 1192 and I 16 produced $285{\pm}55mg$ and $157{\pm}18mg$ of PHB g (dry weight)$^{-1}$. To further understand why chickpea bacteroids contained little PHB, the enzyme activities of PHB metabolism (3-ketothiolase, acetoacetyl-CoA reductase, PHB depolymerase, and 3-hydroxybutyrate dehydrogenase), the TCA cycle (malate dehydrogenase, citrate synthase, and isocitrate dehydrogenase), and related reactions (malic enzyme, pyruvate dehydrogenase, and glutamate:2-oxoglutarate transaminase) were compared in extracts from chickpea and cowpea bacteroids and the respective free-living bacteria. Significant differences were observed between chickpea and cowpea bacteroids and between the bacteroid and free-living forms of CC 1192, with respect to the capacity for some of these reactions. It is indicated that a greater potential for oxidizing malate to oxaloacetate in chickpea bacteroids could be a factor that favors the utilization of acetyl-CoA in TCA cycle rather than for PHB synthesis.

• 생명과학회지
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• 제31권8호
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• pp.778-785
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• 2021

오이 씨앗의 발아는 세포의 지방체내 저장지방의 분해 결과인 acyl-CoA의 글라이옥시좀으로 이동 후 베타 산화의 결과물인 acetyl-CoA의 글라이옥실산 회로로의 유입과 지방의 유동으로 촉발된다. Acetyl-CoA는 글라이옥실산 회로의 가동을 위한 탄소원을 제공하며 시트르산과 말산을 생성하며 글라이옥실산 회로의 작동을 유도한다. 지방 저장 종자의 발아에 있어서 글라이옥실산 회로는 필수적 요소이며, 그 결과물인 말산 및 숙신산의 미토콘드리아로의 이동은 TCA 회로의 가동과 옥살초산의 생성 및 세포질로의 유동으로 PEPCK에 의한 당신생을 가능하게 한다. 즉, 저장 지방을 원료로 여러 대사물질의 생산 및 이동과 다중의 대사경로를 통하여 발아 시 사용 가능한 에너지원인 포도당의 형태로 전환이 이루어진다. 이에 동반하여 많은 유전자의 발현 조절이 이루어지고, 세포내 소기관 특히 미소체로 대표되는 글라이옥시좀은 말산 합성효소(malate synthase)와 이소 시트르산 분해효소(isocitrate lyase)로 특화된다. 또 다른 acetyl-CoA의 유동은 carnitine을 매개로 하는 BOU (A BOUT DE SOUFFLE)의 작동이다. 이것은 카니틴의 대사와 관련하여 고등식물의 발달과 대사과정에서의 중요성이 확인된 것으로 사료된다.

• Journal of Ginseng Research
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• 제9권2호
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• pp.221-231
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• 1985

The effect of ginseng saponin on the activities of isocitrate lyase, palate synthase, succinate dehydrogenase, malate dehydrogenase and lipase have been investigated at the early phase of germinating soy-bean sprout and found that all the above enzymes were stimulated when the bean was rinsed for 24 hours with 10-4% saponin solution. The length of the saponin treated group was not longer than that of control group but the weight of the former was heavier (15%) than the latter. Total sugar content of test group was always much higher than that of control. From the above results, it was concluded that ginseng saponin might stimulate several enzymes of Soybean sprout during germination resulting in rapid growth of the Soybean sprout.