• 한국미생물·생명공학회지
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• 제34권1호
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• pp.35-39
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• 2006

높은 C4 대사활성을 보이는 반추위미생물이 가지는 포도당 발효대사 조절양식의 한가지를 대장균에서 모사하였다. 대장균은 glycolytic condition에서는 phosphoenolpyruvate(PEP) ${\leftrightarrow}$ oxaloacetate(OAA)간 반응을 phosphenolpyruvate carboxylase(PPC)에 의해, gluconeogenetic condition에서는 phosphoenolpyruvate carboxykinase(PCK)에 의해 촉매하도록 조절한다. 반면 반추위미생물은 glycolytic condition에서 PCK를 통하여 반응이 촉매된다. 이러한 조절양식의 차이점이 C4 대사활성에 미치는 영향을 조사하기 위하며 ppc가 돌연변이되고 대신 인위적으로 PCK를 발현할 수 있는 대장균을 제조하였다. 이렇게 PEP-OAA간 대사조절이 변이된 대장균 K12 ppc-/pck+는 야생형 K12보다 2.5배의 높은 C4대사활성을 보였다. 대장균에서의 C4 대사생리를 증가시키는 연구는 대사공학을 이용한 여러가지 유용물질(i.e. 숙신산, ALA)생산에 응용하기 위한 기초자료로 활용될 수 있을 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1385-1389
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• 2007

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the formation of EPSP and inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in the biosynthesis of aromatic amino acids. To delineate the domain-specific function, we successfully isolated the discontinuous C-terminal domain (residues 1-21, linkers, 240-427) of EPSP synthase (427 residues) by site-directed mutagenesis. The engineered C-terminal domains containing no linker (CTD), or with gly-gly ($CTD^{GG}$) and gly-ser-ser-gly ($CTD^{GSSG}$) linkers were purified and characterized as having distinct native-like secondary and tertiary structures. However, isothermal titration calorimetry (ITC), $^{15}N-HSQC$,\;and\;^{31}P-NMR$ revealed that neither its substrate nor inhibitor binds the isolated domain. The isolated domain maintained structural integrity, but did not function as the half of the full-length protein.

• Journal of Microbiology and Biotechnology
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• 제6권1호
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• pp.43-49
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• 1996

Modulation of the catabolic PEP-pathway of Anaerobiospirillum succiniciproducens was tried using some enzymatic inhibitors such as gases and chemicals in order to enhance succinic acid production. 10$\%$ CO increased the succinic acid/acetic acid (S/A) ratio but inhibited growth as well as production of succinic and acetic acid. Hydrogen gas also increased the S/A ratio and inhibited the synthesis of pyruvate: ferredoxin oxidoreductase when used in mixture with $CO_2$, Catabolic repression by acetic, lactic and formic acid was not recognized and other modulators such as glyoxylate, pyruvate derivatives, arsenic salt, phosphate and sulfate were shown not to be effective. Magesium carbonate was shown effective for repressing acetate production. Palmitic acid, myristic acid and phenylalanine did not affect acetate production but carprylic acid completely inhibited growth.

• Journal of Microbiology and Biotechnology
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• 제10권4호
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• pp.496-501
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• 2000

Escherichia cole NZN111, which is known as a pfl ldhA double mutant strin, was metabolically engineered to produce succinic acid by overexpressing malic enzyme into the E. coli controlled by a trc promoter. Fermentation studies were carried out in a LB medium by first growing cells aerobically to an $OD_{600}$ of 5. At this point, 0.01 mM IPTG was added to induce the overexpression of malic enzyme and the agitation speed was gradually lowered. When the culture $OD_{600}$ reached 11, a complete anaerobic condition was achieved by flushing with a $CO_3-H_2$ gas mixture. When NZN111(pTrcML) was cultured at $37^{\circ}C$, the final succinic acid concentration of 2.8 g/l could be obtained after 30 h of anaerobic cultivation. The fermentation results were analyzed by the calculation of metabolic fluxes. Metaolic flux analysis showed that about 85% of phosphoenolpyruvate (PEP) was converted to pyruvate, and further converted to malic acid by malic enzyme.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1582-1589
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• 2009

Bacterial UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridine diphospho-N-acetylglucosamine (UNAG), which is the first step of bacterial cell wall synthesis. We identified thimerosal, thiram, and ebselen as effective inhibitors of Haemophilus influenzae MurA by screening a chemical library that consisted of a wide range of bioactive compounds. When MurA was preincubated with these inhibitors, their 50% inhibitory concentrations ($IC_{50}s$) were found to range from 0.1 to $0.7\;{\mu}M$. In particular, thimerosal suppressed the growth of several different Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium at a concentration range of $1-2\;{\mu}g/ml$. These inhibitors covalently modified the cysteine residue near the active site of MurA. This modification changed the open conformation of MurA to a more closed configuration, which may have prevented the necessary conformational change from occurring during the enzyme reaction.

• Journal of Microbiology and Biotechnology
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• 제18권4호
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• pp.746-753
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• 2008

The hprK gene encoding bifunctional HPrK/P (kinase/phosphorylase) was cloned from L. mesenteroides SY1, a strain isolated from kimchi. hprK was transcribed as a monocistronic gene. His-tagged HPrH16A and HPrK/P were produced in E. coli BL21 (DE3) using pET26b(+) and purified. HPrK/P phosphorylation assay with purified proteins showed that the kinase activity of HPrK/P increased at slightly acidic pHs. Divalent cations such as $Mg^{2+}$ and $Mn^{2+}$ and glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and phosphoenolpyruvate (PEP) increased the kinase activity of HPrK/P, but inorganic phosphate strongly inhibited it. Kinetic studies for the kinase activity of HPrK/P showed that the apparent $K_m$ values were 0.18 and $14.57{\mu}M$ for ATP and HPr, respectively. The $K_m$ value for the phosphorylase activity of HPrK/P was $14.16{\mu}M$ for P-Ser-HPr (HPr phosphorylated at the serine residue).

• Journal of Microbiology and Biotechnology
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• 제16권6호
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• pp.870-879
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• 2006

The present study describes the formation of succinic acid by a nonvirulent, highly osmotolerant Klebsiella pneumoniae strain SAP (succinic acid producer), its profile of metabolites, and enzymes of the succinate production pathway. The strain produced succinate along with other metabolites such as lactate, acetate, and ethanol under aerobic as well as anaerobic growth conditions. The yield of succinate was higher in the presence of $MgCO_3$ under $N_2$ atmosphere as compared with that under $CO_2$ atmosphere. Analysis of intracellular metabolites showed the presence of a smaller PEP pool than that of pyruvate. Oxaloacetate, citrate, and $\alpha$-ketoglutarate pools were considerably larger than those of isocitrate and fumarate. In order to understand the synthesis of succinate, the enzymes involved in end-product formation were studied. Levels of phosphoenolpyruvate carboxykinase, fumarate reductase, pyruvate kinase, and acetate kinase were higher under anaerobic growth conditions. Based on the profiles of the metabolites and enzymes, it was concluded that the synthesis of succinate took place via oxaloacetate, malate, and fumarate in the strain under anaerobic growth conditions. The strain SAP showed potential for the bioconversion of fumarate to succinate under $N_2$ atmosphere in the presence of $MgCO_3$. At an initial fumarate concentration of 10 g/l, 7.1 g/l fumarate was converted to 7 g/l succinate with a molar conversion efficiency of 97.3%. The conversion efficiency and succinate yield were increased in the presence of glucose. Cells grown on fumarate contained an 18-fold higher fumarate reductase activity as compared with the activity obtained when grown on glucose.

• BMB Reports
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• 제38권5호
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• pp.584-590
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• 2005

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Aspergillus aculeatus, a filamentous fungus previously isolated from infected tongue of a patient. The enzyme, apparently homogeneous, had a specific activity of $220\;units\;mg^{-1}$/, a molecular weight of $105,000{\pm}5,000$ Dal by gel filtration and subunit size of $52,000{\pm}1,100$ Dal by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The substrate specificity was extremely strict, with glucose 6-phosphate (G6P) being oxidized by nicotinamide adenine dinucleotide phosphate (NADP) only. At assay pH of 7.5, the enzyme had $K_m$ values of $6\;{\mu}m$ and $75\;{\mu}m$ for NADP and G6P respectively. The $k_{cat}$ was $83\;s^{-1}$. Steady-state kinetics at pH 7.5 produced converging linear Lineweaver-Burk plots as expected for ternary-complex mechanism. The patterns of product and dead-end inhibition suggested that the enzyme can bind NADP and G6P separately to form a binary complex, indicating a random-order mechanism. The enzyme was irreversibly inactivated by heat in a linear fashion, with G6P providing a degree of protection. Phosphoenolpyruvate (PEP), adenosinetriphosphate (ATP), and fructose 6-phosphate (F6P), in decreasing order, are effective inhibitors. Zinc and Cobalt ions were effective inhibitors although cobalt ion was more potent; the two divalent metals were competitive inhibitors with respect to G6P, with $K_i$ values of $6.6\;{\mu}m$ and $4.7\;{\mu}m$ respectively. It is proposed that inhibition by divalent metal ions, at low NADPH /NADP ratio, is another means of controlling pentosephosphate pathway.

• 한국미생물·생명공학회지
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• 제12권4호
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• pp.277-283
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• 1984

제방 효모로 부터 분리 정제한 adenylate kinase 는 한개의 기질에 의해 또 하나의 기질 결합을ADP생성 반응에서는 4배, AMP와 Mg·ATP 생성에서는 2배 촉진되었다. 기질 특이성에 있어서는 nucleotide monophosphale일 경우 dAMP만이 활성을 보여주었으며 nucleotide triphosphate일 경우 ATP이외 UTP, ITP, GTP의 순위로 활성이 높았다. AMP와 Mg·ATP가 기질일 경우 과잉의 AMP는 pH 7.2와 pH8.0에서는 Mg·ATP에 경쟁적으로 저해하였으며 pH가 높을수록 그 Ki정수는 낮았다. Phosphoenolpyruvate는 AMP에 대해 경쟁적 Mg·ATP에 대해서는 비 경쟁적 저해제 이었으며 Adenosine pentaphosphoadenosine은 모든 기질에 대해 경쟁적 저해제로 작용하였다. 제빵 효모로부터의 adenylate kinase는 아미노산 조성에 있어서 동물의 Mitochondria형에 가까우며 Ito등의 결과와 일치하지 않았다. 상기와 같은 효소학적 성질을 종합 고찰한 결과 효모 adenylate kinase는 동물의 Mitochondria형 효소로 분류할 수 있으며 효모 adenylate kinase에 있어 연구자 상호간의 차이점은 사용한 균주의 차이에 기인하는 것으로 생각된다.