• 생명과학회지
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• 제25권11호
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• pp.1290-1297
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• 2015

에탄올 생산 세균 Zymomonas mobilis에서 에탄올 생산 경로의 핵심으로 작용하는 효소인, pyruvate decarboxylase(pdc) 유전자의 불활성 실험을 통해, PDC 활성이 50% 감소된 PDC 활성 변형균주가 분리되었다. 이러한 균주들의 에탄올 탄소대사 흐름이 고부가가치 화합물인 피루브산, 숙신산 및 젖산 등으로 전환되는지를 발효 실험을 통해 평가하였다. 하지만 pdc의 발현을 중지시키기 위해 cat-삽입형-pdc와 pdc-결손형 아형 유전자를 전기천공법을 이용해 야생형 균주 ZM4의 염색체에 이식하기 위한 다수의 시도에도 불구하고, 이러한 방법을 통해 분리된 균주들은 대부분 부분적 유전자 불활성 특성을 보였으며, PDC 활성이 완전히 손실된 삭제 돌연변이 균주를 획득할 수는 없었다. PDC활성이 변형된 돌연변이 균주의 발효 실험에서, 야생형 균주와 비교 시 감소된 PDC 효소 활성의 변화로 인해 기질 흡수율과 에탄올 생산율이 감소되어 피루브산 생산이 약 2.5 g l^-1 정도로 증가함을 확인하였으나, 젖산과 숙신산의 생산에 현저한 농도 변화를 보이지 못했다. 이러한 결과는 Z. mobilis의 산화환원 에너지가 PDC 효소 활성에 의한 에탄올 생산 경로에 전적으로 의존하여 발생한다는 것을 암시하였다. 상기 결과를 토대로 pdc 유전자의 완전한 불활성 유도와 산화환원 에너지의 균형은, 젖산 생산을 위한 lactate dehydrogenase, 숙신산 생산을 위한 pyruvate dehydrogenase와 malic enzyme과 같은 효소의 활성 증가를 통해, 세포내 NAD와 NADH 농도의 산화환원 균형이 이루어져야 발생할 수 있음을 시사하였다.

• BMB Reports
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• 제32권1호
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• pp.39-44
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• 1999

Pyruvate decarboxylase (PDC) catalyzes the conversion of pyruvate to acetaldehyde as the penultimate step in alcohol fermentation. The enzyme requires two cofactors, thiamin diphosphate (ThDP) and $Mg^{2+}$, for activity. Zymomonas mobilis PDC shows a strong preference for pyruvate although it will use the higher homologues 2-ketobutyrate and 2-ketovalerate to some extent. We have investigated the effect of mutagenesis of valine 111 and leucine 112 on the substrate specificity. V111 was replaced by glycine, alanine, leucine, and isoleucine while L112 was replaced by alanine, valine, and isoleucine. With the exception of L112I, all mutants retain activity towards pyruvate with $k_{cat}$ values ranging from 40% to 139% of wild-type. All mutants show changes from wild-type in the affinity for ThDP, and several (V111A, L112A, and L112V) show decreases in the affinity for $Mg^{2+}$. Two of the mutants, V111G and V111A, show an increase in the $K_m$ for pyruvate. The activity of each mutant towards 2-ketobutyrate and 2-ketovalerate was investigated and some changes from wild-type were found. For the V111 mutants, the most notable of these is a 3.7-fold increase in the ability to use 2-ketovalerate. However, the largest effect is observed for the L112V mutation which increases the ability to use both 2-ketobutyrate (4.3-fold) and 2-ketovalerate (5.7-fold). The results suggest that L112 and, to a lesser extent, V111 are close to the active site and may interact with the alkyl side-chain of the substrate.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 춘계학술발표대회
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• pp.81-84
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• 2000

• Journal of Microbiology and Biotechnology
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• 제21권10호
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• pp.1049-1052
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• 2011

In this study, we demonstrated the asymmetric synthesis of L-tert-leucine from trimethylpyruvate using branched-chain aminotransferase (BCAT) from Escherichia coli in the presence of L-glutamate as an amino donor. Since BCAT was severely inhibited by 2-ketoglutarate, in order to overcome this here, we developed a BCAT/aspartate aminotransferase (AspAT) and BCAT/AspAT/pyruvate decarboxylase (PDC) coupling reaction. In the BCAT/AspAT/PDC coupling reaction, 89.2 mM L-tert-leucine (ee>99%) was asymmetrically synthesized from 100 mM trimethylpyruvate.

• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.251-259
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• 2013

An endoxylanase gene (PoxynA) that belongs to the glycoside hydrolase (GH) family 11 was cloned from a xylanolytic strain, Penicillium oxalicum B3-11(2). PoxynA was overexpressed in Trichoderma reesei QM9414 by using a constitutive strong promoter of the encoding pyruvate decarboxylase (pdc). The high extracellular xylanase activities in the fermentation liquid of the transformants were maintained 29~35-fold higher compared with the wild strain. The recombinant POXYNA was purified to homogeneity, and its characters were analyzed. Its optimal temperature and pH value were $50^{\circ}C$ and 5.0, respectively. The enzyme was stable at a pH range of 2.0 to 7.0. Using beechwood as the substrate, POXYNA had a high specific activity of $1,856{\pm}53.5$ IU/mg. In the presence of metal ions, such as $Cu^{2+}$, and $Mg^{2+}$, the activity of the enzyme increased. However, strong inhibition of the enzyme activity was observed in the presence of $Mn^{2+}$ and $Fe^{2+}$. The recombinant POXYNA hydrolyzed birchwood xylan, beechwood xylan, and oat spelt xylan to produce short-chain xylooligosaccharides, xylopentaose, xylotriose, and xylobiose as the main products. This is the first report on the expression properties of a recombinant endoxylanase gene from Penicillium oxalicum. The properties of this endoxylanase make it promising for applications in the food and feed industries.