• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.571-578
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• 2018

Biofuel production using lignocellulosic biomass is gaining attention because it can be substituted for fossil fuels without competing with edible resources. However, because Saccharomyces cerevisiae does not have a ${\text\tiny{D}}$-xylose metabolic pathway, oxidoreductase or isomerase pathways must be introduced to utilize ${\text\tiny{D}}$-xylose from lignocellulosic biomass in S. cerevisiae. To elucidate the biochemical properties of xylose isomerase (XI) from Piromyces sp. E2 (PsXI), we determine its crystal structure in complex with substrate mimic glycerol. An amino-acid sequence comparison with other reported XIs and relative activity measurements using five kinds of divalent metal ions confirmed that PsXI belongs to class II XIs. Moreover kinetic analysis of PsXI was also performed using $Mn^{2+}$, the preferred divalent metal ion for PsXI. In addition, the substrate-binding mode of PsXI could be predicted with the substrate mimic glycerol bound to the active site. These studies may provide structural information to enhance ${\text\tiny{D}}$-xylose utilization for biofuel production.

• KSBB Journal
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• 제19권3호
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• pp.226-230
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• 2004

볏짚 가수분해물로부터 xylitol 생산능력이 가장 우수하다고 알려진 Candida mogii ATCC 18364의 xylose 발효특성에 관한 기초연구를 수행하였다. 상업적인 자일로즈로부터 C. mogii게 의해 xylitol을 생산할 때 수율과 속도에 미치는 여러 영향을 검토한 결과, 포도당을 함유한 기질에서 12시간 배양한 종균을 3.0 g/L의 농도로 접종하여 발효시켰을 때 가장 좋은 결과를 얻었다. 또한 xylose 발효에서 가장 중요한 요인인 용존산소 농도의 영향을 알아보기 위해, 2 L 발효조에서 통기량과 교반속도를 조절해 가면서 xylitol 생산을 비교 검토하였다. 농축균과 10% 접종균을 사용했을 경우 모두, 1 vvm의 통기량과 200 rpm의 교반속도에서 xylitol 생산이 가장 우수하였다.

• 한국수산과학회지
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• 제19권3호
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• pp.212-218
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• 1986

In order to isolate antioxigenic substrates, the browning reaction products of xylose and various amino acids were analysed by TLC and dialysis. Rf values of browning reaction products of xylose and hydrophobic amino acids separated on silica gel TLC plate were shown in the range of 0.38 to 0.56 and that of basic amino acids was around 0.2. Browning reaction products made from xylose and Trp were separated on TLC into four bands with Rf values of 0.25, 0.55, 0.81 and 0.91 respectively. Among these the bands with Rf values of 0.25 and 0.55 appeared having strong antioxidant activity. The band of Rf 0.55 which showed the highest activity was positive to Prochazka reagent and had an absorption maximum at 275 nm. In dialysis of the xylose-Trp browning reaction products, the undialysed fraction (inner solution) was repsponsible to the antioxidant activity, which was separated into two bands with Rf values of 0.25 and 0.55 on TLC. The inner fractions of the browning products of xylose and His or Arg were also apparent in antioxdant activity.

• 펄프종이기술
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• 제44권3호
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• pp.9-14
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• 2012

Formed fermentation inhibitors during acid saccharification leads to poor alcohol production based on lignocellulosic bio-alcohol production process. In this work, it is focused on the formation of fermentation inhibitors from xylan, which is influenced by reaction tempearature and time of acidic sacharifiaction of xylose and glucuronic acid. In second step of concentrated acid hydrolysis, part of xylose and glucuronic acid was converted to furfuraldehyde and formic acid by dehydration and rearrangement reactions. Furfural was form from xylose, which was highly sensitive to reaction temperature. Formic acid was come from both xylose and glucuronic acid, which supposed to main inhibitor in biobutanol fermentation. Reaction temperature of second hydrolysis was main variables to control the furfural and formic acid generation. Careful control of acid saccharification can reduce generation of harmful inhibitors, especially second step of concentrated sulfuric acid hydrolysis process.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.560-564
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• 2013

${\small{D}}$-Ribose is a value-added five-carbon sugar used for riboflavin production. To investigate the effects of oxygen supply and mixed sugar concentration on microbial production of ${\small{D}}$-ribose, a transketolase-deficient Bacillus subtilis SPK1 was cultured batch-wise using xylose and glucose. A change of agitation speed from 300 rpm to 600 rpm at 1 vvm of air supply increased both the xylose consumption rate and ${\small{D}}$-ribose production rate. Because the sum of the specific consumption rates for xylose and glucose was similar at all agitation speeds, metabolic preferences between xylose and glucose might depend on oxygen supply. Although B. subtilis SPK1 can take up xylose and glucose by the active transport mechanism, a high initial concentration of xylose and glucose was not beneficial for high ${\small{D}}$-ribose production.

• Current Research on Agriculture and Life Sciences
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• 제32권4호
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• pp.199-202
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• 2014

The bioconversion of cellulosic biomass hydrolyzates consisting mainly of glucose and xylose requires the use of engineered Saccharomyces cerevisiae expressing a heterologous xylose pathway. However, there is concern that a fungal xylose pathway consisting of NADPH-specific xylose reductase (XR) and $NAD^+$-specific xylitol dehydrogenase (XDH) may result in a cellular redox imbalance. However, the glycerol biosynthesis and glycerol degradation pathways of S. cerevisiae, termed here as the glycerol cycle, has the potential to balance the cofactor requirements for xylose metabolism, as it produces NADPH by consuming NADH at the expense of one mole of ATP. Therefore, this study tested if the glycerol cycle could improve the xylose metabolism of engineered S. cerevisiae by cofactor balancing, as predicted by an in-silico analysis using elementary flux mode (EFM). When the GPD1 gene, the first step of the glycerol cycle, was overexpressed in the XR/XDH-expressing S. cerevisiae, the glycerol production significantly increased, while the xylitol and ethanol yields became negligible. The reduced xylitol yield suggests that enough $NAD^+$ was supplied for XDH by the glycerol cycle. However, the GPD1 overexpression completely shifted the carbon flux from ethanol to glycerol. Thus, moderate expression of GPD1 may be necessary to achieve improved ethanol production through the cofactor balancing.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1944-1949
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• 2020

Mutant sugar transporter ScGAL2-N376F was overexpressed in Kluyveromyces marxianus for efficient utilization of xylose, which is one of the main components of cellulosic biomass. K. marxianus ScGal2_N376F, the ScGAL2-N376F-overexpressing strain, exhibited 47.04 g/l of xylose consumption and 26.55 g/l of xylitol production, as compared to the parental strain (24.68 g/l and 7.03 g/l, respectively) when xylose was used as the sole carbon source. When a mixture of glucose and xylose was used as the carbon source, xylose consumption and xylitol production rates were improved by 195% and 360%, respectively, by K. marxianus ScGal2_N376F. Moreover, the glucose consumption rate was improved by 27% as compared to that in the parental strain. Overexpression of both wild-type ScGAL2 and mutant ScGAL2-N376F showed 48% and 52% enhanced sugar consumption and ethanol production rates, respectively, when a mixture of glucose and galactose was used as the carbon source, which is the main component of marine biomass. As shown in this study, ScGAL2-N376F overexpression can be applied for the efficient production of biofuels or biochemicals from cellulosic or marine biomass.

• Journal of Applied Biological Chemistry
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• 제53권1호
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• pp.1-7
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• 2010

xylA 프로모터는 대장균의 xylose 대사에 관여하는 xylose 오페론 상의 중요한 프로모터이다. 이 프로모터는 xylose에 의해 강하게 조절을 받는다고 알려져 있다. 이러한 특징은 새로운 발현 백터를 구축하는데 충분한 조건을 갖추고 있다고 생각된다. 본 연구에서는 이러한 xylose에 의해 유도 되는 발현벡터를 구축하기 위하여 600 bp의 xylA 프로모터를 증폭하여 pUC18의 AatII와 HindIII 사이에 삽입하여 pXA600을 구축하였다. 또한 조절단백질인 XylR의 영향을 조사하기 위하여 xylR 유전자를 삽입하여 pXAR600을 구축하였다. 발현의 강도를 측정하기 위하여 3,048 bp의 lacZ유전자를 xylA 프로모터의 하류에 연결하여 pXA600-lacZ와 pXAR600-lacZ를 구축하고 대장균 JM109에 형질전환시켰다. 구축된 pXA600-lacZ와 pXAR600-lacZ는 LB 배지에서 배양하였을 때 xylose 유도하에서 각각 1,641 unit와 2,304 unit의 $\beta$-galactosidase 활성을 보였으며, DM 배지상에서 배양했을 때 xylose 유도 시 각각 6,282 unit와 9,320 unit의 $\beta$-galactosidase 활성을 보였다. 또한 왜래 유전자의 발현 가능성을 확인하기 위하여 S. thermocyaneoviolaceus의 내열성 xylanase를 코딩하는 xynA 유전자를 실제로 구축된 pXA600과 pXAR600에서 발현을 확인하여 pXA600 및 pXAR600이 새로운 xylose 유도성 발현벡터로서의 사용 가능성을 확인하였다.

• Applied Biological Chemistry
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• 제39권6호
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• pp.430-436
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• 1996

대장균에서 xylose isomerase(XI) 생산의 조절양상을 밝히기 위한 연구의 일환으로 유도물질인 xylose에 의한 XI 생산유도 및 glucose에 의한 이화물 억제 양상을 조사하였다. XI 생산 유전자인 xylA 유전자의 발현을 조절하는 xylR 유전자가 염색체에 존재하는 상태에서 xylA 유전자가 고복제수 유래의 플라스미드에 존재하는 경우 (pEX202/DH77)와 저복제수 유래의 플라스미드에 존재하는 경우(pEX102/DH77)에는 염색체에 존재하는 경우 (JM109)보다 0.4% xylose 첨가에 의한 XI의 유도생산이 각각 1.9 및 1.7배 정도 증가하였다. 염색체에 존재하는 xylR 유전자에 의해 생산된 xylR유전자 산물이 xylA 유전자가 플라스미드에 존재할 경우에도 염색체에 존재할때와 마찬가지로 작용하는 것으로 나타났다. 형질전환주 pEX202/DH77과 pEX102/DH77 및 친주 JM109에서 다 같이 0.2% glucose 첨가에 의해 완전히 XI 유도생산이 억제되었으며 이와같은 glucose에 의한 이화물 억제는 1 mM cAMP의 첨가로 해제되었다. DM 최소배지에서 xylose에 의한 XI 유도시 1 mM CAMP를 첨가하면 0.4% xylose만 첨가했을때 보다 XI 생산이 1.7 내지 2배 정도 증가하었다. Xylose isomerase와 cAMP 생산 변이주(xyl, cya ; TP2010)에 xylA 유전자를 형질전환시킨 pEX13/TP2010은 xylose 첨가로 Xl가 유도생산되지 않았고 cAMP를 함께 첨가해야만 XI가 유도되었다. 이와같이 대장균의 xylA 유전자에서 XI의 생산조절에는 xylose이외에 cAMP도 필수적인 효과물질임을 알 수 있었다.

• Current Research on Agriculture and Life Sciences
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• 제10권
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• pp.125-135
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• 1992

Xylose 오페론의 조절기구(調節機構)를 밝히고 xyl 유전자(遺傳子)를 가진 재조합유전자(再組合遺傳子)들의 수용세포(受容細胞)로 사용(使用)하기 위해 대장균(大腸菌)에 NTG를 처리(處理)하여 xylose를 이용(利用)할 수 없는 xyl 변이주(變異株)를 최종(最終) 9주(株) 선발(選拔)하였다. MC-Xyl 한천평판배지(寒天平板培地)에서 백색(白色) 콜로니로 분리(分離)된 xyl 변이주(變異株)들은 모두 LB와 DM-Glc 배지(培地)에서는 친주(親株)인 E. coli JM109와 동일(同一)하게 자랐으나, DM-Xyl 배지(培地)에서는 생육(生育)하지 못했다. 그러나 xyl 유전자(遺傳子) 전체(全體)를 가진 pBX1으로 형질변형(形質變形)시킨 결과(結果) 모두MC-Xyl 한천평판배지(寒天平板培地)에서는 적색(赤色) 콜로니를 나타내고 xylose isomerase 활성(活性)도 친주(親株)와 유사(類似)하게 되살아 났다. 이들의 부귀(復歸) 돌연변이빈도(突然變異頻度)는 $10^{-8}{\sim}10^{-11}$ 이하(以下)로 유전적(遺傳的)으로 안정(安定)되었다. 분리(分離)된 xyl 변이주(變異株)와 그들의 형질전환주(形質轉換株)에 대하여 MC-Xyl과 MC-Xylu 한천평판배지(寒天平板培地)에서 콜로니의 색(色)을 관찰(觀察)하였고 xylose isomerase와 xylulokinase의 활성(活性)을 측정(測定)하여 다시 xylT 변이주(變異株) 3주(株)(DH13, DH121, DH125) xylA 변이주(變異株) 1주(株)(DH77), xylB 변이주(變異株) 1주(株)(DH43) 그리고 xylose 존재하(存在下)에서 이들 효소(酵素)들의 생성(生成)을 조절(調節)하는 xylR 변이주(變異株) 3주(株)(DH10, DH53, DH60), 마지막으로 xylR, A 유전자부위(遺傳子部位)에 변이(變異)가 일어난 것(DH35)으로 최종선별(最終選別)하였다.