• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2076-2086
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• 2016

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at $30^{\circ}C$. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

• Clinical and Experimental Reproductive Medicine
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• v.22 no.3
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• pp.301-307
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• 1995

소 체외수정란에 있어서 pentose phosphate pathway (PPP)를 연구하기 위해서, 한개의 체외수정란으로부터 glucose 6-phosphate dehydrogenase (G6PDH)의 활성을 효소증폭방법으로 측정하였다. Glucose 6-phosphate (G6P) 기질을 처리하지 않은 2, 4, 8세포기, 상실배 및 배반포기 수정란에서의 G6PDH 활성치는 각각 $25.5{\pm}3.3$, $27.8{\pm}3.4$, $40.9{\pm}6.2$, $34.9{\pm}3.6$ 및 $52.9{\pm}2.5{\times}10^{-8}mol/embryo/h$ 을 나타내었다. 즉, 8 세포기 이후 수정란들은 2 세포기나 4 세포기보다도 높은 효소활성치를 보여주었다 (P<0.01). 그리고 G6P 기질을 첨가한 2,4,8 세포기, 상실배기 및 배반포기 수정란의 G6PDH 활성치는 각각 $32.3{\pm}3.9$, $29.4{\pm}1.8$, $51.9{\pm}4.2$, $42.6{\pm}2.7$ 및 $52.9{\pm}2.5{\times}10^{-8}mol/embryo/h$ 로서 기질 무처리구와 마찬가지로 유의성이 인정되었다 (P<0.01). 전반적으로 수정란의 발달단계에 있어서 G6P 첨가한 수정란들에 G6PDH의 효소활성치가 기질을 처리하지 수정란들의 것보다도 높은 경향을 보였다. 한편, 소 체외수정란의 G6PDH 효소활성치와 발생능과의 관계를 알아보기 위하여, 4 세포기 수정란들을 효소활성치의 정량적 수준 (low, middle, high)에 따라 3 군으로 분류한 다음 $38.5^{\circ}C$, 5% $CO_2$에서 5일간 난구세포들과 공동배양을 실시하였다. 그 결과, G6PDH 효소활성치 차이에 따른 수정란들의 체외발달율에는 유의성이 인정되지 않았다. 본 실험의 결과를 종합하여 볼 때, 소 체외수정란에 있어서 PPP 대사는 8세포기 이후부터 활발히 이루어지고 있음을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• v.29 no.4
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• pp.577-586
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• 2019

The engineered Aspergillus oryzae has a high NADPH demand for xylose utilization and overproduction of target metabolites. Glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) is one of two key enzymes in the oxidative part of the pentose phosphate pathway, and is also the main enzyme involved in NADPH regeneration. The open reading frame and cDNA of the putative A. oryzae G6PDH (AoG6PDH) were obtained, followed by heterogeneous expression in Escherichia coli and purification as a his6-tagged protein. The purified protein was characterized to be in possession of G6PDH activity with a molecular mass of 118.0 kDa. The enzyme displayed maximal activity at pH 7.5 and the optimal temperature was $50^{\circ}C$. This enzyme also had a half-life of 33.3 min at $40^{\circ}C$. Kinetics assay showed that AoG6PDH was strictly dependent on $NADP^+$ ($K_m=6.3{\mu}M$, $k_{cat}=1000.0s^{-1}$, $k_{cat}/K_m=158.7s^{-1}{\cdot}{\mu}M^{-1}$) as cofactor. The $K_m$ and $k_{cat}/K_m$ values of glucose-6-phosphate were $109.7s^{-1}{\cdot}{\mu}M^{-1}$ and $9.1s^{-1}{\cdot}{\mu}M^{-1}$ respectively. Initial velocity and product inhibition analyses indicated the catalytic reaction followed a two-substrate, steady-state, ordered BiBi mechanism, where $NADP^+$ was the first substrate bound to the enzyme and NADPH was the second product released from the catalytic complex. The established kinetic model could be applied in further regulation of the pentose phosphate pathway and NADPH regeneration of A. oryzae to improve its xylose utilization and yields of valued metabolites.

• Microbiology and Biotechnology Letters
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• v.25 no.5
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• pp.495-500
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• 1997

Xylitol production from xylose and glucose was investigated using Candida tropicalis KFCC-10960. As glucose concentration in xylose medium was increased, ethanol production increased. However, xylitol production was maximum at glucose concentration of 10 g/l. The concentrated cells grown on xylose or glucose were inoculated in xylose medium. The specific activities of xylose reductase and xylitol dehydrogenase, and xylitol production in concentrated cells grown on glucose were the same as those in concentrated cells grown on xylose. The results suggested that cells grown on glucose had the same xylitol producing activity as those grown on xylose. By feeding glucose in xylose medium, cell growth was achieved from glucose and xylitol production was obtained from xylose. By using this technique, a final xylitol concentration of 261 g/l was achieved from 300 g/l xylose in 41 hours which corresponded to a xylitol yield from xylose of 87% and a xylitol productivity of 6.37 g/1-h.

• Journal of Electrochemical Science and Technology
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• v.12 no.2
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• pp.225-229
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• 2021

Commercially available continuous glucose sensors require the operation stability for more than two weeks. Typically, the sensor comprises a sensing layer and an over-coating layer for the stable operation inside the body. In the sensing layer, enzymes and mediators are cross-linked together for the effective sensing of the glucose. The over-coating layer limits the flux of glucose and works as a biocompatible layer to the body fluids. Here, we report the simple preparation of the flux-limiting layer by the condensation of polyethyleneimine (PEI), tri-epoxide linker, and trimethylolpropane triglycidyl ether (PTGE). The sensor is constructed by a layer-by-layer drop-coating of the sensing layer containing glucose dehydrogenase and the PEI-derived blocking layer. It is stable for more than 14 days, which is enough for the sensor in the continuous monitor glucose monitoring (CGM) system.

• Journal of Plant Biology
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• v.12 no.2
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• pp.15-22
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• 1969

By spectrophotometric assay method, the following enzymes could be detected in Dunaliella tertiolecta and Chlorella pyrenoidosa cell-free extracts: Hexokinase; Glucose-6-phosphate, 6-Phosphogluconate and Triosephosphate dehydrogenase; Transketolase; Phosphogluco and Ribosephosphate isomerase; Phosphoglucomutase; Phosphofructokinase; Fructosediphosphate aldorase and Ribulosephosphate 3-epimerase. Such enzymes are in accordance with the proposed pathway of glucose catabolism by D. tertiolecta as well as C. pyrenoidosa. Also, it could be estimated, under the presence of NADP, that pentose phosphate pathway were more active than glycolytic pathway in D. tertiolecta cell-free systems.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.56-59
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• 2000

Thermophilic bacterium, Bacillus stearothermophilus NUB3621, was engineered to produce ethanol from glucose by introducing cloned thermostable pyruvate decarboxylase and alcohol dehydrogenase genes. A novel promoter sequence was screened and used for the enhancement of these two enzymes. Successful redirection of metabolic flux into ethanol was obtained. In addition, gene expression profiling using Bacillus subtilis DNA microarray was analyzed to overcome the intrinsic low glucose utilization of B.stearothermophilus. Many known and unknown genes were identified to be up or down regulated under glucose-containing media.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.6
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• pp.748-756
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• 2000

Effects of xanthine (X), xanthine oxidase (XO), and catalase (C), $H_2O_2$, and carbohydrates on sperm capacitation, acrosome reaction, and fertilizing ability in vitro were examined. Glucose alone, but not fructose, supported the maximum rate of sperm capacitation and acrosome reaction. However, in the combination of X, XO, and C (XXOC) or $H_2O_2$, fructose alone also supported maximum capacitation, acrosome reaction, and fertilization. Either insufficient or excessive amounts of $H_2O_2$ decreased sperm capacitation and the acrosome reaction. In order to understand how glucose generates $H_2O_2$ or other reactive oxygen species in sperm cells, 6-aminonicotinamide, an inhibitor of the pentose-phosphate pathway (PPP), and apocynin, an inhibitor of NADPH oxidase, were added to sperm suspensions in glucose-containing medium. Results appeared that sperm capacitation, acrosome reaction, and fertilization were consequently inhibited by either one of these compounds. These inhibitory effects were nullified by addition of XXOC. These results support the hypothesis that glucose, in addition to being a substrate for glycolysis, facilitates sperm capacitation and the acrosome reaction by generating reactive oxygen species through G-6-P dehydrogenase and NADPH oxidase.

• Journal of the Korean Applied Science and Technology
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• v.32 no.2
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• pp.226-231
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• 2015

This study was carried to investigate the antidiabetic effect of ethanol extract in Streptozotocin(STZ)-induced diabetic rats. Diabetes was induced by intravenous injection of STZ at a dose of 45mg/kg dissolved in citrate buffer. The ethanol extract of Forsythia Koreana(F.K) was orally administrated once a day for 7 days at a dose of 1,000mg/kg. The contents of serum glucose, triglyceride(TG), total cholesterol were significantly decreased in F.K treated group compared to the those of STZ-control group. The content of hepatic glycogen and activity of glucokinase(GK) were significantly increased, and activity of glucose-6-phoshatase(G-6-Pase) was significantly decreased in F.K treated group compared to the those of STZ-control group, but activity of glucose-6-phosphate dehydrogenase(G-6-PDH) was not significantly increased, These results indicated that ethanol extract of F.K would have antidiabetic effect in STZ-induced diabetic rats.

• The Journal of Internal Korean Medicine
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• v.25 no.2
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• pp.288-297
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• 2004

Objective : This study was undertaken to investigate how Bombycis corpus (BC) effects the development and progress of complications occurring in Diabetes Mellitus (DM). Methods : Laboratory rats were seperated into three groups; normal, rats with DM and treated with BC, and rats with DM and not treated. In this study DM was experimentally induced through injection of streptozotocin. The BC treated group was given BC extract p.o. for 15 days. Then, The activities of glucose phosphatic enzymes and polyol pathway channels were observed. Results : The blood glucose level greatly increased in the DM groups after injection of streptoztocin, but it significantly decreased in the BC treated group. Significantly enhanced levels of serum insulin levels were seen in the BC treated group, while supressed levels were seen in the untreated DM group. Weight was recovered by the BC treated group, matching the normal group. Decreased enzyme activity of aldose reductase, sorbitol dehydrogenase and glucose-6-phosphatase were seen in BC treated diabetic rats. Increased enzyme activity, of the glucokinase and hexokinase were seen in BC treated diabetic rats. Conclusions : This study suggests that BC normalized the blood glucose and serum insulin levels destablized by DM. Because increased activity of glucose phosphatic enzymes, glucokinase and hexokinase, and decreased glucose-6-phosphatase activity, and suppression of polyol pathway enzymes, aldose reductase and sorbitol dehydrogenase, were all seen, these observations suggest that BC suppresses blood glucose levels and prevents complications due to DM.