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Yi, Yi,Qiao, Dairong,Bai, Linhan,Xu, Hui,Li, Ya,Wang, Xiaolin,Cao, Yi The Microbiological Society of Korea 2007 The journal of microbiology Vol.45 No.2
5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.
Feiwei Zhang,Dairong Qiao,Hui Xu,Chong Liao,Shilin Li,Yi Cao 한국미생물학회 2009 The journal of microbiology Vol.47 No.3
Xylose reductase (XR) is a key enzyme in xylose metabolism because it catalyzes the reduction of xylose to xylitol. In order to study the characteristics of XR from Candida tropicalis SCTCC 300249, its XR gene (xyl1) was cloned and expressed in Escherichia coli BL21 (DE3). The fusion protein was purified effectively by Ni2+-chelating chromatography, and the kinetics of the recombinant XR was investigated. The Km values of the C. tropicalis XR for NADPH and NADH were 45.5 µM and 161.9 µM, respectively, which demonstrated that this XR had dual coenzyme specificity. Moreover, this XR showed the highest catalytic efficiency (kcat=1.44×104 min-1) for xylose among the characterized aldose reductases. Batch fermentation was performed with Saccharomyces serivisiae W303-1A:pYES2XR, and resulted in 7.63 g/L cell mass, 93.67 g/L xylitol, and 2.34 g/L·h xylitol productivity. This XR coupled with its dual coenzyme specificity, high activity, and catalytic efficiency proved its utility in in vitro xylitol production.
DsLCYB Directionally Modulated β-Carotene of the Green Alga Dunaliella salina under Red Light Stress
Lan Yanhong,Song Yao,Guo Yihan,Qiao Dairong,Cao Yi,Xu Hui 한국미생물·생명공학회 2022 Journal of microbiology and biotechnology Vol.32 No.12
Carotenoids, which are natural pigments found abundantly in wide-ranging species, have diverse functions and high industrial potential. The carotenoid biosynthesis pathway is very complex and has multiple branches, while the accumulation of certain metabolites often affects other metabolites in this pathway. The DsLCYB gene that encodes lycopene cyclase was selected in this study to evaluate β-carotene production and the accumulation of β-carotene in the alga Dunaliella salina. Compared with the wild type, the transgenic algal species overexpressed the DsLCYB gene, resulting in a significant enhancement of the total carotenoid content, with the total amount reaching 8.46 mg/g for an increase of up to 1.26-fold. Interestingly, the production of α-carotene in the transformant was not significantly reduced. This result indicated that the regulation of DsLCYB on the metabolic flux distribution of carotenoid biosynthesis is directional. Moreover, the effects of different light-quality conditions on β-carotene production in D. salina strains were investigated. The results showed that the carotenoid components of β-carotene and β-cryptoxanthin were 1.8- fold and 1.23-fold higher than that in the wild type under red light stress, respectively. This suggests that the accumulation of β-carotene under red light conditions is potentially more profitable.
( Huoxiang Zhou ),( Xi Li ),( Mingyue Guo ),( Qingrui Xu ),( Yu Cao ),( Dairong Qiao ),( Yi Cao ),( Hui Xu ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.7
The endo-polygalacturonase gene (endo-pgaA) was cloned from DNA of Aspergillus niger SC323 using the cDNA synthesized by overlapping PCR, and successfully expressed in Saccharomyces cerevisiae EBY100 through fusing the α-factor signal peptide of yeast. The fulllength cDNA consists of 1,113 bp and encodes a protein of 370 amino acids with a calculated molecular mass of 38.8 kDa. After induction by galactose for 48 h, the activity of recombinant endo-PgaA in the culture supernatant can reach up to 1,448.48 U/mg. The recombinant protein was purified to homogeneity by ammonium sulfate precipitation and gel filtration column chromatography and subsequently characterized. The optimal pH and temperature of the purified recombinant enzyme were 5.0 and 50°C, respectively. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of the enzyme for pectin were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively. The enzyme activity was enhanced by Ca2+, Cu2+, and Na+, and strongly inhibited by Pb2+ and Mn2+. The pectin hydrolysates were mainly galacturonic acid and other oligo-galacturonates. Therefore, these characteristics suggest that the recombinant endo-PgaA may be of potential use in the food and feed industries.