Cloning, Expression, and Characterization of Xylose Reductase with Higher Activity from Candida tropicalis

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.

Cloning, Expression, and Functional Characterization of the Dunaliella salina 5-enolpyruvylshikimate-3-phosphate Synthase Gene in Escherichia coli

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.

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.

Anti‑infammatory and antioxidant activities of acteoside isolated from Acanthus ilicifolius var. xiamenensis

Zhang Yifan,Shen Jinhuang,Ma Xinhua,Yao Mingshuang,Zhang Yonghong,Cao Dairong 한국응용생명화학회 2022 Applied Biological Chemistry (Appl Biol Chem) Vol.65 No.3

Acanthus ilicifolius var. xiamenensis (Acanthaceae), a mangrove found in southeastern China, is an herb with strong antiinflammatory property. Phytochemical study of the mangrove showed that the plant has a high content of phenylethanol glycoside acteoside (AC). In this research, the anti-inflammatory activity of acteoside on dextran sodium sulphate (DSS)-mediated mouse ulcerative colitis model were studied. For DSS- mediated colitis in mice, AC can reduce weight loss and DAI score in UC mice, suppress colon shortening and alleviate colon pathological injury. Moreover, AC treatment notably up-regulates IL-10, down-regulates the levels of IL-1β and TNF-α, and inhibits the protein expression of JAK2/STAT3, NF-κB p65, IKKα/β and IKB of colon. In addition, after AC treatment, the level of MDA and NO in colonic tissue were remarkably decreased, while the levels of GSH, SOD, and Nrf2 and HO-1 protein expression levels were significantly increased. These results indicate that AC can activate the Nrf2 signaling pathway by inhibiting the JAK/STAT, iNOS/eNOS and NF-κB signaling cascades, enhance the intestinal barrier function, and effectively reduce DSS-induced UC in mice.

Secretory Expression and Characterization of an Acidic Endo-Polygalacturonase from Aspergillus niger SC323 in Saccharomyces cerevisiae

( 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.

Anti-Inflammatory Activity of Phenylethanoids from Acanthus ilicifolius var. xiamenensis

Yifan Zhang,Jinhuang Shen,Xinhua Ma,Yubin He,Yonghong Zhang,Dairong Cao 한국식품영양과학회 2023 Journal of medicinal food Vol.26 No.2

Acanthus ilicifolius var. xiamenensis is a traditional herbal medicine in China. In this study, the anti-inflammatory activities of active ingredients of A. ilicifolius var. xiamenensis were investigated in RAW 264.7 cells and Freund's complete adjuvant-induced arthritic rats. Results showed that n-butanol extract exerted antiarthritic potential by reducing paw edema, arthritis score, and altered hematological and biochemical parameters in experimental rats. Phytochemical studies on n-butanol extract resulted in the isolation of five alkaloids (1–5) and five phenylethanoids (6–10). The anti-inflammatory assay of compounds 1–10 on lipopolysaccharide (LPS)-treated RAW 264.7 cells indicated that phenylethanoids 9 and 10 exhibited notable inhibitory activities. The result indicated that compounds 9 and 10 attenuated inflammation by decreasing the production of nuclear factor kappa-B (NF-κB) p65, inhibitory subunit of NF kappa B alpha, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and inducible nitric oxide synthase in LPS-mediated RAW 264.7 macrophages. Phenylethanoids 9 and 10 increased the expression of interleukin-10 and endothelial nitric oxide synthase. Therefore, compounds 9 and 10 showed anti-inflammatory activity by regulation of NF-κB and JAK/STAT signaling pathways.