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The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.
Subject(s)
Biocatalysis , Cloning, Molecular , Escherichia coli , Glucosyltransferases , Streptomyces coelicolor , TrehaloseABSTRACT
To screen the differentially-expressed microRNAs (miRNAs) in mouse models with renal ischemia-reperfusion injury (IRI),aiming to offer foundation for unraveling the molecular mechanism of the incidence and progression of IRI.Methods The mouse models with acute IRI were established by renal artery clamping.Fifteen mice were divided into the IRI group and sham surgery group (E group).The animals in the IRI group were subdivided into the A group (45 min ischemia followed by 24 h reperfusion),B group (25 min ischemia followed by 24 h reperfusion),C group (45 min ischemia followed by 4 h reperfusion) and D group (25 min ischemia followed by 4 h reperfusion) (n=3 for each group).The severity ofIRI was evaluated by histological changes and renal function.The differentially-expressed miRNAs in the IRI mouse models at different ischemia time (25 and 45 min) and reperfusion time (4 and 24 h) were screened by using cluster analysis of miRNAs microarray data.The differential expression of miR-695 and miR-145 was validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).Results Both histological changes and renal function confirmed that the IRI mouse models were successfully established.Compared with the sham surgery group,71 differentially-expressed miRNAs were detected in the IRI group including 30 down-regulated miRNAs and 40 up-regulated miRNAs.The results of qRT-PCR demonstrated that if the standardized expression level of miRNAs in the E group was 1,the relative expression levels of miR-695 and miR-145 were 11.82 and 0.31 in the IRI group (both P＜0.05),which were consistent with the chip results.Conclusions After renal IRI,different changes occur in the gene expression profile of miRNAs.These differentially-expressed miRNAs act as molecular biomarkers for renal IRI with potential clinical and scientific research values.
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AIM To study the chemical constituents from Hosta plantaginea (Lam.) Aschers flowers.METHODS The aqueous extract of H.plantaginea was isolated and purified by Sephadex LH-20 and HPLC column,then the structures of obtained compounds were identified by spectral data.RESULTS Twelve compounds were isolated and identified as 4-hydroxybenzaldehyde (1),4-hydroxylacetophenone (2),5,7-dimethoxy-8-rnethyl-4'-hydroxyflavan (3),5,7-dimethoxy-4'-hydroxyflavan (4),epicatechin (5),catechin (6),epigallocatechin (7),gallocatechin (8),coumaric acid (9),phenethyl-O-β-D-glucoside (10),acetophenone-4-O-β-D-glucoside (11),2-hydroxyl-6-methoxyacetophenone-4-O-β-D-glucoside (12),3,4-dihydroxycinnamyl alcohol-3-O-glucoside (13).CONCLUSION All the compounds are isolated from this plant for the first time.
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ObjectiveTo investigate the effect of protein acetylation in host cells on the replication of hepatitis B virus (HBV) in hepatocytes, since HBV infection greatly threatens human health and the acetylation of encoding proteins in infected cells plays an important role in HBV replication and infection. MethodsThe deacetylase inhibitors trichostatin A (TSA) and nicotinamide (NAM) were used to stimulate HBV replication in HepG2.2.15 and HepAD38 cells, and the HBV replication markers were measured. The pan-acetylysin protein and Ac-H3 were examined by Western Blot. ResultsThe stimulation of cells with TSA and NAM increased the overall acetylation level of proteins in cells, and the acetylation level increased in a time- and dose-dependent manner. In the HepG2.2.15 and HepAD38 cells, stimulation with TSA and NAM reduced HBsAg level in the supernatant of cell culture and increased HBV DNA level in a time- and dose-dependent manner, while HBeAg in the supernatant of cell culture and DNA in cells did not change significantly. ConclusionAcetylation of host proteins may be involved in and affect HBV replication in cells, and further analysis and determination of host proteins whose acetylation affects HBV replication in cells help to learn more about the regulation of HBV replication and provide new thoughts for the development of specific antiviral strategies.
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To study the relationship between serum Pre-S1 antigen and HBV DNA, serum Pre-S1 antigen and HBV DNA marker in 229 patients positive for HbsAg were detected by using ELISA method and compared. It was found that both Pre-S1 antigen and HbeAg were correlated with HBV DNA with the coefficient being 0. 9812 and could be used to reflect the existence or reproduction of HBV DNA well. Although there was statistical correlation between them, their clinical implication was not completely the same. Therefore, a conclusion was drawn that combined detection of Pre-S1 antigen, HBV and HBV DNA in the patients with different kinds of HBV infection was helpful to clinical diagnosis, therapeutic effect evaluation and prognosis judgement.