Search

A series of mutants of transglutaminase (TGase) gene from Streptomyces fradiae strains were obtained by low energy N+ ion beam irradiation, and the irradiation dose response curve was also investigated. Both the wild type and the mutants of TGase were compared by PCR-SSCP, and specific fragments were sequenced. Then the types of TGase gene mutation induced by N+ were characterized. The results show that the types of DNA base mutation included the transition, the transversion and the deletion. In all of the 24 bases of mutants, the base replacement occupies about 87.5% of the total mutants with only a small portion of gene deletion (12.5%). Interestingly, the frequency of the base transition (58.3%) is obviously higher than that of the base transversion (29.2%) among the base replacements. The base substitutions induced by N+ ion beam irradiation are C-->T transition, A-->G transition, G-->T transversion, C-->G transversion. Furthermore, mutations can be induced in all of the four DNA bases by low energy N+ ion beam irradiation, and cytosine shows the highest frequency of mutation.

With PVA as the carrier, the frozen beer yeast cells were immobilized for production of CTP from CMP. we explored the optimal condition of the immobilization from the aspects of the type, concentration of the PVA, and the immobilizing methods of cells In all 8 continuous batch of fermentation under the reactional condition of the immobilized cells, the conversion rate of CTP were maintained about 85% - 95%. Moreever, the storage stability of immobilized cells were investigated, and the products was also isolated and identifided by HPLC.

Two manganese peroxidase (MnP) active fractions D1 and D2 were got from the extracellular culture of Trametes versicolor by using ammonium sulfate precipitation, DEAE-cellulose DE52 chromatography. MnP1 was purified to electrophoretic homogeneity from the D2 by Phenyl Sepharose 6 Fast Flow chromatography and MnP2 was purified to electrophoretic homogeneity from the D1 by Sephacryl S-200HR chromatography and Phenyl Sepharose 6 Fast Flow chromatography. The specific activities of two MnP isozymes are 579.1U/mg and 425.0U/mg; purification folds are 17.51 and 12.85 and the yields are 6.17% and 2.47%, respectively. MnP1 and MnP2 have approximate molecular masses of 46.3kD and 43.0kD respectively, as determined by SDS-PAGE. The isoenzymes differed in optimum temperature (60degreesC and 65degreesC) and optimum pH(5.8 and 6.2) for oxidation of DMP (2,6-dimethoxyphenol). MnP1 and MnP2 are stable below 45degreesC and ranging from pH4.0 to pH7.0. DMP is the best substrate, the Km values of MnP1 and MnP2 for DMP are 13.43micromol/L and 12.45micromol/L respectively. Catalysis doesn't occur in the complete absence of Mn. EDTA inhibites the activities of MnP1 and MnP2 at the higher concentration and DTT inhibites the enzyme activities completely.

An endoinulinase produced by Chaetomium sp. C34 was purified to electrophoretic homogeneity, with recovery of 7.7% activity and purification factor of 30.8 fold by five steps including ammonium sulfate precipitation, DEAE-cellulose, Q-sepharose Fast Flow, Sephacryl S-200 and Pre-Packed Hydrophobic Column. Its subunit molecular weight was estimated to be about 66kD by SDS-PAGE. The optimum temperature and pH of the enzyme activity were 50 approximately 55 degrees C and 6.0 respectively. The K(m) and V(max) values for inulin were 0.199 mmol/L and 115 micromol/(mg x min) respectively. Cu2+ completely inhibited inulinase activity. An appreciable loss of activity was observed in presence of NBS, Mn2+, Zn2+, Fe2+ and EDTA. A ratio of inulinase activity to invertase activity (I/S) of 20 was found in purified inulinase. The endoinulinase hydrolyzed inulin and liberated inulooligosaccharides. But it lacked activity toward melezitose or raffinose.

The uracil in DNA comes from either the misincorporation of dUTP in place of dTTP or deamination of cytosine. In the latter case, it can result in a GC to AT transition mutation if the uracil is not removed before DNA replication. Base excision repair (BER) is a major pathway for removing DNA lesions arising from endogenous processes as well as those induced by exposure to exogenous chemicals or irradiation. BER is initiated by DNA glycosylases that excise aberrant bases from DNA by cleavage of the N-glycosidic bond linking to the base of its deoxyribose sugar. Uracil N-glycosylase (UNG) is the enzyme responsible for the first step in the BER pathway that specifically removes uracil from DNA. The UNG gene undergoes both temporal and spatial regulation mainly at the level of transcription. Normally cancer cells undergo over-proliferation and up-regulate their UNG during tumorigenesis. In this study we examine the correlation between UNG level and carcinogenesis, and explore the possibility of using UNG as a marker for cancer diagnosis. Human UNG gene was amplified from the total RNA of the human choriocarcinoma cell line, JEG-3, by RT-PCR. After purification, the 942bp full-length UNG cDNA coding sequence was digested with EcoR I and Sal I, and cloned into the digested pET-21 to construct a recombinant vector, pUNG. The UNG protein was expressed under the control of T7 promoter in E. coli BL21 (DE3) cells induced with IPTG. After ultrasonic treatment, the cell lysate and precipitate were analyzed by SDS-PAGE and a 39kD band was detected. The plasmid was serially diluted at appropriate concentrations and employed as standards in the subsequent quantification. Total RNAs were extracted from 18 pairs of clinical samples, each pair contains a sample of esophageal squamous cell carcinoma (ESCC) tissue and its surrounding normal esophageal epithelia. The copy numbers of UNG mRNA in these RNA samples were determined by real-time quantitative RT-PCR using a Lightcycler (Roche). UNG was present in 13 cases of ESCC (13/18, n = 18) but absent in all of the normal tissues. The results indicated that there was a correlation between high level of UNG expression and the carcinogenesis of ESCC.