湖羊瘤胃微生物GH9家族葡聚糖酶基因IDSGLUC9-25的表达与功能表征

【目的】葡聚糖酶是饲用添加剂的重要成分,本研究旨在从湖羊消化道微生物中挖掘性质优良的GH9家族葡聚糖酶基因,用于研发新型饲用酶制剂。【方法】从湖羊瘤胃微生物cDNA中扩增IDSGLUC9-25基因,在大肠杆菌中进行异源表达,对重组蛋白进行诱导表达和纯化,研究重组蛋白的酶学性质和底物水解模式。【结果】IDSGLUC9-25基因编码527个氨基酸,包含一个CelD_N结构和一个GH9家族催化结构域;重组蛋白rIDSGLUC9-25分子量约为62.7 kDa,最适反应温度和pH分别为40℃和6.0,在30-50℃下活性较高,在pH 4.0-8.0范围内能够保持较高的稳定性,经pH 4.0-8.0缓冲液处理1 h后残余活性均大于90%;底物谱分析表明,rIDSGLUC9-25能催化大麦β-葡聚糖、苔藓地衣多糖、魔芋胶和木葡聚糖,比活性分别为(443.55±24.48)、(65.56±5.98)、(122.37±2.85)和(159.16±7.73) U/mg;利用薄层色谱法(thin layer chromatography, TLC)和高效液相色谱法(high performance liquid chromatography, HPLC)分析水解产物发现,rIDSGLUC9-25降解大麦葡聚糖主要生成纤维三糖(占总还原糖64.19%±1.19%)和纤维四糖(占总还原糖26.24%±0.12%),催化地衣多糖主要生成纤维三糖(占总还原糖78.46%±0.89%)。【结论】本研究报道了一种来自密螺旋体属细菌的内切β-1,4-葡聚糖酶IDSGLUC9-25 (EC 3.2.1.4),能高效催化多糖底物生成纤维三糖和纤维四糖,为研发饲用酶制剂和制备低聚寡糖建立基础。     

Removal of nitrate from water by cells ofParacoccus denitrificans in a membrane flow reactor

A new type of flow bioreactor designed to remove nitrate from water was developed. Denitrification activity of native Paracoccus denitrificans cells was used, the cells being separated from the refined medium by a semipermeable membrane. Relationships between the degree of nitrate conversion and the denitrification rate, on the one hand, and the volume flow rate and the amount of biomass, on the other, together with the results concerning denitrification during closed-circuit recirculation of the medium are discussed.     

Effect of actinomycin D on viability,sporulation and nucleotide pool ofBacillus megaterium

A transient 7-fold rise of ppGpp concentration, 2-3-fold increase of pppGpp concentration and 50 % drop of the concentration of GTP inBacillus megaterium cells immediately after their transfer to the sporulation medium were observed. Actinomycin D, in concentrations inhibiting RNA synthesis by 95%, blocked the rise of the (p)ppGpp pool and caused an instant several-fold increase of the GTP level. When the cells were exposed to actinomycin D in the sporulation medium for a 1-h period (time 0–1 h, 1–2 h or 2.20–3.20-h), they were able to form colonies on nutrient agar after being kept, in addition for 1–2 h in the sporulation medium free of the antibiotic. The ability of sporulation was, however, markedly limited. The share of cells that could sporulate increased when the irreversible sporulation phase was reached.     

Recovery from ethyl methanesulphonate-induced genetical damage in barley. Independence of sodium azide treatment

Barley seeds were treated for 3 h at 25°C with 240 mM ethyl methanesulphonate (EMS), washed for 18 h, treated with various concentrations of unbuffered sodium azide (pH 6.7–7.3) for 3 h at 25°C, re-dried to 30% water content and either sown immediately or stored at 25°C for 12 days and then sown. The synergistic action of sodium azide post-treatment has been demonstrated only for the EMS-induced M₁ germination reduction, while the EMS-induced M₁ sterility and the yield of M₂ chlorophyll mutants were unaffected. The ?storage” recovery from EMS-induced mutagenic effects was insensitive to sodium azide post-treatment. The 12 day-seed storage at 25°C brought about an improvement of M₁ germination, M₁ survival, M₁ fertility and a decrease in the amount of M₂ mutants, regardless of whether sodium azide post-treatment was applied or not.     

Biochemical dehydrogenations of saccharides

Fermentation of a medium containing 5% 2-deoxy-D-glucose and barium carbonate by a strain ofPseudomonas aeruginosa yielded barium 2-deoxy-d-gluconate. The yield was 77% theoretical. The strain in question makes it possible to prepare directly calcium, magnesium, manganese and ferrous salts of 2-deoxy-d-glueonic acid. A treatment of 6% solution of 2-deoxy-d-glucose with commercial glucose oxidase preparation caused also a complete dehydrogenation.     

Different protective roles in vitro of alpha- and beta-domains of growth inhibitory factor (GIF) on neuron injuries caused by oxygen free radicals.

It was well known that beta-amyloid (Abeta) and tau protein play an important role in pathological procedure of Alzheimer's disease (AD), a senile dementia. The growth inhibitory factor (GIF, also named metallothionein-3, MT-3) had been demonstrated to inhibit the outgrowth of cortex neurons in the medium with extract of the AD patient brain. In our experiments, it was found that the neurons of cortex and the PC12 (pheochromocytoma) cells could be protected from the cytotoxicity of beta-amyloid 25-35 in presence of GIF and its domains. Additionally, GIF can scavenge the hydroxyl radical efficiently in CytC-VitC radical producing system and its alpha-domain shown more effective potentials than its beta-domain. The electron paramagnetic resonance spectra also show that the alpha-domain has more potential ability for eliminating reactive oxygen free radicals than its beta-domain. The results suggest that GIF could act as an efficient scavenger against free radicals in vitro and the alpha-domain in GIF molecule shows more potential in protecting against reactive oxygen species injury than the beta-domain.