嗜碱性芽孢杆菌产高碱碱性蛋白酶发酵培养基及发酵条件的研究

对一株高产高碱碱性蛋白酶的嗜碱性芽孢杆菌的发酵培养基及发酵工艺进行了优化。确定了发酵培养基所采用的棉籽饼粉最适粒度为 80目 ,麦芽糊精的最佳DE值为 3 0 %。并确定了该菌株的最适发酵培养基配方为 ( g/1 0 0mL) :棉籽饼粉 3 ,酵母浸粉 1 75 ,麦芽糊精 1 0 ,柠檬酸钠 0 3 ,CaCl2 0 3。K2 HPO4 1 ;最适摇瓶发酵条件为 :种龄 1 2h ,接种量 2 %,装液量 5 0mL/2 5 0mL ,摇床转速 2 0 0r/min ,3 4℃ ,发酵 5 4h ,碱性蛋白酶的发酵单位可达 3 3 985u/mL ,比优化前提高了5 4 48%。此外 ,还进行了 5L罐放大实验 ,在 5L罐所确定的最适工艺条件下 ,发酵单位可达3 65 79u/mL。     

Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease

Angiotensin-I converting enzyme inhibitory activities were measured after hydrolysis of casein by 9 different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest angiotensin-I converting enzyme inhibitory activity per peptide. The A. oryzae peptide also showed the highest antihypertensive effect in spontaneously hypertensive rats when the systolic blood pressure was measured 5 h after oral administration of 32 mg/kg of various enzymatic hydrolysates. Significant antihypertensive effects were observed with dosages of 9.6, 32, and 96 mg of the A. oryzae peptide/kg of body weight (BW), and the effects were dependent on these peptide dosages.Analysis of peptide length showed the A. oryzae hydrolysate was the shortest of all tested casein hydrolysates; the peptide mixture had an average value of 1.4 amino acids (AA) in the sequence. To further characterize the A. oryzae hydrolysate, we analyzed the AA sequence of the whole peptide mixture. Various AA were detected at the first AA position, however, an increased number of Pro residues were observed at the second and third position of the A. oryzae hydrolysate. No strong signals were detected after the fourth AA position of the A. oryzae hydrolysate. These results suggest that the casein hydrolysate of A. oryzae, which expressed potent antihypertensive effects in spontaneously hypertensive rats, mainly contain short peptides of X-Pro and X-Pro-Pro sequences.     

新疆传统乳制品中产蛋白酶乳酸菌的筛选及产酶条件优化

以新疆传统发酵乳制品为分离源,分离筛选产蛋白酶的乳酸菌,并通过形态学观察及16S rDNA序列比对鉴定菌株。以碳源、氮源、金属盐、磷酸盐的种类和添加量为影响因素,以蛋白酶活作为评价指标,通过单因素试验和正交试验优化乳酸菌产蛋白酶的产酶条件。结果表明,从样品中分离筛选出乳酸菌16株,产蛋白酶的乳酸菌4株（编号为A1～A4）,分别被鉴定为戊糖片球菌（Peciococcus pentosaceus）、发酵乳杆菌（Lactobacillus fermentum）、短乳杆菌（Lactobacillxus brevis）、乳酸明串珠菌（Leuconostoc lactis）。在以4.0%葡萄糖为碳源、4.0%牛肉膏为氮源、0.02%的镁离子为金属盐、0.7%的K2HPO4为磷酸盐的条件下,菌株A2产蛋白酶活最高为65.92 U/mL。     

Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity

This study aimed to determine the activity of one Mycoplasma bovis nuclease encoded by MBOV_RS02825 and its association with cytotoxicity. The bioinformatics analysis predicted that it encodes a Ca²⁺-dependent nuclease based on existence of enzymatic sites in a TNASE_3 domain derived from a Staphylococcus aureus thermonuclease (SNc). We cloned and purified the recombinant MbovNase (rMbovNase), and demonstrated its nuclease activity by digesting bovine macrophage linear DNA and RNA, and closed circular plasmid DNA in the presence of 10 mM Ca²⁺ at 22–65 °C. In addition, this MbovNase was localized in membrane and rMbovNase able to degrade DNA matrix of neutrophil extracellular traps (NETs). When incubated with macrophages, rMbovNase bound to and invaded the cells localizing to both the cytoplasm and nuclei. These cells experienced apoptosis and the viability was significantly reduced. The apoptosis was confirmed by activated expression of phosphorylated NF-κB p65 and Bax, and inhibition of Iκβα and Bcl-2. In contrast, rMbovNase^Δ181–342 without TNASE_3 domain exhibited deficiency in all the biological functions. Furthermore, rMbovNase was also demonstrated to be secreted. In conclusion, it is a first report that MbovNase is an active nuclease, both secretory and membrane protein with ability to degrade NETs and induce apoptosis.     

不同C/N下SBBR脱氮过程N2O释放及胞外多聚物变化

在(20±2.0)℃条件下,利用序批式生物膜反应器（sequencing batch biofilm reactor,SBBR）,考察不同碳氮比（C/N=3.0、5.0、8.0和10.0）下同步脱氮（simultaneous nitrification and denitrification,SND）过程N₂O释放及胞外聚合物（extracellular polymeric substance,EPS）变化。C/N由3.0增至10.0,异养菌大量增殖,曝气阶段DO降低,系统硝化性能受到抑制,SBBR系统出水NH₄⁺由0.5 mg/L以下增至（7.85±1.42）mg/L,N₂O产量由（2.68±0.17）mg/L降至（1.02±0.12）mg/L。C/N=8.0,TN去除率最大为80.4%±3.5%。反应初期,微生物体内聚β-羟基烷酸酯（PHA）增加,可为后续反硝化过程提供电子供体。AOB好氧反硝化和低氧条件下异养菌反硝化过程均可导致N₂O产生。C/N降低,SBBR内部缺氧区域减少,N₂O还原过程减弱,释放量增加;C/N增加,N₂O扩散进入生物膜内缺氧区域,促进其减量。C/N由3.0增至10.0,微生物EPS分泌由（57.6±5.6）mg / g VSS降至（32.7±3.2）mg / g VSS,其中,TB-EPS含量占65.8%~68.8%。低C/N下,紧密型EPS（TB-EPS）中多糖（PS）含量增加,生物膜更加密实,N₂O扩散进入缺氧区阻力增加,释放量增加。     

Inhibition of extracellular signal-regulated kinases ameliorates hypertension-induced renal vascular remodeling in rat models

The aim of this study is to investigate the effect of the extracellular signal-regulated kinases 1/2 (ERK1/2) inhibitor, PD98059, on high blood pressure and related vascular changes. Blood pressure was recorded, thicknesses of renal small artery walls were measured and ERK1/2 immunoreactivity and erk2 mRNA in renal vascular smooth muscle cells (VSMCs) and endothelial cells were detected by immunohistochemistry and in situ hybridization in normotensive wistar kyoto (WKY) rats, spontaneously hypertensive rats (SHR) and PD98059-treated SHR. Compared with normo-tensive WKY rats, SHR developed hypertension at 8 weeks of age, thickened renal small artery wall and asymmetric arrangement of VSMCs at 16 and 24 weeks of age. Phospho-ERK1/2 immunoreactivity and erk2 mRNA expression levels were increased in VSMCs and endothelial cells of the renal small arteries in the SHR. Treating SHR with PD98059 reduced the spontaneous hypertension-induced vascular wall thickening. This effect was associated with suppressions of erk2 mRNA expression and ERK1/2 phosphorylation in VSMCs and endothelial cells of the renal small arteries. It is concluded that inhibition of ERK1/2 ameliorates hypertension induced vascular remodeling in renal small arteries.     

Inhibition of p53 deSUMOylation Exacerbates Puromycin Aminonucleoside-Induced Apoptosis in Podocytes

Apoptosis is a major cause of reduced podocyte numbers, which leads to proteinuria and/or glomerulosclerosis. Emerging evidence has indicated that deSUMOylation, a dynamic post-translational modification that reverses SUMOylation, is involved in the apoptosis of Burkitt’s lymphoma cells and cardiomyocytes; however, the impact of deSUMOylation on podocyte apoptosis remains unexplored. The p53 protein plays a major role in the pathogenesis of podocyte apoptosis, and p53 can be SUMOylated. Therefore, in the present study, we evaluated the effect of p53 deSUMOylation, which is regulated by sentrin/SUMO-specific protease 1 (SENP1), on podocyte apoptosis. Our results showed that SENP1 deficiency significantly increases puromycin aminonucleoside (PAN)-induced podocyte apoptosis. Moreover, SENP1 knockdown results in the accumulation of SUMOylated p53 protein and the increased expression of the p53 target pro-apoptotic genes, BAX, Noxa and PUMA, in podocytes during PAN stimulation. Thus, SENP1 may be essential for preventing podocyte apoptosis, at least partly through regulating the functions of p53 protein via deSUMOylation. The regulation of deSUMOylation may provide a novel strategy for the treatment of glomerular disorders that involve podocyte apoptosis.     

动物蛋白水解复合酶的研究

以酶解牛骨蛋白为对象,研究动物蛋白水解复合酶的配制并考察其适应性.实验结果表明,由中性蛋白酶、胰蛋白酶、风味酶等3种酶按酶活性1∶3∶2组合配制而成的复合酶,其酶解动物蛋白效果高于单一酶.     

产功能性胞外多糖及DL-乳酸的植物乳杆菌的高效发酵和应用

植物乳杆菌(Lactobacillus plantarum)是一种多功能乳酸菌,所产胞外多糖(exopolysaccharides, EPS)具有很多优良功能特性。但发酵生产EPS时,原料和操作成本高,EPS产量低限制了其工业化应用。该研究在5 L罐中,使用常规流加发酵、生物催化、生物催化结合液液萃取和基于pH-Stat自动流加葡萄糖法的反复生物催化等策略发酵,旨在提高EPS产量、降低原料和操作成本。其中,生物催化法仅使用葡萄糖即可将EPS质量浓度提升至3.34 g/L,较摇瓶发酵水平提高110%。使用基于pH-Stat自动流加葡萄糖法的反复生物催化策略,可以连续回用各反复发酵中的残存细胞,第2次反复发酵批次的EPS质量浓度达到3.33 g/L。由于在收集处理细胞时,细胞总量下降,导致EPS产量下降,但EPS/细胞量不变,细胞活性稳定。可以通过加大首批次发酵的装液量、提高细胞总量,解决EPS产量不断下降的问题。用基于pH-Stat法的反复生物催化策略进行EPS发酵,提升了EPS产量,省去了昂贵MRS培养基的使用,降低了原料成本,实现了自动化控制。该发酵策略还可联产具有一定价值的副产物4...     

蛋白酶的生产和应用(上)

为充分了解蛋白酶,该文从蛋白酶的分类与底物专一性、微生物蛋白酶的生产等方面介绍了蛋白酶。蛋白酶按活性中心可分为丝氨酸蛋白酶、天门冬氨酸蛋白酶、半胱氨酸蛋白酶和金属蛋白酶;按最适pH值来分又分酸性蛋白酶、中性蛋白酶和碱性蛋白酶。微生物蛋白酶的产生与微生物生长有关,合成受分解代谢阻遏的调控和底物厦其类似物的诱导。并介绍了生产蛋白酶的微生物。