An Atomistic Understanding of Allosteric Inhibition of Glutamate Racemase: a Dampening of Native Activation Dynamics

Glutamate racemases (GR) are members of the family of bacterial enzymes known as cofactor-independent racemases and epimerases and catalyze the stereoinversion of glutamate. D-amino acids are universally important for the proper construction of viable bacterial cell walls, and thus have been repeatedly validated as attractive targets for novel antimicrobial drug design. Significant aspects of the mechanism of this challenging stereoinversion remain unknown. The current study employs a combination of MD and QM/MM computational approaches to show that the GR from H. pylori must proceed via a pre-activation step, which is dependent on the enzyme's flexibility. This mechanism is starkly different from previously proposed mechanisms. These findings have immediate pharmaceutical relevance, as the H. pylori GR enzyme is a very attractive allosteric drug target. The results presented in this study offer a distinctly novel understanding of how AstraZeneca's lead series of inhibitors cripple the H. pylori GR's native motions, via prevention of this critical chemical pre-activation step. Our experimental studies, using SPR, fluorescence and NMR WaterLOGSY, show that H. pylori GR is not inhibited by the uncompetitive mechanism originally put forward by Lundqvist et al.. The current study supports a deep connection between native enzyme motions and chemical reactivity, which has strong relevance to the field of allosteric drug discovery.     

Permeabilization of Escherichia coli with ampicillin for a whole cell biocatalyst with enhanced glutamate decarboxylase activity

The activity of whole-cell biocatalysts is strongly compromised by the cell envelope, which is a permeability barrier against the diffusion of substrates and products. Although common chemical or physical permeabilization methods used in cultured cells enhance cell permeability, these methods inevitably add several extra processing steps after cell cultivation, as well as impede large scale processing. To increase membrane permeability and cellbound glutamate decarboxylase(GAD) activity of recombinant Escherichia coli(BL21(DE3)-p ET28a-gad B) cells without the need for an additional permeabilization step, we investigated the permeabilizing effects of adding cell wall synthesis inhibitors or surfactants to the culture media. Ampicillin was the most effective at improving cell-bound GAD activity of the BL21(DE3)-p ET28a-gad B, although it decreased the cell biomass yield. The best permeabilization effect was observed using an ampicillin concentration of 5 μg·ml-1. Using this concentration,the cell biomass did decrease by 40.58%, but the cell-bound GAD activity of BL21(DE3)-p ET28a-gad B and total cell-bound GAD activity per milliliter of culture was enhanced by 6.24- and 3.64-fold, respectively. Treatment of BL21(DE3)-p ET28a-gad B cells with 5 μg·ml-1ampicillin resulted in structural changes to the cell envelope,but did not substantially affect GAD expression. By entrapping the ampicillin-treated cells in an open pore gelation matrix, which is a polymer derived from polyvinyl alcohol(PVA), alginate, and boric acid, the transformation rate of γ-aminobutyric acid(GABA) at the 10 th cycle produced by immobilized and permeabilized cells remained 46% of the first cycle. GAD activity of the immobilized, permeabilized cells remained over 90% after30 days of storage at 4 °C.     

糖蜜味精废水饲料化的生产实践

主要介绍了利用糖蜜味精废水采用干燥法试制速溶蛋白产品的依据及其禽畜饲养效果，并对存在问题进行了讨论。     

L－谷氨酸脱羧酶电极的研制与性能

将谷氨酸脱羧酶包埋于聚乙烯醇（ＰＶＡ）中，并吸附于棉布支持物上，再用饱和硼酸溶液交联固化，制成酶膜。将其与ＣＯ_２气敏电极组成电位型谷氨酸酶电极，电极对测定谷氨酸的浓度表现出良好的线性响应性能，其线性范围为１．５×１０~（－４）～７．５×１０~（－３）ｍｏｌ／Ｌ，斜率为５４．８ｍｖ，响应时间为６～９ｍｉｎ，使用稳定性为１３天（活性下降１０％），贮存稳定性为１５天（活性下降１０％）。该酶电极对谷氨酸有很高的专一性（赖氨酸对电极有少许影响），盐酸吡哆辛存在下，活性有所增加。将酶电极用于味精发酵液样品中的Ｌ－谷氨酸的测定，获得令人满意的结果。     

A non-plasticized chitosan based solid state electrode for flow injection analysis of glutamate in food samples

This report describes the development of solid state glutamate sensor based on chitosan. The electrode has linearity range of 0.01–1 mM glutamate with detection limit of 0.008 mM. The presence of other conventional food additives at physiological level does not interfere. The interfering effect is, however, minimized through prior dilution of the sample. Recovery values of 88.9–99.2% are obtained throughout. The proposed electrode has been applied in flow injection analysis (FIA) glutamate in food samples. In the validation experiment, the proposed electrode is found to be comparable with the standard method.     

Immunolocalization of multiple membrane proteins on a carbon replica with STEM and EDX

We present a method for immunolabeling of multiple species of membrane proteins with high spatial resolution. It allows differentiation of equally sized very small markers with different chemical compositions, which leads to high labeling efficiency and reduces steric hindrance of closely spaced immunolabeled biomolecules. Markers such as CdSe/ZnS semiconductor quantum dots and colloidal gold particles are distinguished by differential contrast in high-angle annular detector dark-field STEM mode or by EDX microanalysis of their elemental contents. This method was tested by observation of labeled AMPA- and NMDA-type glutamate receptors on sodium-dodecyl-sulfate-digested replica prepared from rat hippocampus. To improve particle visibility and detectability, the replica films were made exclusively with carbon to avoid the high background of conventional platinum/carbon replica. Extension of the method is suggested by detection of 1.4 nm nanogold particles and its potential application in the biological imaging research.     

天麻素通过抑制细胞凋亡保护谷氨酸诱导的PC12细胞损伤

目的: 研究天麻素(Gastrodin)对谷氨酸诱导的大鼠肾上腺嗜铬细胞瘤PC12细胞损伤的影响及可能机制。方法: 以谷氨酸建立体外培养PC12细胞损伤模型并采用MTT比色法测定细胞存活率;AO/EB双染法经荧光显微镜观察细胞凋亡形态;采用流式细胞术检测细胞内活性氧含量以及Annexin V/PI染色后的细胞凋亡率;Western blot法检测细胞内Caspase-3蛋白表达。结果: 天麻素可明显抑制谷氨酸诱导的PC12细胞凋亡,在 0.1~10 μmol/L 剂量呈一定的量效关系;同时,天麻素可明显抑制谷氨酸引起的活性氧(ROS)的累积,降低谷氨酸诱导的活性Caspase-3蛋白的表达,降低PC12细胞的凋亡率,在 0.1~10 μmol/L 剂量呈量效相关性。结论: 在一定剂量范围内,天麻素对谷氨酸损伤的PC12细胞具有保护作用,其机制可能与减少ROS的生成,阻止氧化损伤的发生,抑制Caspase-3途径依赖的细胞凋亡相关。     

快速鉴别味精中的硫酸镁

MgSO4与味精极其相似,肉眼很难分辨,利用硫酸盐能使大豆蛋白凝固的特点,从而寻找一种快速简便的鉴别味精中MgSO4的方法,以便初步判断味精中是否掺入MgSO4。     

A hybrid support vector machine and fuzzy reasoning based fault diagnosis and rescue system for stable glutamate fermentation

In industrial glutamate fermentation by biotin-auxotroph Corynebacterium glutamicum, biotin content variation in corn slurry greatly affects fermentation performance. To maintain the fermentation stability, a hybrid support vector machine (SVM) and fuzzy reasoning based fault diagnosis and rescue system was developed. The system uses SVM outputs as the inputs of the fuzzy reasoning classifier having a couple of production rules and condition membership functions related with SVM outputs, to categorize multiple faults. The effectiveness of the proposed system was verified in a normal fermentation run and two abnormal runs with different biotin initial-content faults with the aid of using on-line measurable data such as ammonia consumption rate and CO₂ evolution rate. The results indicated that the proposed faults-diagnosis system could cluster multiple fermentation faults quickly, accurately and stably, and faults and their types could be identified at the earliest fermentation stage. Based on the diagnosis results, the proposed system was further applied for real fault-rescue in two fermentations with different biotin initial-content faults. In both cases, by immediately taking relevant rescue measures after identifying the faults and their types, glutamate fermentations with initial faults were restored to normal, and final glutamate concentrations reached a normal level of 75–80 g/L at 34 h.