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.     

酶法分离制备γ-氨基丁酸和L-天冬氨酸

以L-谷氨酸(L-G lu)和L-天冬氨酸(L-Asp)两种混合酸性氨基酸〔m(L-G lu)∶m(L-Asp)=1∶1〕为原料,利用大肠杆菌菌体内脱羧酶对L-谷氨酸的专一脱羧作用,酶法分离制备了γ-氨基丁酸和L-天冬氨酸。考察了转化体系温度、pH等影响L-谷氨酸脱羧酶活力的主要因素,实验表明,最佳工艺条件为:温度35℃,转化体系pH=5.0,ρ(菌体)=6 g/L,ρ(Tween80)=0.15 g/L,菌龄16 h,ρ(底物)=60 g/L。L-谷氨酸脱羧酶在最适转化条件下比酶活高达15 036 U。1 g湿菌体可重复使用3次共转化L-谷氨酸和L-天冬氨酸混合物30 g,其中L-谷氨酸完全转化为γ-氨基丁酸。γ-氨基丁酸及L-天冬氨酸的总收率可分别达到理论收率的88%和90%。     

唾液链球菌嗜热亚种Y-2产谷氨酸脱羧酶的影响因子确立

从产酶和细胞生长较好的MRS培养基出发,对Streptococcus salivarius ssp.thermophilus Y-2产谷氨酸脱羧酶(glutamate decarboxylase,GAD)的影响因子进行探讨,结果当培养基组成和培养条件为蛋白胨15g/L、牛肉膏12.5g/L、蔗糖12.5g/L、柠檬酸二铵2.0g/L、乙酸钠5.0g/L、K2HPO42.0g/L、CaCl2 2.0g/L、Tween 80 1.0ml、pH7.0、接种量2%(V/V)、发酵温度37℃、发酵时间12h时,较有利于菌株Y-2产GAD。Plackett-Burman设计法研究表明培养基初始pH值和K2HPO4为影响菌株Y-2产GAD的主要影响因素。经对菌株Y-2产GAD影响因素的筛选,新获得的培养基在组成上与MRS培养基相比已发生显著变化,GAD活力提高了1.3倍。     

L-鸟氨酸生产菌的选育及其发酵条件的研究

以谷氨酸棒杆菌为出发菌株 ,经硫酸二乙酯、紫外线诱变 ,定向选育出一株鸟氨酸生产菌A1157,其遗传标记为Arg- +D Argr+SGr,并能耐受高浓度葡萄糖。对菌株A1157的发酵条件进行了研究 ,在最佳培养条件下 ,该菌株L 鸟氨酸产量可达 9.85g/L。     

Purification and biochemical characterisation of a novel glutamate decarboxylase from rice bran

BACKGROUND: Glutamate decarboxylase (GAD) is a useful enzyme whose main function is to catalyse the irreversible α‐decarboxylation of L ‐glutamate to produce γ‐aminobutyric acid. The cheap and abundant rice‐processing by‐product rice bran contains a high amount of GAD, the purification and characterisation of which have not yet been reported. In this study, research on rice bran GAD was initiated. RESULTS: Rice bran GAD was purified to homogeneity via a combined purification protocol of ammonium sulfate fractionation, ion exchange chromatography and two gel filtrations, with a purification fold of 128.6 and an activity recovery of 21.3%. The enzyme was active at pH 5.5 and 40 °C and retained 80% of its original activity in the pH range 5–9 and the temperature range 30–50 °C. GAD activity was significantly enhanced in the presence of Ca²⁺ but strongly inhibited by Ag⁺, Hg²⁺, sodium dodecyl sulfate and CH₃COOH. Kinetic determination of the apparent K_m for L ‐glutamate and pyridoxal 5′‐phosphate gave values of 27.4 mmol L^?1 and 1.16 µmol L^?1 respectively. CONCLUSION: Considering that rice bran is cheap and commercially available and that rice bran GAD is relatively stable, the development of cost‐effective rice bran GAD‐related functional foods would seem to be feasible. Copyright © 2010 Society of Chemical Industry     

Ethyl carbamate formation regulated by ornithine transcarbamylase and urea metabolism in the processing of Chinese yellow rice wine

Ethyl carbamate (EC) is a potential carcinogenic compound present in most of the fermented foods. In this work, EC was inhibited through different strategies during vinification of Chinese yellow rice wine. EC can be inhibited by the use of ornithine in contrast to the control at peak point. However, the utilisation of urease resulted in little inhibitive effect on EC. The comparative data of intracellular ornithine transcarbamylase (OTCase) and arginine deiminase (ADI) among four experiments showed that EC was positively regulated by intracellular OTCase, but ADI was not determined. Extracellular urea and citrulline content was significantly increased by adding ornithine (P < 0.05), whereas ethanol played a minor role in EC formation. The correlation analysis between EC and OTCase or urea revealed a linear association (correlation coefficients above 0.8). These findings suggested that OTCase may be a required factor regulating EC formation during the brewing of Chinese yellow rice wine.     

α—乙酰乳酸脱羧酶应用研究

从细菌AD6发酵产生并纯化的α-乙酰乳酸脱羧酶活性成份加入前酵开始的12°啤酒麦汁中，添加量为200u／L，对照样品的总双酰量降到0．15mg／L以下的总发酵时间为45天，而加入α-乙酰乳酸脱羧酶的样品则为9天，并且该提取物对发酵麦汁中的酵母数12°麦汁PH无影响．     

α─乙酸乳酸脱羧酶在啤酒酿造中的应用

本文介绍了在啤酒酿造中应用α─乙酰乳酸脱羧酶的新方法。采用该方法较有效地控制了成品啤酒中的双乙酰含量，克服了装瓶后啤酒中双乙酰的回升现象，既缩短了啤酒发酵周期，又保证了啤酒质量。     

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。