BACKGROUND: Gamma(γ )-aminobutyric acid (GABA) has been used extensively in pharmaceuticals and functional foods and is also a building block for bioplastics. GABA is produced from glutamate through decarboxylation catalyzed by glutamate decarboxylase (GAD). The reaction medium should be kept acidic because a pH rise resulting from the reaction inactivates the enzyme catalyst, which is active only at acidic pH. The use of conventional buffers and acids inevitably accompanies salts, which cause serious problems in separation and purification of GABA. In this work, we have applied heterogeneous solid acids for the first time. RESULTS:The GAD-catalyzed reaction was conducted in 0.2 mol L −1 sodium acetate buffer (pH 4.6) with 1 mol L −1 monosodium glutamate at 37 • C. When commercial cation-exchange resins as solid acids were simply added to the reaction medium, the conversion improved from 13% to 67% without salt formation. Even when water was used as the reaction medium, acidic ion-exchange resins enhanced the reaction conversion significantly. CONCLUSION: In a salt-free manner, acidic resins suppress the pH rise during the reaction so that they can enhance the reaction conversion. In addition, they can be recovered and reused easily after the reaction. Heterogeneous solid acids make the GABA production process more economical and eco-friendly.

Redox enzymes are widely used as powerful biocatalysts owing to their high selectivity and sustainability. Since most of the enzymatic reactions require the expensive coenzyme (the reduced form of nicotinamide adenine dinucleotide, NADH) in a stoichiometric amount, efficient NADH regeneration is essential for various biocatalytic syntheses. Even though an electroenzymatic recycling method has many advantages, it requires low-molecular-weight redox mediators to transfer electrons from electrodes to an enzymes' active site, which are generally toxic and make it difficult to purify products after the reaction. In the present study, we have conjugated a mediator to enzyme catalyst to solve the problem of the electroenzymatic NADH regeneration. Mediators bound to enzymes can be separated from the reaction medium and then reused readily. Moreover, the conjugation can promote a faster electron transfer between mediators and enzymes. For the conjugation, a viologen with a carboxyl group, ethyl carboxyethyl viologen (ECV) was synthesized and covalently linked to diaphorase (DI) using a cross-linker. Cyclic voltammetry showed that the ECV conjugated to DI displayed electrochemical behavior similar to free ECV. In marked contrast to native DI, the DI-ECV conjugate reduced NAD + to NADH without any mediator, and this bioelectrocatalyst regenerated NADH successfully for enzymatic lactate synthesis.Redox enzymes are widely used as selective and sustainable catalysts not only in various organic synthesis processes but also in electrochemical applications such as biosensors and biofuel cells. 1,2 About 90% of them require nicotinamide coenzymes [e.g., nicotinamide adenine dinucleotide (NAD + ) and its reduced form, NADH], which form reversible redox pairs to accept or donate electrons in redox reactions. 1,3 In particular, NADH participates in important and challenging reactions including CO 2 fixation, alkane hydroxylations, and chiral alcohol syntheses in stoichiometric quantities; 1,4 however, NADH is too expensive to use on a large scale. Hence, the efficient regeneration of NADH from NAD + is essential for industrial applications of these biocatalysts. 3,[5][6][7][8] There are three popular methods to recycle NADH: enzymatic, electrochemical, and electroenzymatic. Even though enzymatic regeneration of NADH where a second enzyme (e.g., formate dehydrogenase) and an auxiliary substrate (e.g., formate) are used as a catalyst and a sacrificial donor, respectively, is the most practical method owing to its high total turnover number and selectivity, the co-substrate (e.g., formate) makes it difficult to isolate reaction products. 3,6 In this respect, an electrochemical method in which water is the sole electron and proton source for NAD + reduction has been intensively investigated. 7,8 Although electron supply from water electrolysis is attractive, a direct NAD + reduction on bare electrodes yields enzymatically inactive forms such as NAD dimers and 1,6-NADH isomer due to the formation of NAD free radicals followed by radical c...

BACKGROUND: Gamma( )-aminobutyric acid (GABA) is produced through an -decarboxylation reaction of L-monosodium glutamate (MSG) using glutamate decarboxylase (GAD). The pH rise caused by the reaction inactivates the enzyme catalyst, which is active only under acidic conditions, and consequently leads to low reaction conversions. Employment of conventional acids and buffers inevitably forms salts, which result in serious problems in separation and purification of GABA. It is essential to render GAD active even at neutral and alkaline pHs. In the present study, we first apply a cross-linked aggregation method in order to extend the active range of GAD toward alkaline pH. RESULTS: GAD from Escherichia coli was prepared as cross-linked enzyme aggregate (CLEA) in which the enzyme was precipitated using ammonium sulfate (60% saturation) and then cross-linked with glutaraldehyde (2%) in sodium acetate buffer (0.2 mol L −1 , pH 4.6). The cross-linked aggregation extended an active pH range of GAD from 5.5 up to 8.0; as a result, the reaction conversion of 1 mol L −1 MSG into GABA was improved from 13% to 22%. Moreover, the CLEA of GAD was easily recovered after the reaction and reused retaining >95% of its initial activity during the first four cycles and >60% activity at the 10th cycle. CONCLUSION: Cross-linked aggregation could make GAD active even at neutral and alkaline pHs. It is shown to be a useful method capable of facilitating recovery and reuse of the enzyme as well as increasing the reaction conversion by extending the active pH range of GAD. AnalysisThe concentrations of substrate and product were determined using a HPLC system (Waters 2960 series) equipped with a UV/Vis dual absorbance detector (Waters 2487). After removal of contaminants by centrifugation, samples were injected into the HPLC J Chem Technol Biotechnol 2015; 90: 2100-2105

Improving bacterial membrane permeability in a controlled manner using BPEIs can improve biosensing of toxic compounds, as well as be used in biofuel and bioenergy applications where membrane permeability to a solute is important.

No abstract