Characterization and immobilization on nickel-chelated Sepharose of a glutamate decarboxylase A from Lactobacillus brevis BH2 and its application for production of GABA

Abstract

A gene encoding glutamate decarboxylase A (GadA) from Lactobacillus brevis BH2 was expressed in a His-tagged form in Escherichia coli cells, and recombinant protein exists as a homodimer consisting of identical subunits of 53 kDa. GadA was absolutely dependent on the ammonium sulfate concentration for catalytic activity and secondary structure formation. GadA was immobilized on the metal affinity resin with an immobilization yield of 95.8%. The pH optima of the immobilized enzyme were identical with those of the free enzyme. However, the optimum temperature for immobilized enzyme was 5 °C higher than that for the free enzyme. The immobilized GadA retained its relative activity of 41% after 30 reuses of reaction within 30 days and exhibited a half-life of 19 cycles within 19 days. A packed-bed bioreactor with immobilized GadA showed a maximum yield of 97.8% GABA from 50 mM l-glutamate in a flow-through system under conditions of pH 4.0 and 55 °C.

Cross‐linked aggregation of glutamate decarboxylase to extend its activity range toward alkaline pH

“…Figure shows the recyclability of GAD CLEA; the CLEA was reused with <5% loss of activity during the initial four cycles and <40% loss at the tenth cycle. This recyclability of GAD CLEA was similar to that of GAD immobilized by adsorption or entrapment . This demonstrates that cross‐linked aggregation is capable of facilitating recovery and reuse of enzymes as well as extending the pH range of enzyme activity.…”

“…Because the pH dependence of GAD activity is related to protein conformational changes, we have reasoned that decreasing the flexibility of this enzyme molecule through immobilization will enable GAD to be active even at neutral to alkaline pHs. GAD has been immobilized through various methods such as adsorption, covalent binding to supports, and entrapment; however, extension of its active pH range has not been observed or reported. Herein, we immobilize GAD using a cross‐linked enzyme aggregate (CLEA) technique, which consists of two subsequent steps: (1) precipitation of soluble enzymes by addition of salts, water‐miscible organic solvents, or nonionic polymers; and (2) cross‐linking of the formed aggregates with bifunctional reagents such as glutaraldehyde .…”

Genome Sequence of Lactobacillus plantarum KB1253, a Gamma-Aminobutyric Acid (GABA) Producer Used in GABA-Enriched Tomato Juice Production

“…Many microorganisms can produce GABA, including bacteria, fungi, and yeasts, although lactic acid bacteria (LAB) are the most utilized GABA producers (5–8). GABA-producing LAB have been a focus of research in recent years because they possess unique physiological properties and are regarded as safe for use in the food industry (6, 9–11). Many food-derived lactobacilli produce GABA, including several strains of Lactobacillus brevis (12–16), L.…”

Genome Sequence of Lactobacillus curieae CCTCC M 2011381 T , a Novel Producer of Gamma-aminobutyric Acid

“…Many microorganisms can produce GABA, including bacteria, fungi, and yeasts, among which lactic acid bacteria (LAB) are the main GABA producers ( 5 – 8 ). GABA-producing LAB have been a focus of research in recent years, because LAB possess special physiological activities and are generally regarded as safe organisms, which have been extensively used in the food industry ( 6 , 9 – 11 ).…”