Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum / 生物工程学报

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2

Background: GABA (γ-aminobutyric acid) is a four-carbon nonprotein amino acid that has hypotensive, diuretic, and tranquilizing properties. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple and economical method of preparing and immobilizing GAD would be helpful for GABA production. In this study, the GAD from Lactobacillus fermentum YS2 was expressed under the control of a stress-inducible promoter and was purified and immobilized in a fusion form, and its reusability was investigated. Results: The fusion protein CBM-GAD was expressed in Escherichia coli DH5α carrying pCROCB-gadB, which contained promoter PrpoS, cbm3 (family 3 carbohydrate-binding module from Clostridium thermocellum) coding sequence, the gadB gene from L. fermentum YS2 coding for GAD, and the T7 terminator. After a one-step purification of CBM-GAD using regenerated amorphous cellulose (RAC) as an adsorbent, SDS-PAGE analysis revealed a clear band of 71 kDa; the specific activity of the purified fusion protein CBM-GAD reached 83.6 ± 0.7 U·mg-1. After adsorption onto RAC, the immobilized GAD with CBM3 tag was repeatedly used for GABA synthesis. The protein-binding capacity of RAC was 174 ± 8 mg·g-1. The immobilized CBM-GAD could repeatedly catalyze GABA synthesis, and 8% of the initial activities was retained after 10 uses. We tested the conversion of monosodium glutamate to GABA by the immobilized enzyme; the yield reached 5.15 g/L and the productivity reached 3.09 g/L·h. Conclusions: RAC could be used as an adsorbent in one-step purification and immobilization of CBM-GAD, and the immobilized enzyme could be repeatedly used to catalyze the conversion of glutamate to GABA.

Atorvastatin protects GABAergic and dopaminergic neurons in the nigrostriatal system in an experimental rat model of transient focal cerebral ischemia / La atorvastatina protege las neuronas gabérgicas y dopaminérgicas del sistema nigroestriatal en un modelo experimental de isquemia cerebral focal transitoria en ratas

Introduction: Cerebral ischemia is the third leading cause of death and the primary cause of permanent disability worldwide. Atorvastatin is a promising drug with neuroprotective effects that may be useful for the treatment of stroke. However, the effects of atorvastatin on specific neuronal populations within the nigrostriatal system following cerebral ischemia are unknown. Objective: To evaluate the effects of atorvastatin on dopaminergic and GABAergic neuronal populations in exofocal brain regions in a model of transient occlusion of the middle cerebral artery. Materials and methods: Twenty-eight male eight-week-old Wistar rats were used in this study. Both sham and ischemic rats were treated with atorvastatin (10 mg/kg) or carboxymethylcellulose (placebo) by gavage at 6, 24, 48 and 72 hours post-reperfusion. We analyzed the immunoreactivity of glutamic acid decarboxylase and tyrosine hydroxylase in the globus pallidus, caudate putamen and substantia nigra. Results: We observed neurological damage and cell loss in the caudate putamen following ischemia. We also found an increase in tyrosine hydroxylase immunoreactivity in the medial globus pallidus and substantia nigra reticulata, as well as a decrease in glutamic acid decarboxylase immunoreactivity in the lateral globus pallidus in ischemic animals treated with a placebo. However, atorvastatin treatment was able to reverse these effects, significantly decreasing tyrosine hydroxylase levels in the medial globus pallidus and substantia nigra reticulata and significantly increasing glutamic acid decarboxylase levels in the lateral globus pallidus. Conclusion: Our data suggest that post-ischemia treatment with atorvastatin can have neuro-protective effects in exofocal regions far from the ischemic core by modulating the GABAergic and dopaminergic neuronal populations in the nigrostriatal system, which could be useful for preventing neurological disorders.

Introducción. La isquemia cerebral es la tercera causa de muerte y la primera de discapacidad permanente en el mundo. La atorvastatina es un fármaco neuroprotector prometedor para el tratamiento de la apoplejía; sin embargo, su acción sobre las poblaciones neuronales del sistema nigroestriatal después de la isquemia aún se desconoce. Objetivo. Evaluar el efecto de la atorvastatina sobre poblaciones gabérgicas y dopaminérgicas en regiones exofocales en un modelo de oclusión transitoria de la arteria cerebral media. Materiales y métodos. Se utilizaron 28 ratas Wistar macho de ocho semanas de edad. Los ejemplares con isquemia simulada y los ejemplares sometidos a isquemia fueron tratados con atorvastatina (10 mg/kg) y carboximetilcelulosa (placebo) administrados por medio de sonda a las 6, 24, 48 y 72 horas después de la reperfusión. Se analizó la inmunorreacción de la descarboxilasa del ácido glutámico y de la tirosina hidroxilasa en el globo pálido, el putamen caudado y la sustancia negra. Resultados. Los datos confirmaron el daño neurológico y la pérdida celular en el putamen caudado. Se incrementó la inmunorreacción de la tirosina hidroxilasa en el globo pálido medial y la sustancia negra pars reticulata , disminuyendo la inmunorreacción de la descarboxilasa del ácido glutámico en el globo pálido lateral de los animales isquémicos tratados con placebo; sin embargo, el tratamiento con atorvastatina pudo revertirla, lo que logró una disminución significativa de la tirosina hidroxilasa en el globo pálido medial y la sustancia negra pars reticulata y aumentando los niveles de descarboxilasa del ácido glutámico en el globo pálido lateral. Conclusión. Nuestros datos sugieren que la atorvastatina en el tratamiento posterior a la isquemia ejerce neuroprotección en las zonas exofocales, modulando las poblaciones neuronales gabérgicas y dopaminérgicas del sistema nigroestriatal, lo que podría prevenir trastornos neurológicos.

Association of genetic polymorphisms of glutamate decarboxylase 2 and the dopamine D2 receptor with obesity in Taiwanese subjects

It has been proposed that glutamate decarboxylase 2 and the dopamine D2 receptor are involved in the brain reward cascade to increase carbohydrate craving and cause eating disorders. We investigated the association between the polymorphisms of the CAD2 and DRD2 genes and obesity with a higher body mass index [BMI] in Taiwanese patients. A retrospective, case-control study at Antai Tian-Sheng Memorial Hospital from 1 January to 31 December 2009. Of 300 subjects enrolled in the study, 132 were obese [BMI>30 kg/m[2]] and 168 controls were not obese [BMI

Influence of progesterone on GAD65 and GAD67 mRNA expression in the dorsolateral striatum and prefrontal cortex of female rats repeatedly treated with cocaine

Female rats are intensely affected by cocaine, with estrogen probably playing an important role in this effect. Progesterone modulates the GABA system and attenuates the effects of cocaine; however, there is no information about its relevance in changing GABA synthesis pathways after cocaine administration to female rats. Our objective was to investigate the influence of progesterone on the effects of repeated cocaine administration on the isoenzymes of glutamic acid decarboxylase (GAD65 and GAD67) mRNA in brain areas involved in the addiction circuitry. Ovariectomized, intact and progesterone replacement-treated female rats received saline or cocaine (30 mg/kg, ip) acutely or repeatedly. GAD isoenzyme mRNA levels were determined in the dorsolateral striatum (dSTR) and prefrontal cortex (PFC) by RT-PCR, showing that repeated, but not acute, cocaine decreased GADs/β-actin mRNA ratio in the dSTR irrespective of the hormonal condition (GAD65: P < 0.001; and GAD67: P = 0.004). In the PFC, repeated cocaine decreased GAD65 and increased GAD67 mRNA ratio (P < 0.05). Progesterone replacement decreased both GAD isoenzymes mRNA ratio after acute cocaine in the PFC (P < 0.001) and repeated cocaine treatment reversed this decrease (P < 0.001). These results suggest that cocaine does not immediately affect GAD mRNA expression, while repeated cocaine decreases both GAD65 and GAD67 mRNA in the dSTR of female rats, independently of their hormonal conditions. In the PFC, repeated cocaine increases the expression of GAD isoenzymes, which were decreased due to progesterone replacement.