scholarly journals Food-grade γ-aminobutyric acid production by immobilized glutamate decarboxylase from Lactobacillus plantarum in rice vinegar and monosodium glutamate system

2021 ◽  
Author(s):  
Li-Li Yao ◽  
Jia-Ren Cao ◽  
Chang-Jiang Lyu ◽  
Fang-Fang Fan ◽  
Hong-Peng Wang ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Glutamate Decarboxylase ◽  
Specific Activity ◽  
Monosodium Glutamate ◽  
Reaction System ◽  
Catalytic Efficiency ◽  
Aminobutyric Acid ◽  
Protein Amino Acid ◽  
Food Grade ◽  
Rice Vinegar

Abstract Objectives γ-Aminobutyric acid (GABA) is a non-protein amino acid, considered a potent bioactive compound. This study focused on biosynthesis of food-grade GABA by immobilized glutamate decarboxylase (GAD) from Lactobacillus plantarum in the rice vinegar and monosodium glutamate (MSG) reaction system.Results The gene encoding GadB from L. plantarum has been heterologously expressed in Lactococcus lactis and biochemically characterized. Recombinant GadB existed as a homodimer, and displayed maximal activity at 40℃ and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of GadB for L-Glu was 22.33 mM and 1.04 L/(mmol·s), respectively, with a specific activity of 24.97 U/mg protein. Then, purified GadB was encapsulated in gellan gum beads. Compared to the free enzyme, immobilized GadB showed higher operational and storage stability. Finally, 9.82 to 21.48 g/L of GABA have been acquired by regulating the amounts of catalyst microspheres ranging from 0.5 to 0.8 g (wet weight) in 0.8 mL of the designed rice vinegar and MSG reaction system. Conclusions The method of production GABA by immobilized GadB microspheres mixed in the rice vinegar and MSG reaction system is introduced herein for the first time. Especially, the results obtained here meet the increased interest in the harnessing of biocatalyst to synthesize food-grade GABA.

scholarly journals Active-site engineering of ω-transaminase from Ochrobactrum anthropi for preparation of L-2-aminobutyric acid

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhiwei Zhang ◽  
Yang Liu ◽  
Jing Zhao ◽  
Wenqiang Li ◽  
Ruiwen Hu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reaction System ◽  
Catalytic Efficiency ◽  
Unnatural Amino Acid ◽  
Aminobutyric Acid ◽  
Saturation Mutagenesis ◽  
High Catalytic Activity ◽  
Pharmaceutical Drugs ◽  
Ochrobactrum Anthropi ◽  
Threonine Deaminase

Abstract Background The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering. Results Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g. Conclusion This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.

Enzymatic Production of γ-Aminobutyric Acid Using Rice Bran

2012 ◽  
Vol 586 ◽  
pp. 85-91
Author(s):  
Ying Guo Lü ◽  
Hui Zhang ◽  
Hui Yuan Yao
Keyword(s):  
Reaction Time ◽  
Rice Bran ◽  
Glutamate Decarboxylase ◽  
Monosodium Glutamate ◽  
Aminobutyric Acid ◽  
Enzymatic Method ◽  
Enzymatic Production ◽  
Gaba Production ◽  
Gaba Content ◽  
Time Buffer

An enzymatic method for γ-aminobutyric acid (GABA) production was invested. With this method, rice bran was used as glutamate decarboxylase (GAD) source and exogenous monosodium glutamate(MSG) was used as substrate. We stimulated the rice bran GAD via regulating the temperature, pH, reaction time, buffer and adding PLP, Ca2+ and substrate. In the existence of PLP and Ca2+, the GABA content of rice bran had been improved by about 45 fold. The GABA production reached 2.3g/100g bran, and the Glu conversion reached 100%. As rice bran is a by-product in rice processing and a large quantity of rice bran is commercially available, our study illuminated a safe and efficient way to produce GABA and GABA enriched food.

scholarly journals Microbial Production and Enzymatic Biosynthesis of γ-Aminobutyric Acid (GABA) Using Lactobacillus plantarum FNCC 260 Isolated from Indonesian Fermented Foods

Processes ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 22
Author(s):  
Ida Bagus Agung Yogeswara ◽  
Suwapat Kittibunchakul ◽  
Endang Sutriswati Rahayu ◽  
Konrad J. Domig ◽  
Dietmar Haltrich ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Culture Medium ◽  
Monosodium Glutamate ◽  
Aminobutyric Acid ◽  
Fermented Foods ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Gaba Production ◽  
Rdna Sequencing ◽  
Layer Chromatography

In the present study, we isolated and screened thirty strains of GABA (γ-aminobutyric acid)-producing lactic acid bacteria (LAB) from traditional Indonesian fermented foods. Two strains were able to convert monosodium glutamate (MSG) to GABA after 24 h of cultivation at 37 °C based on thin layer chromatography (TLC) screening. Proteomic identification and 16S rDNA sequencing using MALDI-TOF MS identified the strain as Lactobacillus plantarum designated as L. plantarum FNCC 260 and FNCC 343. The highest yield of GABA production obtained from the fermentation of L. plantarum FNCC 260 was 809.2 mg/L of culture medium after 60 h of cultivation. The supplementation of 0.6 mM pyridoxal 5’-phosphate (PLP) and 0.1 mM pyridoxine led to the increase in GABA production to 945.3 mg/L and 969.5 mg/L, respectively. The highest GABA production of 1226.5 mg/L of the culture medium was obtained with 100 mM initial concentration of MSG added in the cultivation medium. The open reading frame (ORF) of 1410 bp of the gadB gene from L. plantarum FNCC 260 encodes 469 amino acids with a calculated molecular mass of 53.57 kDa. The production of GABA via enzymatic conversion of monosodium glutamate (MSG) using purified recombinant glutamate decarboxylase (GAD) from L. plantarum FNCC 260 expressed in Escherichia coli was found to be more efficient (5-fold higher within 6 h) than the production obtained from fermentation. L. plantarum FNCC 260 could be of interest for the synthesis of GABA.

scholarly journals Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Hui-Hui Su ◽  
Fei Peng ◽  
Pei Xu ◽  
Xiao-Ling Wu ◽  
Min-Hua Zong ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Rational Design ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Value Added ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Glucaric Acid ◽  
Triple Mutant ◽  
Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

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