Enhancing the Activity of Glutamate Decarboxylase from Lactobacillus brevis by Directed Evolution

Li, Gang; Hu, Sheng; Yu, Kai; Huang, Jun; Yao, Shan‐Jing; Lei, Yinlin; Hu, Guixiang; Mei, Lehe

doi:10.1016/j.cjche.2014.09.025

Cited by 11 publications

(10 citation statements)

References 29 publications

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“…Among the eight mutations predicted by this way, two mutants with higher thermostability had been verified, which manifested that such strategy was efficient and useful for the molecular reinforcement of protein. Compared to other strategies which have been applied to enhance the thermostability of GAD, including site-directed saturation mutagenesis [37] and introduction of proline [29], our method greatly reduced the workload of screening. We believe the parallel strategy including consensus design and free energy calculation designed in this study provides an effective way to improve thermostability of other enzymes.…”

Section: Discussionmentioning

confidence: 99%

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase

Qingfei

Wang

et al. 2020

Molecules

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Glutamate decarboxylase (GAD; EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculation was applied to identify critical amino acid sites affecting thermostability of GAD and select proper mutation contributing to improve structure rigidity of the enzyme. Two mutant enzymes, D203E and S325A, with higher thermostability were obtained, and their semi-inactivation temperature (T5015) values were 2.3 °C and 1.4 °C higher than the corresponding value of the wild-type enzyme (WT), respectively. Moreover, the mutant, S325A, exhibited enhanced activity compared to the wild type, with a 1.67-fold increase. The parallel strategy presented in this work proved to be an efficient tool for the reinforcement of protein thermostability.

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Section: Discussionmentioning

confidence: 99%

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase

Qingfei

Wang

et al. 2020

Molecules

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“…Several studies have reported individual Lb. brevis GadB tested either in acetate buffer or citrate-phosphate buffer exhibiting its activity spectrum toward acidic conditions (pH 3.5–5.5) but not near-neutral pH (pH 5.5–6.5; Yu et al, 2012 ; Seo et al, 2013 ; Lin et al, 2014 ), but the activity of only one GadA from Lb. brevis IFO 12005 has been reported twice focusing on its activation of enzymatic activity during in vitro conditions (Ueno et al, 1997 ; Hiraga et al, 2008 ).…”

Section: Discussionmentioning

confidence: 99%

Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH

Tun

Law

et al. 2017

Front. Microbiol.

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Many strains of lactic acid bacteria (LAB) and bifidobacteria have exhibited strain-specific capacity to produce γ-aminobutyric acid (GABA) via their glutamic acid decarboxylase (GAD) system, which is one of amino acid-dependent acid resistance (AR) systems in bacteria. However, the linkage between bacterial AR and GABA production capacity has not been well established. Meanwhile, limited evidence has been provided to the global diversity of GABA-producing LAB and bifidobacteria, and their mechanisms of efficient GABA synthesis. In this study, genomic survey identified common distribution of gad operon-encoded GAD system in Lactobacillus brevis for its GABA production among varying species of LAB and bifidobacteria. Importantly, among four commonly distributed amino acid-dependent AR systems in Lb. brevis, its GAD system was a major contributor to maintain cytosolic pH homeostasis by consuming protons via GABA synthesis. This highlights that Lb. brevis applies GAD system as the main strategy against extracellular and intracellular acidification demonstrating its high capacity of GABA production. In addition, the abundant GadA retained its activity toward near-neutral pH (pH 5.5–6.5) of cytosolic acidity thus contributing to efficient GABA synthesis in Lb. brevis. This is the first global report illustrating species-specific characteristic and mechanism of efficient GABA synthesis in Lb. brevis.

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“…Similarly, Naser (Naser et al, 2015) reported that the favorable initial pH for GABA production by LAB was 5 to 5.5. And Komatsuzaki (Komatsuzaki et al, 2008) have demonstrated an optimal pH value for maintaining the activity of glutamic acid decarboxylase (GAD) which is responsible for the synthesis of the GABA, and the high or low pH may lead to partial loss of GAD activity (Lin et al, 2014). Effect of different culture temperature from 20 to 40℃ on GABA production results showed…”

Section: Resultsmentioning

confidence: 99%

An Efficient γ-Aminobutyric Acid (GABA) Producing and Nitrite Reducing Ability of <i>Lactobacillus plantarum</i> BC114 Isolated from Chinese Paocai

Zhang

Zeng

Tan

et al. 2017

FSTR

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Some γ-aminobutyric acid (GABA) producing and nitrite reducing lactic acid bacteria were isolated from Sichuan paocai, a Chinese traditional fermented vegetable. Determination of GABA and sodium nitrite residue was carried out by thin layer chromatography combined with HPLC and colorimetric analysis, respectively. It was found that BC114 strain which was identified and designated as Lactobacillus plantarum BC114 possessed the highest GABA producing and nitrite degradation capacity in MRS broth with 5 g/L monosodium glutamate (MSG) and 100 mg/L sodium nitrite. Then, GABA production and nitrite degradation ability were enhanced by optimizing culture conditions, such as time, pH, temperature and MSG concentration. The greatest yield (3.45 g/L) of GABA was determined after 72 h fermentation in MRS with 20 g/L MSG at 30℃ and initial pH 5.5. Furthermore, nitrite degradation ability reached the maximum value of 90.17%. These results suggested that L. plantarum BC114 with GABA producing and nitrite reducing ability has the potential to be starter culture and promote the development of functional fermented foods. Keywords: γ-aminobutyric acid, Nitrite degradation, Lactobacillus plantarum, Chinese paocai et al., 2013) and Marcha of Sikkim (Lee et al., 2010) containedGABA. The material basis of beneficial properties is closely linked and dominated by the fermentation strains of the genera Lactobacillus from the old salt brine or the fresh vegetable materials (Anadon et al., 2006). Recently, some of Lactobacillus spp. such as Lactobacillus brevis, Lactobacillus plantarum and Lactococcus fermentum which isolated from traditional fermented food sources has shown the ability to produce GABA (Sasimar et al., 2016). Therefore, the traditional fermentation Sichuan paocai is believed to be a good candidate for the isolation of GABA producing LAB.In addition to conducting the desirable properties, the spontaneous fermentation of traditional paocai is easily influenced by bacterial contamination which may results in the accumulation of nitrite in products, produce inconsistent and unsafe product. (Zahra et al., 2016). The paocai with high nitrite consumption has been associated with the cancer risk. Extensive experimental and some epidemiological data have suggested that humans are susceptible to carcinogenesis by N-nitroso compounds resulted from endogenous nitrosation reaction of nitrite (Fatemeh et al., 2016). Moreover, Seel (Seel et al., 1994) have found that the high levels of compounds in kimchi after nitrosation may play a role in gastric carcinogenesis in Southwest Korea. To overcome the limitation by nitrite overproduction in paocai associated with spontaneous fermentation, inoculation with starter culture fermentation is currently being investigated. Oh (Oh et al., 2004) have reported that Leuconostoc mesenteroides isolated from Kimchi samples depleted more than 90.0% of 150 mg/L nitrite after 48 h at 30℃. Additionally, several species of LABs isolated from naturally fermented products have been used as starter cultures fo...

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Enhancing the Activity of Glutamate Decarboxylase from Lactobacillus brevis by Directed Evolution

Cited by 11 publications

References 29 publications

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase

Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH

An Efficient γ-Aminobutyric Acid (GABA) Producing and Nitrite Reducing Ability of <i>Lactobacillus plantarum</i> BC114 Isolated from Chinese Paocai

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