Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study

Tavakoli, Yasaman; Esmaeili, Abolghasem; Saber, Hossein

doi:10.1016/j.compbiolchem.2016.05.006

Cited by 12 publications

(5 citation statements)

References 24 publications

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“…Huang et al [ 30 ] demonstrated that residues (Ser126, Ser127, Cys168, Ile211, Ser276, His278, and Ser321) in GAD from Lactobacillus brevis CGMCC 1306 play important roles in anchoring the PLP cofactor to the active site, thereby supporting its catalytic reactivity. Tavakoli et al [ 31 ] identified, through a stimulated docking study, that performing mutations separately at positions Ile164, Asn302, Asp304, Tyr393, Ser396, Arg398, and Thr410 could increase binding affinity to substrate. The corresponding residues in GADZ11 are Asp126, Cys127, Arg164, Ala168, His211, Ala276, Phe278, Tyr302, Leu304, Pro321, Asn393, Asp396, Pro398, and Ser410, which are totally different to those in the two enzymes mentioned above.…”

Section: Discussionmentioning

confidence: 99%

Characterization of three glutamate decarboxylases from Bacillus spp. for efficient γ-aminobutyric acid production

et al. 2021

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Background Gamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts l-glutamate (l-Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use. Results Three GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards l-Glu (2.19 mM− 1 s− 1). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 14 times, the cells produced GABA with an average molar conversion rate of 98.6% within 14 h. Conclusions Three recombinant GADs from Bacillus strains have been conducted functional identification. The engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of l-Glu to GABA in a buffer-free reaction at a high l-Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA.

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Section: Discussionmentioning

confidence: 99%

Characterization of three glutamate decarboxylases from Bacillus spp. for efficient γ-aminobutyric acid production

et al. 2021

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“…The higher the B -factor value of an amino acid in a protein, the less that amino acid contributes to protein stability . The negative value of ΔΔ G indicates the positive effect of the mutant amino acids on the thermal stability of the protein . The three-dimensional structure of AlyMc was submitted to the HotSpot Wizard 3.0 and PoPMuSiC web servers to predict mutation sites.…”

Section: Resultsmentioning

confidence: 99%

“…39 The negative value of ΔΔG indicates the positive effect of the mutant amino acids on the thermal stability of the protein. 40 The three-dimensional structure of AlyMc was submitted to the HotSpot Wizard 3.0 and PoPMuSiC web servers to predict mutation sites. The mutation sites were screened based on the values of the Bfactor (>22) and ΔΔG (<0).…”

Section: Mutation Site Selection and Mutant Expressionmentioning

confidence: 99%

Computer-Aided Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase AlyMc

Cui,

Yang,

Liu

et al. 2024

J. Agric. Food Chem.

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Alginate lyase degrades alginate by the β-elimination mechanism to produce unsaturated alginate oligosaccharides (UAOS), which have better bioactivities than saturated AOS. Enhancing the thermal stability of alginate lyases is crucial for their industrial applications. In this study, a feasible and efficient rational design strategy was proposed by combining the computer-aided ΔΔG value calculation with the B-factor analysis. Two thermal stability-enhanced mutants, Q246V and K249V, were obtained by site-directed mutagenesis. Particularly, the t 1/2, 50 °C for mutants Q246V and K249V was increased from 2.36 to 3.85 and 3.65 h, respectively. Remarkably, the specific activities of Q246V and K249V were enhanced to 2.41-and 2.96-fold that of alginate lyase AlyMc, respectively. Structural analysis and molecular dynamics simulations suggested that mutations enhanced the hydrogen bond networks and the overall rigidity of the molecular structure. Notably, mutant Q246V exhibited excellent thermal stability among the PL-7 alginate lyase family, especially considering the heightened enzymatic activity. Moreover, the rational design strategy used in this study can effectively improve the thermal stability of enzymes and has important significance in advancing applications of alginate lyase.

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“…GADs have been identified in a variety of microorganisms and plants. Among them, GADs from prokaryotes, such as E. coli, B. megaterium, Lactobacillus sp., Streptomyces sp., and Enterococcus, exhibit optimal activity at pH less than 5.0 (Tavakoli et al 2016); GADs derived from eukaryotic microorganisms, including Aspergillus oryzae and Alternaria crassa, have optimal activity at pH less than 5.5 (Xu et al 2017); GADs derived from A. thaliana, petunia, potato, wheat, etc., have optimal activity within a pH range of 5.5-5.8 (Baum et al 1996). They show enzymatic activity only under acidic conditions and their enzymatic activity sharply declines when pH exceeds 6.0.…”

Section: Discussionmentioning

confidence: 99%

Characterization and mutagenesis of a novel Mycobacterium smegmatis-derived glutamate decarboxylase active at neutral pH

Chen

et al. 2022

World J Microbiol Biotechnol

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γ-aminobutyric acid (GABA) has various physiological functions and is widely used in medicine, food, and other fields. Glutamate decarboxylase (GAD) is a key enzyme that catalyzes the decarboxylation of L-glutamate to synthesize GABA. However, the industrial application of microorganism-derived GAD is limited by its rapid loss of enzymatic activity with pH approaching neutrality. In this study, a novel glutamate decarboxylase, GADMSM, from Mycobacterium smegmatis was overexpressed and purified. On the basis of homologous modeling and substrate molecular docking, several GADMSM mutants were constructed, and their enzymatic properties were analyzed. The results showed that the optimal pH of wild-type GADMSM is 5.4; at pH 6.2, 22.8% enzymatic activity was retained; the T211 amino acid residue and C-terminal deletion mutant GADMSMΔC showed relatively high catalytic activity in a pH range of 5.0-7.0. The Vmax and Km values of GADMSMΔC were 14.69 and 5.70, respectively, at pH 5.5, and 9.87 and 6.17, respectively, at pH 7.0. Compared with the wild-type GAD, GADMSMΔC still maintained higher affinity and enzymatic activity of the substrate, maintaining 78.5% of the highest enzymatic activity even at pH 7.0, which is the highest reported activity retention for GAD under neutral pH condition. Therefore, GADMSMΔC can be used for the transformation of high-yielding strains and industrial production of GABA.

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Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study

Cited by 12 publications

References 24 publications

Characterization of three glutamate decarboxylases from Bacillus spp. for efficient γ-aminobutyric acid production

Characterization of three glutamate decarboxylases from Bacillus spp. for efficient γ-aminobutyric acid production

Computer-Aided Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase AlyMc

Characterization and mutagenesis of a novel Mycobacterium smegmatis-derived glutamate decarboxylase active at neutral pH

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