Increasing thermal stability of glutamate decarboxylase from Escherichia. coli by site-directed saturation mutagenesis and its application in GABA production.
Abstract
Gamma-amino butyric acid (GABA) is an important bio-product used in pharmaceuticals, functional foods, and a precursor of the biodegradable plastic polyamide 4 (Nylon 4). Glutamate decarboxylase B (GadB) from Escherichia. coli is a highly active biocatalyst that can convert l-glutamate to GABA. However, its practical application is limited by the poor thermostability and only active under acidic conditions of GadB. In this study, we performed site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability. A triple mutant (M6, Gln5Ile/Val6Asp/Thr7Gln) showed higher thermostability, with a 5.6 times (560%) increase in half-life value at 45 °C, 8.7 °C rise in melting temperature (Tm) and a 14.3 °C rise in the temperature at which 50% of the initial activity remained after 15 min incubation (T
Submission Details
ID: Bb3EiQLv
Submitter: Shu-Ching Ou
Submission Date: Dec. 28, 2018, 10:50 a.m.
Version: 1
Publication Details
Fan LQ;Li MW;Qiu YJ;Chen QM;Jiang SJ;Shang YJ;Zhao LM,J Biotechnol (2018) Increasing thermal stability of glutamate decarboxylase from Escherichia. coli by site-directed saturation mutagenesis and its application in GABA production. PMID:29660473Additional Information
Study Summary
| Number of data points | 33 |
| Proteins | Glutamate decarboxylase beta |
| Unique complexes | 5 |
| Assays/Quantities/Protocols | Experimental Assay: T15_50 ; Experimental Assay: t1/2 at 45 °C ; Experimental Assay: Thermal-Inactivation Rate Constant at 45 °C ; Experimental Assay: Tm ; Derived Quantity: SD of Tm ; Derived Quantity: t1/2 at 45 °C: Enhanced Fold ; Derived Quantity: △T15_50 |
| Libraries | Variants for GadB |
Structure view and single mutant data analysis
Study data
| Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
|---|
Data Distribution
Studies with similar sequences (approximate matches)
Correlation with other assays (exact sequence matches)
Relevant PDB Entries
| Structure ID | Release Date | Resolution | Structure Title |
|---|---|---|---|
| 2DGK | 2006-06-20 | 1.9 | Crystal structure of an N-terminal deletion mutant of Escherichia coli GadB in an autoinhibited state (aldamine) |
| 2DGM | 2006-06-20 | 1.95 | Crystal structure of Escherichia coli GadB in complex with iodide |
| 1PMM | 2004-02-17 | 2.0 | Crystal structure of Escherichia coli GadB (low pH) |
| 1XEY | 2004-10-05 | 2.05 | Crystal structure of the complex of Escherichia coli GADA with glutarate at 2.05 A resolution |
| 1PMO | 2004-02-17 | 2.3 | Crystal structure of Escherichia coli GadB (neutral pH) |
| 3FZ7 | 2009-02-03 | 2.5 | Crystal structure of apo glutamate decarboxylase beta from Escherichia coli |
| 3FZ6 | 2009-02-03 | 2.82 | Crystal structure of glutamate decarboxylase beta from Escherichia coli: complex with xenon |
| 3FZ8 | 2009-02-03 | 3.0 | Crystal structure of glutamate decarboxylase beta from Escherichia coli: reduced Schiff base with PLP |
| 2DGL | 2006-06-20 | 3.15 | Crystal structure of Escherichia coli GadB in complex with bromide |
Relevant UniProtKB Entries
| Percent Identity | Matching Chains | Protein | Accession | Entry Name |
|---|---|---|---|---|
| 600.0 | A,B,C,D,E,F | Glutamate decarboxylase beta | P69911 | DCEB_ECO57 |
| 600.0 | A,B,C,D,E,F | Glutamate decarboxylase beta | P69910 | DCEB_ECOLI |
| 600.0 | A,B,C,D,E,F | Glutamate decarboxylase beta | P69912 | DCEB_SHIFL |
| 597.6 | A,B,C,D,E,F | Glutamate decarboxylase beta | Q8FHG5 | DCEB_ECOL6 |
| 593.4 | A,B,C,D,E,F | Glutamate decarboxylase beta | P69909 | DCEA_ECOL6 |
| 593.4 | A,B,C,D,E,F | Glutamate decarboxylase beta | P69908 | DCEA_ECOLI |
| 592.2 | A,B,C,D,E,F | Glutamate decarboxylase beta | P58228 | DCEA_ECO57 |
| 592.2 | A,B,C,D,E,F | Glutamate decarboxylase beta | Q83PR1 | DCEA_SHIFL |