Strains, media, plasmids and chemicals
The three Bacillus spp. were isolated from desert sand samples obtained from Ningxia Province, China, and deposited in the Agricultural Culture Collection of China under registration numbers ACCC 61750, 61747, and 61748, respectively. These strains were cultured in Luria–Bertani medium at 30 °C. The E. coli XL10 was used for gene cloning. E. coli BL21 and plasmid pET-28a were used as the expression host and vector, respectively. The DNA purification kit, LA Taq DNA polymerase, and restriction endonucleases were purchased from TaKaRa (Tsu, Japan). T4 DNA ligase was purchased from New England Biolabs (Hitchin, UK). All chemicals were of analytical grade and were commercially available.
Cloning of gad genes from Bacillus strains and plasmid construction
Genomic DNA from Bacillus strains grown in LB medium was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The GAD genes, gadz1, gadz11, and gadz20 (GeneBank accession number: MW703457, MW703456 and MW703455) were amplified by PCR from the genomic DNA of Bacillus strains using suitable primer pairs (GADF: 5’-CTGAATTCATGTCCAAGGATCGAAAAGCAG-3’ and GADR: 5’-TTCGCCGGCGAAGCGGCCGCCTAATGATGAAACCCATT-3’). The amplified DNA fragment was purified from a 1.0% agarose gel using the Wizard SV Gel and PCR Clean-Up System (Promega) after gel electrophoresis. The purified 1470 bp gad fragment was digested with EcoRI and NotI and ligated (T4 DNA ligase) into pET-28a (+) to generate pET-28a-gadz1, pET-28a-gadz11, and pET-28a-gadz20. The constructed plasmids were used for expression in E. coli BL21 (DE3).
Expression and enzyme purification
E. coli BL21(DE3) transformed with the plasmids pET-28a-gadz1, pET-28a-gadz11, and pET-28a-gadz20 were cultured in LB medium containing kanamycin (50 μg/mL) at 37 °C for 12 h. Then, the culture was transferred to 400 mL LB broth at 37 °C (1% by volume of inoculant). When suitable bacterial concentration was achieved (OD600 of 0.6–0.8), protein expression was induced by adding 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and shaking at 200 rpm (16 °C, 16 h). The cells were collected at 8000 ×g for 10 min and resuspended in lysis buffer (20 mM Tris-HCl buffer, 500 mM NaCl, pH 7.6). Then, cells were disrupted by ultrasonic waves. After centrifugation, the protein was separated by Ni-NTA affinity chromatography using elution buffer (20 mM Tris-HCl buffer, 500 mM NaCl, 200 mM imidazole, pH 7.6), and then, the proteins in the supernatant and pellet were resolved SDS-PAGE . The final protein concentrations were determined using the Bradford assay (BSA was used as a standard) .
Determination of enzyme activity and GABA formation
Enzyme activity was determined by measuring GABA production using HPLC analysis, with some modifications [40,41]. The reaction mixture comprised 400 μL Na2HPO4-citric acid buffer (80 mM, pH 6.0), 500 μL l-Glu (50 mM), 50 μL PLP (0.02 mM), and 50 μL purified enzyme. Ice-chilled 80% ethanol was added at an equal amount (1 mL) to stop the reaction after 30 min at 40 °C. The reaction supernatant (500 μL) was mixed with 100 μL of NaHCO3 (2.5 g/L) and 200 μL of 4-N, N-dimethylaminoazobenzene-4’-sulfonyl chloride (DABS-Cl) (0.25 g/L, dissolved in acetonitrile), and incubated it at 70 °C for 20 min. This was followed by analysis on a SHIMADZU 20A series instrument (Shimadzu, Kyoto, Japan) and Agilent ZORBAX SB-C18 column (5 μm, 4.6 × 150 mm) (Agilent, Santa Clara, CA, USA). The mobile phase was a solution of 35% (v/v) acetonitrile solution and 65% 50 mM sodium acetate. The flow rate and column temperature were 1 mL/min and 30 °C, respectively; the injection volume was 10 μL; and the detection wavelength was 436 nm. The GABA content in the test solutions was calculated by taking into consideration of the peak areas with the standard. The definition of one enzyme activity unit is the amount of enzyme required for the release of 1 mM free GABA in 1 min. Three parallel wells were set per group.
Reaction conditions and kinetic parameters of enzymes
Optimum pH and temperature assay
The optimum pH properties of the GADs were determined at reaction pH values from 3.0 to 7.0. The pH-activity profiles were examined for 30 min at 37 °C. The optimum temperature was examined at pH 5.0 over a temperature range of 25–55 °C. Each experiment was performed in triplicate.
Enzyme kinetic assays
Enzyme kinetic assays were performed in Na2HPO4-citric acid buffer containing 5–150 mM l-Glu at 40 °C for 15 min. The pH of the mixture containing l-Glu and Na2HPO4-citric acid buffer was 5.0. Each experiment was performed in triplicate. The Km and Vmax values were nonlinearly fitted using the GraphPad Prism 5 software.
Whole-cell bioconversion process
Recombinant E. coli cells harboring GADZ1, GADZ11, and GADZ20 were cultured at 37 °C. Then, protein expression in the engineered bacteria was induced at 16 °C for 16 h. After this, the cells were centrifuged at 8000 ×g for 10 min, washed, and resuspended it in water containing MSG or l-Glu at appropriate concentrations. The OD600 was measured to indicate the cell concentration. The reaction of the 20 mL mixture in 100 mL flasks was performed at 37 °C at 120 rpm. GABA production of the reaction was analyzed and calculated by HPLC.
To optimize the reaction conditions, the effects of substrate specificities, substrate concentration, cell concentration, PLP concentration, time-course analysis of single-batch reactions, and the recycling ratio of batch reactions were analyzed simultaneously in this study.
Substrate specificity and optimum substrate concentration
The substrate specificities of whole-cell biosynthesis were investigated in an assay system containing the following substrates: MSG or l-Glu. The whole-cell biotransformation reactions were conducted in 0.1 M sodium acetate buffer (pH 4.6) or water by single factor and orthogonal experiments. The reaction condition was obtained as follows: reaction time, 1 h; OD600, 20; and l-Glu or MSG concentration, 1 M.
To determine the effects of the l-Glu concentration, the following procedure was performed: after 16 h of preculture of E. coli GADZ11, the cells were centrifuged and suspended in water. The reaction system consisted of 20 mL water containing resuspended cells in a 100 mL Erlenmeyer flask. To this, different amounts of l-Glu (0.5, 1, 2, 3, and 4 M) were added. The reaction mixture contained 0.02 mM PLP. The reaction was stopped by adding 30 mL ethanol after 1 h, and the volume was made up to 100 mL with water. The reaction supernatants were collected to measure the GABA content.
Optimum cell and PLP concentration
To determine of the optimal cell concentration, the following procedure was performed. The 20 mL reaction system was contained 6 M l-Glu, 0.02 mM PLP, and a predetermined quantity of cells (OD600 2, 5, 10, 15, 20, 30). The reaction mixture was incubated in a shaker at a specified shaking rate (120 rpm) at 37 °C for 2 h. Aliquots (500 μL) were withdrawn into an equal volume of ice-chilled 80% ethanol after 2 h to stop the reaction. The reaction supernatants were collected to measure the content of GABA via HPLC.
To determine the effects of the coenzyme PLP, 0 (control), 0.02, 0.05, 0.1, 0.2, 0.5, and 1 mM PLP was added to the 20 mL mixture. The l-Glu and cell concentrations were 1 M and OD600 20, respectively. After 2 h of culture at 37 °C, 500 μL of the reaction mixtures were taken and measured with HPLC.
The upper bound estimation of E. coli BL21 (DE3)/GADZ11 to convert GABA from l-Glu
To optimize the reaction conditions and decrease the manufacturing cost, reactions with different concentrations of l-Glu were conducted for the time-course assays. PLP at a final concentration of 0.4 mM and cells at a final concentration of OD600 20 were added to the reaction system, and the l-Glu concentrations tested were 1, 2, 3, and 4 M. To stop the reaction, 30 mL ethanol was added to the mixtures (when the solid l-Glu was fully consumed), and water was added to make up the volume to 100 mL. The reaction mixtures were subjected to HPLC.
The concentrations of l-Glu and PLP were 1 M and 0.1 mM, respectively, and the cell concentration was OD600 20 in the batch reaction. The reaction was conducted at 37 °C for 1 h, and the cells were collected by centrifugation for the next batch when the solid l-Glu was fully consumed. There were eight batches in total. The samples were tested when the reactions were complete.
Conversion of high concentrations of l-Glu and the purification and crystallization of GABA
To prepare GABA crystals, the mixture from the previous eight batches was separated and concentrated by rotary evaporation. GABA crystals were collected from the concentrated solution. The crystals were oven-dried at 65 °C until they were converted into a white powder . The purity of the powder was determined by HPLC.