Microbial transformation of quinic acid to shikimic acid by Bacillus megaterium
© Ghosh et al. ; Licensee Springer 2014
Received: 17 April 2014
Accepted: 27 June 2014
Published: 31 July 2014
Biotransformation of quinic acid to shikimic acid was attempted using whole cells of Bacillus megaterium as a biocatalyst.
Physico-chemical parameters such as temperature (37°C), pH (7.0), agitation (200 rpm), substrate (5 mM) and cell mass concentrations (200 kg/m 3) and reaction time (3 h) were found optimum to enhance the bioconversion. Maximum conversion (89%) of quinic acid to shikimic acid was achieved using the above optimized parameters. Shikimic acid was extracted from the reaction mixture by a pH-dependent method and maximum recovery (76%) was obtained with petroleum ether.
Biotransformation of quinic acid to shikimic acid seems to be a better alternative over its fermentative production.
KeywordsShikimic acid Quinic acid Biotransformation
Material and methods
Quinic acid, shikimic acid and dehydroshikimic acid were obtained from Sigma Aldrich chemical company (Milawukee, WI, USA). Growth media components were purchased from Hi-Media Inc. (Mumbai, India). All other chemicals were of analytical grade.
Microorganisms and culture conditions
Bacillus megaterium MTCC 428 was obtained from Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India. The organism was cultured at 37°C in modified nutrient broth (NB) medium (pH 7.0) for 24 h. The media components were as follows (kg/m3): peptone 10, beef extract 1.5, yeast extract 1.5, and glucose 10.
The strain was revived from glycerol stock and grown in a 20 mL medium and transferred (5%, v/v) to 100 mL production medium at 37°C (200 rpm) in an incubator shaker (Kuhner shaker, Germany). After 24 h, cells were harvested by centrifugation at 10,000 × g (Sigma 6 K15, GmbH, Germany). Cells were thoroughly washed with 100 mM phosphate buffer (pH 7.0), and resuspended in the same buffer with a cell concentration of 100 kg/m3 and used for the biotransformation reaction.
Optimization of biotransformation parameters
Various physico-chemical parameters for the biotransformation of quinic acid to shikimic acid were optimized in 5 mL reaction mixture by varying one parameter at a time. Temperature was varied between 25°C to 45°C and pH values were adjusted between pH 4.0 to 9.0. An increased substrate concentration (2 to 20 mM) in the reaction mixture was used. The cell mass concentration was varied between 100 to 400 kg/m3. To optimize the cofactor recycling, different glucose concentrations (0.05 to 0.4 M) were used in the reaction mixture. The effect of reaction time on the yield of shikimic acid was observed by carrying out the reaction up to 6 h.
High-performance liquid chromatography
An analytical HPLC method was developed using high-performance liquid chromatography system (Shimadzu 10 AD VP, Kyoto, Japan). Shikimic acid was analyzed on an Alltech OA-2000 organic acid column (100 × 6.5 mm, 6.5 μm) (Grace Davison Discovery Science, Deerfield, IL, USA) using 2.5 mM H2SO4 as mobile phase at a flow rate of 0.3 mL/min and detected at 215 nm by UV detector. A standard curve of shikimic acid was made to quantify the biotransformation.
Reaction mixture was analyzed by LC-MS (Model: LTQ-XL, Thermo Scientific, USA) to further confirm the product formation. Alltech OA-2000 organic acid column (100 × 6.5 mm, 6.5 μm) (Grace Davison Discovery Science, Deerfield, IL, USA) was used with 5 mM formic acid as mobile phase at a flow rate of 0.5 mL/min and detected by UV detector at 215 nm.
Samples were analyzed in MALDI-TOF/TOF mass spectrometer (Bruker Ultraflex-TOF/TOF, Madison, WI, USA) for detecting the molecular mass of the product. After the reaction, sample was extracted by petroleum ether with pH adjustment and dried under vacuum. The dried sample was solubilized in methanol and used for the analysis.
After the completion of the reaction, the mixture was extracted with petroleum ether by pH adjustment and dried under vacuum. The dried sample was solubilized in DMSO and 1H NMR and 13C NMR spectra were obtained with Bruker AVANCE 400 MHz (1H 400 and 13C 100 MHz). Chemical shifts were expressed in δ units relative to the tetramethylsilane (TMS) signal as an internal reference in DMSO.
Extraction of shikimic acid from reaction mixture
Being a highly polar compound, it is very hard to isolate shikimic acid from the reaction mixture by solvent extraction. Therefore, pH-dependent extraction method was selected where, at a particular pH, the compound gets deionized and it comes into the organic non-polar phase during solvent extraction. Before the extraction, the pH of the solution was adjusted to 4.48 and extracted with different solvents such as ethyl acetate, dichloromethane, n-butanol, petroleum ether, etc.
Results and discussion
Biotransformation of quinic acid to shikimic acid using whole cells of B. megaterium
Quinic acid is converted to shikimic acid by two enzymatic systems in Gluconobacter oxydans, as reported by Adachi et al. ,. Along with the classical shikimate pathway, starting from glucose, quinic acid can also be a potential entry point with the formation of 3-dehydroquinate by the enzyme quinate dehydrogenase. The reaction was carried out with quinic acid as the substrate and whole cells of B. megaterium as the catalyst. The reaction conditions were as follows: quinic acid, 2.5 mM; cell mass concentration, 100 kg/m3; temperature, 30°C; mixing rate 200 rpm. Under the un-optimized conditions 42% conversion of quinic acid to shikimic acid was obtained (Additional file 1: Figure 1-4 of Supporting Information).
Various physico-chemical parameters for the biotransformation of quinic acid to shikimic acid using whole cells of B. megaterium were optimized.
Effect of reaction time
Effect of reaction temperature
Effect of reaction pH
Effect of substrate concentration
Effect of cell mass concentration
Effect of glucose concentration
Optimization of downstream processing of shikimic acid from reaction mixture
Extraction of shikimic acid from reaction mixture using different solvents
Amount recovered (mM)
Identification of shikimic acid
After the completion of the reaction, product was extracted by pH-dependent method. The product was characterized as shikimic acid by 1H and 13C NMR and MALDI. Analytical data were as follows: 1H NMR (400 MHz, MeOD): δ 2.18 to 2.22 (days, 1H), 2.69 to 2.73 (days, 1H), 3.69 to 3.70 (days, 1H), 4.01 (s, 1H), 4.38 (s, 1H), 6.81 (s, 1H); 13C NMR (100 MHz, MeOD): δ 31.69, 67.35, 68.41, 72.78, 130.82, 138.81, 170.20; MALDI-TOF-TOF: m/z 197.32, 213.32 (Additional file 1: Figure 12-16 of Supporting Information).
A potential biotransformation process is reported for the production of shikimic acid using whole cells of B. megaterium as biocatalyst. Various reaction parameters (reaction time, temperature, pH, substrate, biocatalyst concentration, etc.) were optimized. Maximum conversion (89%) was achieved under the optimized condition. Biotransformation of quinic acid to shikimic acid using purified enzymes are also reported; however, it has many limitations such as the use of co-factors (NADH/NAD+) in the reaction mixture and the quick denaturation of the enzyme during the course of reaction. Although a major advantage of mass transfer limitations are omitted in a cell-free system, due to inherent problems of enzyme denaturation, which makes this method difficult. The main limitation of transformation of quinic acid to shikimic acid is the use of higher substrate concentration. The enzyme titre may be increased by the well-developed techniques of recombinant DNA technology, directed evolution, etc. Shikimic acid production from quinate seems to be a better alternative over its fermentative production.
high-performance liquid chromatography
matrix assisted laser desorption/ionisation
microbial type culture collection
nicotinamide adenine dinucleotide phosphate
reduced form of nicotinamide adenine dinucleotide phosphate
nuclear magnetic resonance
revolutions per minute
One of the authors SG acknowledges the financial support by Indian Council of Medical Research, India.
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