Feruloyl esterase (FAE; EC 126.96.36.199) is a member of the alpha/beta hydrolase family that cleaves ester or ether linkages between ferulic acid (FA) and sugars in order to facilitate plant cell wall degradation and release FA [1
]. FA, a phenolic acid, is a natural and safe antioxidant, approved by the Japan Food Chemical Research Foundation and the US Food and Drug Administration for use as a food additive in 2010 [3
]. FA possesses several benefits, such as the delayed progression of fatty liver disease, improved vascular function [4
], and anti-diabetic and anti-cancer activities [5
]. To meet sustainable development objectives, biological catalysis for the purpose of degrading abundant biomass waste is promising [6
]. For instance, FAE has been used with cellulase and hemicellulase for the production of fuel ethanol, fermentable sugar, and other high-value by-products [7
]. Moreover, FAE has also been used in paper processing and bleaching, and as an additive in animal feed to improve nutrient digestibility [3
Owing to their utility, strains that secrete FAEs continue to be identified, and some FAEs from fungi have been studied extensively, including their classification [7
], biochemical properties, three-dimensional structures, and catalytic mechanisms [9
]. However, there is the demand to identify more novel FAEs because of lower enzyme activity and poor thermal stability [7
]. Gopalan et al. [10
] pointed out that most FAEs were usually less than 1 U/mL of culture medium, while Meng et al. [12
] mentioned that most FAEs were mesophilic enzymes, suggesting that they lose their catalytic activity when the temperature exceeds 50 °C. Natural environments and metagenomic libraries have been used to screen and identify novel FAEs [6
]. To boost the industrial application and simplify the purification of FAEs to facilitate the efficient production of FA, genes are cloned in different hosts in order to overexpress recombinant enzymes [14
]. Escherichia coli
, a common and highly efficient expression system, is often a poor choice for industrial food production since it produces endotoxins and is not a “Generally Regarded as Safe” (GRAS) host. In recent years, an increasing number of researchers have begun to use Bacillus subtilis
as a host to express enzymes, mainly because it is a food-grade microorganism and suitable for the industrial production of FAEs. B. subtilis
is also amenable to a simple fermentation process and lacks obvious codon bias [15
To date, there have been few studies on the heterogeneous expression of FAE using the B. subtilis system. In this study, the FAE from B. pumilus SK52.001 (BpFAE) was identified, cloned, and heterogeneously expressed in B. subtilis for the first time. The studies were conducted to characterize the biochemical properties and liberate FA from de-starched wheat bran (DSWB) by synergy action of BpFAE and commercial xylanase.
2. Materials and Methods
2.1. Chemicals and Reagents
Lysozyme, ampicillin, and kanamycin sulfate were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). FA (content > 99%) was obtained from J&K Scientific Ltd. (Shanghai, China). Methyl ferulate (MFA) (content > 99%) was purchased from ThermoFisher Scientific (Shanghai, China). Commercial xylanase (10,000 U/g) was obtained from Macklin Co., Ltd. (Shanghai, China). Wheat bran was obtained from Auchan Supermarket (Wuxi, China). Other chemicals were of analytical grade and were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Strain Screening
The soil samples from Wuxi, Jiangsu Province were suspended in sterile saline at a final concentration of 1 g/mL at 30 °C and 200 r/min for 30 min. The strains were isolated using gradient dilution in a non-selective isolation medium (200 g/L potato extract, 20 g/L source, and 20 g/L ager). Subsequently, the colonies were picked up from the selective medium (2 g/L NaNO3, 1 g/L K2HPO4, 0.5 g/L KCl, 0.5 g/L MgSO4·7H2O, 0.01 g/L FeSO4·7H2O, and 20 g/L ager), with ethyl ferulate (10 g/L) as the sole carbon source at 30 °C for 48 h. The resultant colonies surrounded by halo were purified in a non-selective isolation medium 3 times and inoculated in a fermentation medium (2 g/L NaNO3, 1 g/L K2HPO4, 0.5 g/L KCl, 0.5 g/L MgSO4·7H2O, 0.01 g/L FeSO4·7H2O, and 20 g/L wheat bran) at 30 °C, with shaking at 200 r/min for 26 h. The enzyme activity of FAE was estimated and the strain with the highest activity was identified as the target strain for further study.
2.3. Engineered Strain, Plasmid, and Culture Media
E. coli DH5α (Sangon Biotech, Shanghai, China) and B. subtilis WB800 (preserved in our laboratory) were hosts for cloning and expression, respectively. The shuttle plasmid pMA5 with NdeI and BamHI sites (Talen Biotech, Shanghai, China) was used as the expression vector. Luria-Bertani (LB) broth (5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl) was prepared for the routine growth of E. coli. Ampicillin was dissolved in sterile water and used at a final concentration of 100 μg/mL. A super-rich medium (SR) containing 50 μg/mL kanamycin consisted of 3 g/L K2HPO4, 20 g/L yeast extract, 25 g/L tryptone, and 30 g/L glucose for B. subtilis WB800 cultivation.
Genomic DNA from the B. pumilus SK52.001 strain was extracted using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China). The fae gene fragment was amplified with primers, Bpfae forward: AAAAGGAGCGATTTACATATGATGAACTTACAAGAGCAAATCAAAATCGCTGC and Bpfae reverse: GAGCTCGACTCTAGAGGATCCTTAATGGTGATGGTGATGATGTTCAAATGCCTTT. The FAE sequence fused with a 6 × histidine tag at C-terminus was ligated with the expression vector pMA5 between the NdeI and BamHI sites, resulting in pMA5-fae. The recombinant plasmids were transformed into competent E. coli DH5α cells via heat shock. Positive transformants were cultured overnight in 5 mL LB medium supplemented with ampicillin, and plasmids pMA5-fae were extracted by FastPure Plasmid Mini Kit (Vazyme Biotech Co., Ltd., Nanjing, China) and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).
2.5. Heterogeneous Expression and Purification
For the expression of fae, the resultant plasmids pMA5-fae were introduced into B. subtilis WB800 to obtain the recombinant strains, B. subtilis WB800/pMA5-fae. A colony positive for kanamycin resistance was incubated in LB broth supplemented with 50 μg/mL kanamycin at 37 °C, with shaking at 200 r/min for 12 h as seed. To express the target enzyme, the engineered B. subtilis cells were cultured with 3% inoculation in 50 mL SR medium, with 50 μg/mL kanamycin at 37 °C, with shaking at 200 r/min for 14 h.
Following overexpression, all purification steps of the target protein were carried out at 4 °C. Cells expressing BpFAE were harvested by centrifugation at 8000× g for 15 min. The cells were resuspended in a sample buffer (50 mmol/L Tris-HCl, pH 8.0) containing 100 mmol/L NaCl and disintegrated by biological lysis with 20 mg/mL lysozyme in a water bath at 37 °C for 30 min, with gentle shaking every 10 min. Then, cells were ruptured by ultrasonication for 10 min (ultrasound 1 s, pause 2 s) and centrifuged at 6000× g for 15 min. To purify the enzyme, the nickel affinity column was washed with deionized water and pre-equilibrated with a binding buffer (500 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 8.0). The supernatant was loaded into the column, which was then washed with a binding buffer and a washing buffer (500 mmol/L NaCl, 50 mmol/L Tris-HCl, 20 mmol/L imidazole, pH 8.0) to remove unbound or weakly bound proteins. Subsequently, the target enzyme was eluted with an elution buffer (500 mmol/L NaCl, 500 mmol/L imidazole, 50 mmol/L Tris-HCl, pH 8.0) and dialyzed against a sample buffer, with three buffer exchanges to remove imidazole.
2.6. Molecular Mass Determination
The subunit and native molecular mass (Mm) of BpFAE were evaluated by sodium dodecyl sulfate (SDS) and native polyacrylamide gel electrophoresis (PAGE, 12% separating gel), respectively. The protein was stained with Coomassie brilliant blue R250 and de-stained with a decoloring solution (ethanol:acetic acid:distilled water = 1:2:17).
2.7. Hydrolytic Activity Assay and Protein Concentration Determination
The hydrolytic activity of BpFAE was measured using MFA as substrate. The enzymatic reaction was conducted in a 1 mL mixture containing 990 μL MFA (3 mmol/L, dissolved in 50 mmol/L Tris-HCl, pH 9.0) and 10 μL enzyme solution at 50 °C for 10 min, and terminated by placing this mixture in a boiling water bath for 10 min.
The concentration of FA was analyzed with a high-performance liquid chromatography (HPLC) system (Agilent 1200, Agilent Technologies Inc., Santa Clara, CA, USA), equipped with an ultraviolet detector at 320 nm and a ZORBAX Eclipse Plus C18 column (Agilent, 150 mm × 4.6 mm, 3.5 μm). The injection volume was 10 μL, and the flow rate and temperature were controlled at 1 mL/min and 30 °C. The mobile phase consisted of 1% (v/v) acetic acid in H2Odd (eluent A) and methanol (eluent B). The elution gradient protocol was: 0 min (90% A/10% B); 0–0.23 min (70% A/30% B); 0.23–1.66 min (50% A/50% B); 1.66–4.97 min (100% B); 4.97–5.57 min (85% A/15% B); 5.57–7.52 min (90% A/10% B). A standard curve was generated by linear fitting based on the retention time and FA peak area. One unit of FAE hydrolytic activity was defined as the amount of enzyme required to release 1 μmol FA per minute under standard assay conditions.
The protein concentration of a purified enzyme was evaluated using the Lowry method with bovine serum albumin as a standard [16
2.8. Biochemical Characterization of BpFAE
To determine the optimal pH of BpFAE, experiments were carried out under five buffer conditions: acetate buffer (50 mmol/L, pH 4.0–5.5), 2–(N–morpholino) ethanesulfonic acid (MES) buffer (50 mmol/L, pH 5.5–6.5), sodium phosphate buffer (50 mmol/L, pH 6.5–7.5), Tris-HCl (50 mmol/L, pH 7.5–9.0), and glycine-NaOH buffer (50 mmol/Lol/L, pH 9.0–10.0). The relative activity was normalized to the maximum activity set at 100%. In order to study pH stability, the enzyme was pretreated at pH 8.5, 9.0, and 9.5, at 4 °C for 28 h. The activity at the beginning was set at 100%.
To investigate the optimal temperature, enzyme activity was assayed at different temperatures ranging from 35 to 90 °C. The highest enzyme activity was set at 100%. For thermal stability, the enzyme was incubated at 50, 55, 60, and 65 °C prior to the residual enzyme activity assayed. The activity of enzyme stored at 4 °C was specified as 100%. The half-life time and melting temperature (Tm) were determined. The slope defined as k was obtained according to a linear fitting of the natural logarithm of the relative activity versus time. The half-life was calculated by ln2/k. The molar ellipticity at 222 nm of the enzyme was recorded as a function of variable temperature ranging from 25 to 90 °C on a circular dichroism spectropolarimeter (CD) (Chirascan V100, Applied Photophysics Ltd., Leatherhead, UK), with a bandwidth of 1.0 nm, sensitivity of 20 mdeg, heating rate of 0.2 °C/min, and a 0.1 cm quartz cuvette. The Tm was calculated by the acquisition of maximum slope.
2.9. Kinetic Parameters
The kinetic parameters of the purified BpFAE were measured at different concentrations of MFA (0.1–8 mmol/L) at 50 °C and pH 9.0. The Km, Vmax, and Kcat values were calculated by fitting the nonlinear Hill function using OriginPro 9.1 (Origin Lab Inc., Northampton, MA, USA).
2.10. Production of FA from DSWB
DSWB was incubated with 3 g/L potassium acetate at 95 °C for 1 h with constant stirring, followed by the removal of soluble starch using distilled water. Subsequently, the treated DSWB was dried to a constant weight at 55 °C for later use.
The total amount of FA in the DSWB above was chemically extracted by incubation in 2 mol/L NaOH at 50 °C, with shaking at 150 r/min for 4 h, and supplementing 2 mol/L HCl at 85 °C for 1 h. Then, the suspension was centrifuged at 8000× g for 10 min. The supernatant was analyzed by HPLC-UV, as described above.
The FA release from DSWB was carried out by enzymatic degradation. The 75 mg DSWB was suspended in 3 mL of the Tris-HCl buffer (50 mmol/L, pH 9.0), which was enzymatically degraded by adding 0.18 mg of purified BpFAE, 0.31 mg of commercial xylanase, and 0.18 mg of BpFAE along with 0.31 mg xylanase. It was conducted at 50 °C for 4 h, with shaking at 150 r/min. The reaction mixture was terminated by boiling for 10 min and centrifuged at 8000× g for 10 min. The analysis of the released FA amount was conducted by HPLC.
2.11. Statistical Analysis
The data are presented as the mean ± standard deviation (SD, n = 3). Statistical analyses were performed using OriginPro 9.1 (Origin Lab Inc., Northampton, MA, USA).