Strains, plasmids, culture conditions and chemicals
The Lactobacillus strains of Lb. acidophilus, Lb. amylovorus, Lb. crispatus, Lb. farciminis, Lb. fermentum, Lb. gasseri, Lb. helveticus, Lb. johnsonii, and Lb. reuteri were anaerobically cultured at 37 °C in MRS (De Man, Rogosa and Sharpe) medium, which is composed (per liter) of typtone, 10 g; yeast extract, 5 g; glucose, 20 g; ammonium citrate, 0.58 g; MnSO4, 0.25 g; CH3COONa·3H2O, 3.12 g; Na2HPO4, 1.63 g; CH3COOK, 2.25 g; beef extract, 10 g; Tween-80, 1 mL. The strain E. coli DH5α was used for recombinant plasmids construction. The strain E. coli BL21 (DE3) was used for heterologous feruloyl esterases expression. These two strains were aerobically cultivated in LB (Luria-Bertani) broth containing 10 g/L typtone, 5 g/L yeast extract, and 10 g/L NaCl. Plasmid pET-22b was used for the ligation of feruloyl esterase genes. When the pET-22b-derivative plasmids were transformed into E. coli, the LB medium supplemented with ampicillin at a final concentration of 100 µg/mL was used. To produce ferulic acid from the agricultural waste, the recombinant E. coli BL21 (DE3) strains were cultured in LB broth supplemented with 2 % (w/v) de-starched wheat bran.
Ferulic acid and ethyl ferulate were bought from Sigma Chemicals Industries., Ltd. (SanFrancisco, USA). Para-nitrophenyl ferulate (ρNPF) was procured from Shandong Chambroad Holding Co., Ltd. (Shandong, China). Bacterial genomic DNA extraction kit was purchased from Tiangen biotech Co., Ltd. (Beijing, China), and gel extraction kit, plasmid extraction kit, and cycle pure kit were purchased from Omega Bio-tek (Atlanta, USA). These kits were used by following the manufacture’s recommended protocols. Phanta max super-fidelity DNA polymerase and Exnase®II were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). Restriction enzymes were purchased from TaKaRa Biotech Co., Ltd. (Tokyo, Japan). The wheat bran was procured from a local mill of Jinan city (Shandong, China). All other regents were bought from Solarbio Science & Technology Co., Ltd. (Beijing, China).
Cloning and expression of Lactobacillus feruloyl esterases
The Lactobacillus strains were cultivated in MRS medium at 37 °C for 12 h. The cells were collected by centrifugation at 6000×g for 5 min and washed twice by sterile water. Then the genomic DNAs of these Lactobacillus strains were extracted by using bacterial genomic extraction kit as described above. The amount and quality of the obtained DNAs was determined with a microspectrophotometer (Eppendorf, Hamburg, Germany), and then stored at -20 °C until to use. Based on the related reports and the genome sequences deposited in NCBI database, the primer sets were designed for amplification of these Lactobacillus feruloyl esterase coding genes, respectively. As shown in Table S1, the nucleotides pairing with the feruloyl esterase gene sequence are in uppercase letter, and the nucleotides pairing with the pET-22b vector are in lowercase letters. The PCR amplification procedure contained an initial denaturation at 95 °C for 3 min, followed by 30 cycles each of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 45 s, and then a final extension at 72 °C for 5 min. The obtained feruloyl esterase genes were extracted from the gel after electrophoresis. The pET-22b vector was digested by Nde I and Xho I, and then purified. To ligate the gene into pET-22b, these two fragments were mixed with the molar ratio of 2:1, and the Exnase®II was used to activate the homologous recombination. After incubated at 37 °C for 30 min, the reaction mixture was transformed into the E. coli DH5α competent cells by heat shock method. The correct transformants were selected by colony PCR, and their plasmids were extracted and sequenced in Sangon Biotechnology Co. Ltd. (Shanghai, China). The putative signal peptides of these feruloyl esterases were predicted by using the signalP program (http://www.cbs.dtu.dk/services/SignalP/) and TatP program (http://www.cbs.dtu.dk/services/TatP/).
These generated pET-22b-derivative plasmids were further transformed into E. coli BL21 (DE3) cells. The transformants were picked up and inoculated into LB medium supplemented with 100 µg/mL ampicillin and cultivated at 37 °C in a shaker with 200 rpm. To express the feruloyl esterases, the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM was added into the cultures when the growth of cells reached an OD600 of 0.5, and then the incubation was continued for an additional 72 h at 37 °C.
Feruloyl esterase activity determination
The LB plate-based assay was conducted to detect the feruloyl esterase activity of the recombinant strains and extracellularly secretory component. At the plate-pouring stage of LB medium, 100 µg/mL ampicillin, 0.5 mM IPTG, and 6.7 mM ethyl ferulate (dissolved in dimethylformamide) were added and fully mixed. The recombinant E. coli BL21 (DE3) strains were inoculated in the plates to detect the expression and activity of heterologous feruloyl esterases. In addition, a volume of 200 µL cell-free culture supernatant was added into the Oxford cup placed in the plate to preliminarily detect the extracellular feruloyl esterase activity. All these plates were incubated at 37 °C, and the formed transparent circle was observed and photographed.
The substrate ρNPF, which could be hydrolyzed by feruloyl esterase to produce the ρ-nitrophenol with a yellow color, was used to quantitatively determine the feruloyl esterase activity. The 1 mM substrate solution was prepared by adding 25 mM ρNPF (dissolved in dimethyl sulfoxide) into sodium phosphate buffer (100 mM, pH 7.0) which was supplemented with Tween-80 (1 %, v/v) previously. Then, 100 µL of sample was mixed with 900 µL substrate solution to initialize the reaction. After incubation at 37 °C for 10 min, 1 mL of acetic acid solution (50 %, v/v) was added into the mixture to terminate the reaction. Meanwhile, the control experiments were performed by using the inactivated sample. The released ρ-nitrophenol was determined at 410 nm using a spectrophotometer (Eppendorf, Hamburg, Germany). At the conditions described above, the required enzyme amount to produce 1 µmol ρ-nitrophenol in 1 min was calculated as one unit (U) of feruloyl esterase activity.
The extracellular protein contents of E. coli cells cultured in LB medium were estimated by the Bradford protein assay in which the bovine serum albumin was used as standard . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the protein bands of the whole cell, cytoplasmic component, and extracellular component of E. coli. The gel was composed of a 5 % stacking gel and a 12 % separating gel. After electrophoresis, the Coomassie brilliant blue staining solution was applied to visualize the protein bands. The molecular mass was indicated by the standard protein molecular weight marker.
The whole cell, cytoplasmic component, and extracellular component of E. coli BL21 (DE3) were prepared as follows. The recombinant strain cultures after induction were centrifugated at 6,000×g, 4 °C for 5 min to separate the cells and supernatant. The supernatant was filtered through a 0.22-μm filter, and represented the extracellular component. The harvested cells were resuspended in an equal volume of sodium phosphate buffer (100 mM, pH 7.0). This represented the whole cell of recombinant E. coli. A ultrasonic breaker (Tenlin, Jiangsu, China) was used to break the cell suspension with the conditions of power 400 w, pulse 5 s, pause 5 s, cycle 49 at 4 °C. The cell-free extract was obtained by centrifugation at 17,400×g, 4 °C for 10 min and filtering through a 0.22-μm filter, representing the cytoplasmic component. For electrophoretic analysis, all the components were mixed with 5×SDS-PAGE loading buffer and then boiled for 10 min.
Release of ferulic acid from de-starched wheat bran
The ferulic acid releasing ability by these recombinant strains expressing Lactobacillus feruloyl esterases was investigated using de-starched wheat bran as substrate. A previously reported method was performed to prepare the de-starched wheat bran . In brief, 100 g fresh wheat bran was treated with amylase (0.3 %, w/v) at 65 °C for 30 min, and then with papain (0.3 %, w/v) at 55 °C for 45 min. The reaction mixture was boiled for 20 min to inactivate these enzymes. After centrifugation, the wheat bran was collected and washed repeatedly using distilled water for removing starch completely. Subsequently, the de-starched wheat bran was dried to constant weight at 80 °C and milled to passing a 60-mesh sieve. For ferulic acid production, the medium was prepared by adding 0.1 g de-starched wheat bran into a tube containing 5 mL LB broth, and then treated by autoclave. The recombinant E. coli strains were inoculated in the medium and cultivated at 37 °C in a shaker of 200 rpm. The culture samples were taken out after 72 h induction by IPTG, and analyzed by high performance liquid chromatography (HPLC) as described below.
The HPLC (Shimadzu, Kyoto, Japan) was equipped with a CBM-20A communications bus module, a LC-20AT pump, a SIL-20A auto sampler, CTO-10A column oven, a reversed-phase WondaCract ODS-2 C18 cartridge, and a SPD-M10Avp photodiode array detector. This system was eluted by a mobile phase (methanol, water, and acetic acid as a ratio of 50:49.5:0.5) with a flow rate of 1 mL/min at 30 °C. Absorbance of the eluent was monitored at 320 nm. The cultures were boiled for 30 min and centrifuged at 10,000×g for 15 min. The supernatant was harvested and filtered through a 0.22-µm filter before HPLC analysis. The standard ferulic acid was used to qualitative and quantitative analysis of the samples.
Each experiment was done in triplicate. All statistical procedures were performed using the statistical packages for the social sciences (SPSS).