Bacterial strain, plasmids and media
G. stearothermophilus CAU209 was deposited in Key Laboratory of Food Bioengineering (China National Light Industry) in Beijing, China. B. subtilis WB600 (Δbpr, Δepr, Δmpr, ΔnprB, Δvpr, ΔwprA), a six extracellular protease deficient strain gifted from Guangxi University, Nanning, China, was used as the expression host. E. coli DH5α [F− supE44 Ф80 δlacZ ΔM15 Δ (lacZYA-argF) U169 endA1 recA1 hsdR17 (rK−, mK+) deoR thi-1 λ- gyrA96 relA1] (Biomed, Beijing, China) was used as the host strain for DNA manipulation, and was cultured in Luria-Bertani (LB) medium composed of 10 g/L peptone, 5 g/L yeast extract, and 5 g/L NaCl. Plasmid pWB980 (TaKaRa Corporation, Dalian, China) was used as the expression vector. Whey protein (80% of protein content on dry basis) was obtained from Ausnutria Dairy Corporation (Changsha, China). All other chemicals and reagents were of analytical grades and commercially available from Sigma-Aldrich (St. Louis, MO, USA).
Cloning of the alkaline serine protease gene (GsProS8)
Genomic DNA from G. stearothermophilus CAU209 (GenBank: MW084977) was extracted as described previously [52]. According to the genome sequence of G. stearothermophilus CAU209, a pair of oligonucleotide primers, named GsProS8-F and GsProS8-R (Table S1), were designed to amplify the coding region of the alkaline serine protease. Polymerase chain reaction (PCR) was performed at 94 °C for 5 min, 30 cycles with denaturing at 94 °C for 30 s, annealing at 56 °C for 30 s and extension at 72 °C for 1.5 min, and the last cycle was at 72 °C for 10 min. The PCR product was ligated with pMD-19 T and transformed into E. coli DH5α for sequencing. Sequence analysis was performed using DNAMAN 6.0. The ORF was found using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi/). The signal peptide was predicted by SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/).
Plasmid construction for expression of GsProS8
To express the protease gene in B. subtilis WB600, two pairs of primers (GsProS8-IF and GsProS8-IR, pWB980-VF and pWB980-VR, Table S1) were designed to amplify the ORF region of the GsProS8 gene on the chromosome DNA and the vector, respectively. The overlapping PCR was carried out using 0.0002 mol/L each of the four dNTPs and 0.04 U Phusion DNA polymerase. P43 promoter was located at the upstream of SacB signal peptide in the plasmid pWB980. PCR conditions were as follows: 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 1.5 min (the last cycle at 72 °C for 10 min). The purified PCR product was digested with EcoRI and SmaI restriction endonucleases, and then inserted into the plasmid pWB980. The recombinant plasmid pWB980-GsProS8 was transformed into the B. subtilis WB600 by electroporation. The transformants were cultured on LB plates and incubated at 37 °C until colonies appeared. The colonies were then suspended in the LB medium for further growth in a rotary shaker at 37 °C for 18–24 h. The cultures were centrifuged at 10,000×g for 10 min to collect the supernatant. The protease activity as well as the expression in the supernatant were analyzed by the Folin-Ciocalteu reagent and SDS-PAGE, respectively.
High cell density fermentation
To scale up GsProS8 production, high cell density fermentation was carried out in a 5.0 L fermentor (Guoqiang, Shanghai, China) with a 1.5 L working volume at 37 °C. 1% (v/v) inoculum was initially incubated at 37 °C for 12 h. Subsequently, the fermentor was inoculated with 750 mL of the inoculum and cultivated at 37 °C for 114 h. The fermentation medium was composed of glucose (10 g/L), yeast extract powder (10 g/L), peptone (20 g/L), corn steep powder (5 g/L), sodium chloride (10 g/L), magnesium sulfate (0.3 g/L), sodium phosphate (6 g/L), and dipotassium phosphate (3 g/L). The cultivation was maintained at pH 7.0–7.2 with the aid of ammonium hydroxide and phosphoric acid. The dissolved oxygen level was maintained at 30% air-saturation. Samples were withdrawn at 6 h interval to analyze enzyme activity, cell mass, and protein concentration, and SDS-PAGE was also performed.
Purification of GsProS8
The fermented culture was purified through ammonium sulfate precipitation and a two-step chromatographic method. In short, the crude enzyme was initially precipitated by ammonium sulphate (40–70% w/v saturation) and dialyzed against 0.02 mol/L Tris-HCl at pH 8.0 (buffer A) for overnight. Thereafter, the dialyzed sample was subjected to a HiTrap SP Fast Flow (SPFF) column (GE Healthcare, Wuxi, China), which was pre-equilibrated with buffer A. After washing with buffer A until the absorbance (at 280, OD280) < 0.05, the bounded proteins were then eluted with a linear gradient (0–0.25 mol/L) of NaCl at a flow rate of 1 mL/min. The fraction with high protease activity was collected and dialyzed against 0.05 mol/L N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) at pH 10.3 (buffer B) for overnight. The sample was loaded onto a HiTrap Q-Sepharose Fast Flow (QSFF) column (GE Healthcare, Wuxi, China) that was pre-equilibrated with buffer B. Subsequently, the proteins were eluted with buffer B until OD280 < 0.05, followed by a gradient elution of NaCl (0–0.15 mol/L) at 1 mL/min. The purity of the fractions was verified by SDS-PAGE.
SDS-PAGE, zymogram and molecular mass determination
SDS-PAGE was used to evaluate the purity and molecular mass of the protease. The protein bands were stained by Coomassie Brillaint Blue R-250. The molecular weight markers for electrophoresis included α-lactalbumin (14.4 kDa), trypsin inhibitor (20.1 kDa), carbonic anhydrase (29.0 kDa), ovalbumin (44.3 kDa), BSA (66.4 kDa) and phosphorylase B (97.2 kDa). Zymogram of the protease was analyzed using the SDS-polyacrylamide gel co-polymerized with 0.1% gelatin as described by Sun and co-workers [53]. The native molecular mass of the purified GsProS8 was estimated by a gel filtration chromatography via a Sephacryl S-100 column (1.0 × 100 cm). The purified protease and protein markers were eluted by 20 mM Tris-HCl buffer (at pH 8.0) containing 500 mM NaCl at a flow rate of 0.33 mL/min.
Protease assay and protein determination
Protease activity was determined using casein as a substrate. Briefly, the protease solution (100 μL) was incubated with 2% (w/v) casein (100 μL) in 20 mM phosphate buffer (700 μL, pH 7.5) at 37 °C for 30 min. The reaction was terminated by trichloroacetic acid (10% w/v, 200 μL). After centrifugation at 10,000×g for 5 min, the supernatant (100 μL) was mixed with 600 μL alkaline reagent (0.4 mol/L Na2CO3: Folin-Ciocalteu reagent = 5:1 v/v) and incubated at 40 °C for 20 min. The absorbance was measured at 660 nm using a spectrophotometer (TU1901, Puxi General Instruments Co., Ltd., Beijing, China). The mixture without the protease was used as control. One unit of protease activity (U) was defined as the amount of the protease to liberate 1 μmol tyrosine per minute at the above conditions. Moreover, the protein concentration was measured by the Lowry method using BSA as standard. Specific activity was expressed as units per milligram of protein.
Biochemical properties of GsProS8
The optimal pH for protease activity was evaluated at 40 °C in a pH range of 4.0–11.0. The following buffers (0.05 mol/L) were used: citrate (pH 4.0–5.0), acetate (pH 5.0–5.5), 2-(N-morpholino) ethanesulfonic acid (MES, pH 5.5–7.0), 4-(N-morpholino) propanesulfonic acid (MOPS, pH 7.0–8.5), and N-cyclohexyl-2-aminoethanesulfonic acid (CHES, pH 8.5–11.0). The pH stability was investigated after incubating the protease in various buffers (0.05 mol/L) at 40 °C for 30 min. The residual activity was determined by the standard assay.
The optimal temperature was determined in 0.05 mol/L of MOPS (pH 8.5) buffer at 30-70 °C. The thermostability was measured after incubating the protease at a temperature range of 30-80 °C for 30 min. The residual activity was determined by the standard assay.
The effect of several inhibitors on the protease activity was analyzed. The following inhibitors were used: pepstain A (at 0.01 mM and 0.02 mM), EDTA (at 1 mM and 4 mM), PMSF (at 1 mM and 4 mM), and iodoacetamide (at 1 mM and 4 mM). Moreover, the effect of metal ions on protease activity was examined in the presence of magnesium sulfate, calcium chloride, sodium chloride, nickel chloride, manganese sulfate, cobalt chloride, zinc sulfate, and copper sulfate at the final concentration of 0.001 mol/L.
Substrate specificity was evaluated at pH 8.5 and 50 °C using casein, whey protein, skim milk powder, hemoglobin, soybean protein isolate, BSA, protamine, myoglobin, azocasein, or gelatin (1% w/v). The protease activity was measured as described previously. The protease activity towards casein was determined to be 100%, to calculate the relative protease activities to other substrates.
Kinetic parameters of GsProS8
The kinetic parameters of the purified GsProS8 were determined using Michaelis-Menten plot after measuring the protease activities at various concentrations of casein (4–16 mg/mL) and whey protein (8–25 mg/mL) at pH 8.5 and 50 °C for 5 min. The parameters included Km, Vmax, kcat and kcat/Km, in which Km and Vmax were obtained from non-linear regression analysis by GraphPad Prism V.8 [54].
Hydrolysis of whey protein by GsProS8
According to the preliminary test, pH, temperature, time, and whey protein concentration were identified as four critical factors to impact the hydrolysis of whey protein by GsProS8. Each factor was analyzed at three levels using an orthogonal experimental design (Table S2). In brief, whey protein (7–11%, w/v) was hydrolyzed by GsProS8 (200 U/mL) at designed temperature (40-60 °C) and pH (7.5–8.5) for 4–8 h in a temperature-control shaker (HZQ-X100, Suzhou, China) at 100 rpm, and terminated by heating at 85 °C for 10 min. The hydrolysate solutions were centrifuged (Refrigerated Centrifuge GL-20B, Shanghai, China) at 10,000×g for 10 min to obtain the supernatants for later analysis. Commercial proteases (200 U/mL, e.g., trypsin at pH 8.0 and 37 °C; Flavourzyme at pH 7.0 and 53 °C; Protamex at pH 7.5 and 40 °C; and Alcalase at pH 8.0 and 60 °C) were also applied in the hydrolysis of whey protein.
Peptide content of whey protein hydrolysate
The peptide content in the whey protein hydrolysate was determined by o-phthalaldehyde (OPA) method as described by Church and co-workers [55] with slight modifications. Concisely, the supernatant of whey protein hydrolysate (25 μL) was added into OPA mixture (1 mL), which was prepared by 100 mM sodium tetraborate, 20% (w/w) SDS, and OPA solution (containing 0.02% w/v OPA, methanol, and β-mercaptoethanol). The incubation was then processed at room temperature for 8 min, and the absorbance of resulting solution at 340 nm (TU-1800PC spectrophotometer, Persee General Instrument Co. Ltd., Beijing, China) was recorded. Both distilled water and glutathione were performed as a blank and a standard, respectively. The peptide content was calculated based on the standard curve of glutathione.
Antihypertensive activity of whey protein hydrolysate
Antihypertensive activity of whey protein hydrolysate was expressed as ACE inhibitory activity (%) and measured as described by Cushman and Cheung [56] with some modifications. Namely, the reaction of whey protein hydrolysate (2 mg/mL, 20 μL) with ACE (0.1 U/mL, 10 μL) in 120 μL of 0.1 M sodium borate buffer (containing 5 mM N-Hippuryl-His-Leu hydrate and 0.3 M NaCl at pH 8.3) was performed at 37 °C for 60 min. The reaction was then terminated by HCl (1 M, 150 μL) and ethyl acetate (1 mL), followed by centrifugation (GL-20B Refrigerated Centrifuge, Anting Scientific Instrument Co. Ltd., Shanghai, China) at 4000 rpm for 10 min at room temperature to collect the supernatant, which was dried at 105 °C for 30 min. The liberated hippuric acid was dissolved in the deionized water (0.5 mL) to record the absorbance at 228 nm (TU-1800PC, Persee General Instrument Co. Ltd., Beijing, China). The enzyme reaction without the whey protein hydrolysate and sodium borate buffer (0.1 M) containing substrate were used as a control and a blank, respectively. ACE inhibitory activity (%) was calculated as follows:
$$ \mathrm{ACE}\kern0.5em \mathrm{inhibitory}\kern0.5em \mathrm{activity}\left(\%\right)=\frac{{\mathrm{A}}_{\mathrm{b}}-{\mathrm{A}}_{\mathrm{a}}}{{\mathrm{A}}_{\mathrm{b}}-{\mathrm{A}}_{\mathrm{c}}}\times 100\% $$
where Aa is the absorbance of the sample supernatant, Ab is the absorbance of the control, and Ac is the absorbance of the blank. ACE inhibitory activity (%) was transformed to IC50 (mg/mL, half maximal inhibitory concentration) based on a plot of relative ACE inhibitory activity (%) against different dilutions (0.01–1.00%) of the hydrolyzed supernatant using non-linear regression analysis by GraphPad Prism V.8 (Fig. S5).
Statistics
The experiments were performed in triplicate and expressed as the mean ± one standard deviation. A one-way analysis of variance (ANOVA) was carried out using Systat v10 software (San Jose, CA, USA) to analyze statistical differences on the protease activity, peptide content, and ACE inhibitory activity (IC50). p < 0.05 was considered significant.