Plasmids, bacterial strains and materials
The strain of Bacillus alcalophilus JN21 (CCTCC NO: M 2011229) was an isolate of Lab of Bioprocess and Biosystems Engineering, Jiangnan University. Bacillus subtilis WB600, a strain that contains deletions of all six extracellular protease genes, and the plasmid pMA5 were obtained from Invitrogen. The EZ-10Spin Column Plasmid Mini-Preps kit, agarose gel DNA purification kit, restriction enzymes, and T4 DNA ligase were obtained from TakaRa (Dalian, China). Other chemicals were obtained from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China). The synthesis of DNA primers and DNA sequencing were performed by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, China).
Construction and transformation of the plasmid for secreted alkaline α-amylase expression
The E. coli/B. subtilis shuttle vector (pMA5) was used to clone and express alkaline α-amylase. The mature alkaline α-amylase gene was amplified by polymerase chain reaction (PCR) using gene-specific primers as follows: 5'ACGCGTCGACATGGAGCATASGGCCATGACGA3' (forward primer) and 5'CGGGATCCTTATGACCGCCGAATCAGTGAAGC3' (reverse primer). The forward primer and the reverse primer contained a Sal I and a BamH I restriction sites (underlined), respectively. The amplification was carried out under the following conditions: the first step was at 94°C for 10 min, followed by 30 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 2 min, and the final extension was carried out at 72°C for 10 min. The PCR product was digested with Sal I and BamH I, gel-purified and then ligated into pMA5 which was subjected to a similar treation. The recombinant plasmid was identified by restriction analysis and sequencing.
Expression of alkaline α-amylase in B. subtilis
The recombinant plasmid pMA5/A-amyQ was transformed into the competent cells of Bacillus subtilis WB600. The transformants were selected at 37°C on the LB agar plates containing 100 μg/mL Kanamycin and 1% soluble starch for 8 h. The transformants with transparent rings around colonies were positive. The presence of alkaline α-amylase gene in the transformants was confirmed by PCR. The colonies were grown in 25 mL of LB medium at 37°C for 36 h in conical flasks (250 mL).
Optimization of recombinant alkaline α-amylase production
The effects of culture conditions (starch concentration, peptone concentration, soybean meal concentration, and temperature) on the recombinant production of alkaline α-amylase in B. subtilis were examined. The starch of different concentrations (0, 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, and 4.2% (w/v)) on the production of alkaline α-amylase was added into the culture medium to examine the effects of starch on alkaline α-amylase production. The peptone of different concentrations (0.45, 0.95, 1.45, 1.95, 2.45, 2.95, 3.45, and 3.95% (w/v)) was also added into the culture medium to examine the effects of peptone on alkaline α-amylase production. Soybean meal of different concentrations (0, 0.3, 0.8, 1.3, 1.8, 2.3, and 2.8% (w/v)) was added into the culture medium to investigate the effects of soybean meal on the alkaline α-amylase production. The effects of temperature (25, 28, 31, 34, 37, and 40°C) on the production of alkaline α-amylase were also investigated.
Production of alkaline α-amylase by recombinant B. subtilis in a 3-L fermentor
The recombinant B. subtilis with the highest alkaline α-amylase yield was used to scale up fermentation in a 3 L fermentor (BIOTRON, Korea). The medium was supplemented with 1.45% peptone, 0.6% soluble starch, 1.3% soybean meal, 1.0% NaCl and 100 μg/mL Kanamycin. The fermentation was performed with a working volume of 1.6 L at 37°C for 60 h. The stirring speed of fermentation was 600 rpm, and the volume of ventilation was 1 vvm.
Alkaline α-amylase was determined by measuring the amount of reducing sugar released during enzymatic hydrolysis of 1% soluble starch in glycine/NaOH buffer (pH 10.0) at 55°C for 5 min. A control without enzyme addition was used. The amount of reducing sugar was measured by a modified dinitrosalicylic acid method . One unit of alkaline α-amylase activity was defined as the amount of enzyme that released 1 μmol of reducing sugar as glucose per min at the assay conditions.
Purification and molecular weight determination of alkaline α-amylase
Solid ammonium sulfate was added to the supernatant to 70% saturation at 0°C. The precipitate was collected and dissolved in glycine/NaOH buffer (pH 9.0) and dialyzed overnight against the same buffer. After dialysis, the enzyme solution was filtered using water film. And then the enzyme solution was injected into AKTA purifier (GE Healthcare USA) through the anion exchange (Q-Sepharose HP). The subunit molecular weight of recombinant alkaline α-amylase was determined by SDS-PAGE.
Determination of kinetic parameters
The reaction was performed in glycine/NaOH buffer (pH 10.0) at 55°C for the determination of kinetic parameters. Assays were performed with active enzyme and soluble starch of different concentrations from 1 to 10 g/L. The Eadie-Hofstee plots were used to calculate kinetic parameters K
m and V
max according to the enzyme reactions .
Effects of temperature and pH on activity
To determine the optimal temperature, the enzyme was analyzed from 30 to 90°C in glycine/NaOH buffer (pH 10.0). The thermal stability of alkaline α-amylase was determined at the indicated temperatures in glycine/NaOH buffer (pH 10.0). To estimate the optimal pH for the alkaline α-amylase, the purified protein was incubated in various buffers. The buffer used for determination of optimal pH was as follows: glycine/HCl buffer for pH 3.0-6.0, glycine/NaOH buffer for pH 6.0-11.0. The pH stability of alkaline α-amylase was determined at pH ranging from 3.0 to 11.0 at 25°C for 24 h. After incubation, the alkaline α-amylase activity was measured at pH 10.0 and 55°C.
Effects of metal ions on enzyme activity
Alkaline α-amylase was pre-incubated with 1 mM and 10 mM KCl, NaCl, CaCl2, MgCl2, FeCl3, FeCl2, CoCl2, ZnCl2, MnCl2 and CuCl2, respectively. The relative activity was determined and compared with the activity obtained in glycine/NaOH buffer (pH 10.0) without the addition of any metal ions.
Protein content assay
The protein content of samples was measured by the Bradford method with BSA as a standard .