Peptone and yeast extract were obtained from Oxoid, Thermo Fisher Scientific (Basingstoke, UK). Coomassie Brilliant Blue R-250 staining solution was obtained from Bio-
-Rad (Hercules, CA, USA). The 3,5-dinitrosalicylic acid (DNS) was obtained from Sigma-Aldrich (St Louis, MO, USA). NaCl, CaCl₂, K₂HPO₄, KH₂PO₄, (NH₄)₂SO₄, MgSO₄·7H₂O, sodium acetate, sodium citrate, sodium phosphate, starch, glucose, l-tryptophan, acid-hydrolyzed casein and antibiotics were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, PR China). NuPAGE 12% Bis–Tris Gel and MOPS SDS Running Buffer were obtained from Invitrogen (Carlsbad, CA, USA). PrimerSTAR Max DNA Polymerase and DpnI were purchased from Takara Biotechnology (Dalian) Co., Ltd. (Dalian, PR China).

Plasmids and bacterial strains used in this study are described in Table 1. The pMA5 is an Escherichia coli/B. subtilis shuttle plasmid, which was donated by the Bacillus Genetic Stock Center (Columbus, OH, USA). E. coli DH5α was chosen as a host for molecular cloning and plasmid construction. This strain was cultivated in Luria–Bertani (LB) medium at 37 °C, which contained 5 g/L of yeast extract, 10 g/L of peptone and 5 g/L of NaCl. B. subtilis 1A751 was used as the host strain for AmyS (α-amylase from B. stearothermophilus) expression, and this strain was grown in Super rich (SR) medium (2.5% yeast extract, 1.5% peptone and 0.3% K₂HPO₄, pH=7.2) at 37 °C. Antibiotics were added as follows: kanamycin 25 µg/mL and ampicillin 100 µg/mL.

The components of the medium used for Bacillus subtilis transformation were as follows: Spizizen minimal medium consisting of (in g/L): (NH₄)₂SO₄ 2, KH₂PO₄ 6, K₂HPO₄ 18.3 and sodium citrate 1.2. GMI (growth medium I) was Spizizen minimal medium supplemented with 0.8% glucose, 0.04% acid-hydrolyzed casein, 0.1% yeast extract, 0.02% MgSO₄·7H₂O and 0.005% l-tryptophan, and GMII (growth medium II) was Spizizen minimal medium supplemented with 0.8% glucose, 0.02% acid-hydrolyzed casein and 0.16% MgSO₄·7H₂O.

The Easy Mutagenesis System (TransGen Biotech Co. Ltd., Beijing, PR China) was used to construct the mutant plasmid in accordance with the supplier’s instructions. First, a linear plasmid carrying the desired mutation was PCR-amplified with primers AmyS∆R179-G180-F (5’-TTACATATGAAACAACAAAAACGGCTTTACG-3’) and AmyS∆R179-G180-R (5’-CGCGGATCCTTAAGGCCATGCCACCAGTC-3’) with PrimerSTAR Max DNA Polymerase using the plasmid pMA5-AmyS as a template. The PCR products were then digested with DpnI, which degraded the methylated DNA, removing the template. Finally, the nicked plasmid DNA carrying the desired mutation was transformed into E. coli DH5α, according to the methods described by Green and Sambrook (11). The newly constructed plasmid was designated pMA5-AmyS∆R179-G180 and verified by DNA sequencing (12) (Genewiz, Suzhou, PR China).

First, GMI and GMII were prepared as mentioned previously (13). B. subtilis 1A751 and derivatives were cultivated in GMI overnight at 37 °C. A volume of 0.5 mL of overnight culture was transferred into 4.5 mL of GMI medium and grown at 37 °C for 4.5 h. A volume of 0.75 mL of culture was then mixed with 4.25 mL of GMII medium and the culture was incubated for 1.5 h at 37 °C. A mass of plasmid of 1 μg was mixed with 1 mL of competent cells and incubated for 1 h at 37 °C. The cells were then plated on LB agar plates containing kanamycin (25 μg/mL) and incubated for 12 h at 37 °C.

The plasmids pMA5-AmyS and pMA5-AmyS∆R179-G180 were individually transformed into B. subtilis 1A751 for protein expression. B. subtilis 1A751 cells harbouring pMA5-AmyS and pMA5-AmyS∆R179-G180 were grown overnight and 1-mL culture was transferred into 100 mL of SR medium containing kanamycin (25 μg/mL) and the culture was then incubated at 37 °C for 36 h. The supernatant was collected by centrifugation (4 °C, 5000×g, 10 min) using centrifuge model 5810R (Eppendorf, Hamburg, Germany). The enzyme in the supernatant was precipitated by adding solid ammonium sulfate to 70% (by mass per volume). This was performed at 4 °C with continuous stirring and then kept on ice for 30 min. The precipitated proteins were collected by centrifugation
(4 °C, 12 000×g, 30 min; model 5810R; Eppendorf), dialyzed against 100 mM sodium acetate (pH=6), and then concentrated to minimum volume. The purified enzymes were analyzed by SDS-PAGE using the NuPAGE 12% Bis–Tris Gel together with MOPS SDS Running Buffer. Protein bands were visualized by Coomassie Brilliant Blue R-250 staining.

α-Amylase activity was measured according to the 3,5-dinitrosalicylic acid (DNS) method (14). One unit of α-amylase activity was defined as the amount of enzyme that liberates 1 μmoL of glucose per min under the detection conditions. In our experiment, 40 μL of diluted enzyme solution were mixed with 80 μL of 100 mM sodium acetate (pH=6) and 80 μL of 1% (by mass per volume) soluble starch and then incubated for 20 min at 75 °C. The reaction was stopped by quenching on ice, after which 200 μL of 1% DNS were added and the sample was boiled for 10 min. Absorbance was measured at 540 nm using Microplate Reader SpectraMax M5 (Molecular Devices LLC, San Jose, CA, USA) after cooling to room temperature. A reaction mixture without the enzyme was prepared as a negative control. All measurements were performed in triplicate.

The α-amylase activity at 50, 55, 60, 65, 70, 75, 80, 85 and 90 °C was determined, and the optimal temperature was obtained. α-Amylase activity was measured at pH=4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 and the optimal pH was determined. The buffers used in the experiment were as follows: 100 mM sodium acetate buffer (pH=4.0 to 6.0) and 100 mM sodium phosphate buffer (pH=6.0 to 7.5).

The purified enzyme was incubated with different concentrations of CaCl₂ (0, 0.25, 0.5 and 1 mmol/L) at 100 °C for 0-1 h. Subsequently, the residual enzyme activity was assayed to analyze thermostability.

The homology models of native AmyS and mutant AmyS∆R179-G180 were created with Discovery Studio v. 4.1 (15), using the wild type BStA (1HVX PDB entry) as a template. The structure was viewed using the PyMol editor (16).

B. subtilis 1A751 strain harbouring pMA5-AmyS∆R179-G180 was used for high density fermentation in a 7.5-litre BIO FLO 310 fermentor (New Brunswick Scientific Co Inc., Edison, NJ, USA), with a fed-batch strategy. The medium was supplemented with 2.5% yeast extract, 1.5% peptone, 0.3% K₂HPO₄ and 50 μg/mL of kanamycin. The airflow rate was controlled at 6.0 L/min and, by automatic regulation of stirring speed, dissolved oxygen tension was kept between 20 and 40% air saturation. Temperature and pH were controlled at 37 °C and 7.0, respectively. A silicone-based anti-foaming agent was used to eliminate foam. Fermentation was carried out with a working volume of 3.5 L at 37 °C for 72 h. When cell growth rate reached a constant level, fed-batch cultivation was initiated. Soluble starch (8.0% by mass per volume) was added as the substrate at a constant flow rate until its final mass per volume ratio reached 4.0%. Cell growth was monitored by measuring cell dry mass. α-Amylase activity was analyzed by measuring the cell-free supernatant.

The experiments (α-amylase activity assay and dry cell mass measurement) were performed in triplicates. Data are presented as mean relative value±standard deviation.

In our previous study, we cloned the α-amylase gene (GenBank: AAB86961.1) (17) from a wild type B. stearothermophilus strain and successfully expressed this α-amylase (AmyS) in B. subtilis (18). According to sequence alignment, we found that an Arg-Gly peptide present in AmyS was similar to that in BAA (7) and LAMY (8). The deletion of the Arg-Gly peptide greatly improved the thermostability of BAA and LAMY. Therefore, we attempted to optimize AmyS by deleting amino acid residues Arg179 and Gly180 simultaneously by site-directed mutagenesis. The mutation (∆R179-G180) was introduced into AmyS using the Easy Mutagenesis System (TransGen Biotech) and the altered sequence was confirmed by sequencing. The resultant plasmid pMA5-AmyS∆R179-G180 was transformed into B. subtilis 1A751 cells. The transformants were cultivated in 30 mL of SR medium in 250-mL shake flasks at 37 °C for 36 h. As shown in Fig. 1a, a protein band of approx. 58 kDa was present, which was consistent with the expected size of AmyS. In addition, the proteins of interest, AmyS and AmyS∆R179-G180, were purified to near homogeneity by saturated ammonium sulphate precipitation (Fig. 1b).

After purification of the enzymes, the enzymatic properties of AmyS and AmyS∆R179-G180 were evaluated. The optimal temperature and pH for AmyS and AmyS∆R179-G180 activity were assayed. As shown in Fig. 2a, wild type AmyS maintained biological activity within a temperature range of 50 to 90 °C (pH=6.0) and showed the highest α-amylase activity at 75 °C. The mutant AmyS∆R179-G180 exhibited a similar temperature-activity curve to the wild type AmyS (pH=6.0), indicating that the mutation in AmyS had no significant effect on reaction temperature. In addition, the effect of pH on the wild type and mutant AmyS was measured (Fig. 2b). The wild type AmyS was active within a pH range of 4.0 to 7.5 (at 70 °C) and the optimal pH of AmyS was 6.0. However, it is worth noting that the mutant exhibited its highest activity at pH=5.5, suggesting that the optimal pH decreased by deleting R179-G180 in AmyS. Moreover, the mutant seemed to be more acid-resistant at pH=4.5-6.0. Considering that α-amylases ideally function at pH=4.5 in industry for the liquefaction of starch, the mutant AmyS∆R179-G180 was, therefore, more suitable for future industrial applications.

In order to study the effect of deleting residues R179-G180 on AmyS thermostability, the half-lives of AmyS and AmyS∆R179-G180 were determined at high temperatures. As shown in Fig. 3, AmyS showed a time-dependent decrease in amylolytic activity and the half-life was 24 min at 100 °C. However, AmyS mutant was found to exhibit increased thermostability and the half-life reached 33 min at the same temperature. In order to investigate the cause of the increased thermostability, we created a homology model of AmyS using Discovery Studio v. 4.1 (15) and compared the model with BLA (Fig. 4). We noticed a small extra loop containing Arg179-
-Gly180 located in domain B of AmyS. It has been reported that domain B is likely to be responsible for the differences in substrate specificity and thermostability between different α-amylases. In addition, Suzuki et al. (7) demonstrated that the thermostability of BAA was greatly improved by deletion of Arg176-Gly177, in a similar loop, along with the replacement of Ala269 with Lys269 using site-directed mutagenesis. According to sequence alignment and protein structure comparison, other α-amylases, including LAMY from Bacillus KSM-1378 (8), BStA from B. stearothermophilus US100 (10) and BAC∆NC from Bacillus sp. strain TS-23 (9), also resulted in similar improvements in enzyme properties by the deletion of amino acids in the same region.

We compared the structure of the AmyS model with that of the AmyS∆R179-G180 model and found that the deletion of R179-G180 caused a slight structural rearrangement. Ali et al. (10) confirmed that the CaII site in the AmyUS100∆I214-G215 model lost interactions with Asp105 and His238, which contributes to the maintenance of connections between the A and B domains. They also found that the deletion of I214-G215 in AmyUS100 not only increased enzyme thermostability, but also significantly decreased calcium requirements. It was thus possible that the deletion of R179-G180 affects the calcium-binding sites in AmyS. Therefore, calcium requirements of AmyS and AmyS∆R179-G180 were tested through comparison of enzymatic activity of residue following incubation of the enzyme with different concentrations of calcium at 100 °C. As shown in Fig. 5, the maximal thermostability of AmyS (Fig. 5a) was obtained with 1 mmol/L of CaCl₂, however, the maximal thermostability of AmyS∆R179-G180 (Fig. 5b) was obtained with only 0.25 mmol/L of CaCl₂. This observation confirmed the above hypothesis.

It is well-known that α-amylases usually contain calcium-binding sites to stabilize the interface between the highly homologous domain A and the more variable domain B (19). Declerck et al. (20) reported that the interface was sensitive to structural disruption and confirmed that when BLA was exposed to high temperature, protein unfolding began in this region. Therefore, they speculated that any mutation that altered or reinforced stabilizing interactions in this region may influence the rate of thermal inactivation (21). There are several reasons to illustrate increased thermostability. Suzuki et al. (7) confirmed that increased thermostability of BAA by deletion of Arg176-Gly177 could be attributed to an increase in hydrophobicity and the charged residues changing into non-polar ones. Igarashi et al. (8) reported that the thermostability of LAMY improved with enhanced calcium binding of the enzyme. In the present study, the increased thermostability of AmyS∆R179-G180 may be caused by changing the calcium-binding sites.

Since the AmyS∆R179-G180 exhibited better enzymatic properties and was, thus, able to be applied in the industrial production of sugars, the expression efficiency of B. subtilis 1A751 containing the plasmid pMA5-AmyS∆R179-G180 was evaluated for high density fermentation. A volume of 1 mL of frozen glycerol stock was inoculated into 100 mL of SR medium and incubated for 18 h at 37 °C. Next, 5% (by volume) of seed culture was inoculated into a 7.5-litre fermentor supplemented with fermentation medium. When the rate of cell growth became constant, fed-batch cultivation was initiated. Soluble starch (8% by mass per volume) was added as the substrate at a constant flow rate until its final mass per volume ratio reached 4.0%. For AmyS∆R179-G180 production (Fig. 6), the maximum biomass concentration reached 32.3 g/L after 36 h. The maximum enzyme activity reached 3300 U/mL after 72 h and the productivity level reached 45.8 U/(mL·h).