Purification and Biochemical Characterization of α-Amylase from Newly Isolated Bacillus Cereus Strain and its Application as an Additive in Breadmaking

Starch (99% purity) was purchased from Sigma-Aldrich^® (Merck KGaA, Germany). All chemicals used for cultivation and enzyme assays are analytical grade from Merck KGaA (Germany). Oligonucleotides were synthesized by the Genome Express (France).

The soil samples were collected from the salinity region surrounding Rabigh city (Fig. 1) in the Kingdom of Saudi Arabia. The samples were enriched with various vegetable organic matter and incubated for one week at ambient temperature. The organic soil samples were then transported in sterile plastic bags and stored at 4°C until use.

Fig. 1.

Type of soil used in this study surrounding Rabigh City in the Kingdom of Saudi Arabia (22.803970N, 38.984341E).

α-Amylase-producing bacteria were cultured using starch nitrate agar plates with the following composition (g/l): 20 starch, 15 agar, 0.5 MgSO₄, 1 KH₂PO₄, 3 CaCO₃, 2 KNO₃, 0.5 NaCl, 1 ml Trace solution and pH 7 (Shirling and Gottlieb 1966). After 48 h of incubation at 37°C, amylolytic activity was detected using the starch-iodine test (Gupta et al. 2003). Thirteen isolates were grown on starch nitrate medium with the following composition (g/l): 20 starch, 0.5 MgSO₄, 1 KH₂PO₄, 0.01 FeSO₄, 3 CaCO₃, 2 KNO₃, 0.5 NaCl, 1 ml Trace solution and pH 7. The Trace solution contains (%): 0.1 FeSO₄, 0.1 MnCl₂, and 0.1 ZnSO₄ (Gupta et al. 2003).

One gram of the collected organic soil sample was added to 50 ml of starch nitrate broth medium. The flasks were incubated for 2 days at 37°C. One ml of each broth was spread-plated onto starch nitrate agar plates, which were then incubated at 37°C for 2 days. The screening for amylolytic activity was performed using a starch hydrolysis test on a starch nitrate agar plate (Shirling and Gottlieb 1966). The pure isolated colonies were inoculated onto starch agar plates with starch as the only carbon source. After incubation at 37°C for 48 hr, the individual plates were submerged with Gram’s iodine to produce a blue-colored starch-iodine complex. The appearance of a clearance zone around colonies indicates the presence of α-amylase activity (Gupta et al. 2003). Amylolytic bacteria with the maximum diameter of the clearance zone were retained for further investigations.

The retained amylolytic strain was precultured at 37°C and 230 rpm for 15 h in 250 ml shaking flasks with 50 ml of starch broth medium. Overnight precultures used as inoculum were cultivated in 1 l shaking flasks with 100 ml of the same culture medium. Cells were grown at 37°C on a rotary shaker at 230 rpm. Growth was monitored by measuring the culture’s OD at 600 nm. At the end of the fermentation period, the culture medium was centrifuged at 5,000 rpm for 20 min to obtain the crude extract containing α-amylase activity.

On a starch agar medium, the bacterial isolate was cultured to examine the color and shape of the colonies. Light microscopy was used to conduct morphological analyses (Williams et al. 1989). The following physiological tests, including those for catalase, oxidase, pH, and ideal temperature, were carried out.

Crystal violet was applied, and the bacterial film was allowed to soak for one minute. Then, it was washed off with distilled water after the slide was prepared and the bacterial film was fixed. Ethyl alcohol (95%) was allowed to drip onto the surface of the slide after the iodine was removed from the Gram stain. This process was repeated until the alcohol lost its hue. After being stained with safranin for a minute, the slide was gently cleaned with water. After a brief gentle wash, the slide was dried in the air and inspected with an oil immersion lens while containing a few drops of Cedar oil. Gram-negative bacteria will be red-colored, whereas Gram-positive bacteria will be colored violet.

This examination is performed to detect the catalase enzyme. This enzyme protects against hydrogen peroxide (H₂O₂) toxicity, which can accumulate during aerobic metabolism. A bacterial colony was spread on a glass slide, and a drop of H₂O₂ was added. If bubbles appear, the test is considered successful. If no bubbles are present, it indicates the absence of catalase.

The oxidase test is conducted based on synthesizing the cytochrome oxidase enzyme. When oxygen is present in the air, the colorless substrate tetramethylphenylenediamine dihydrochloride is oxidized by this enzyme to create a dark-purple molecule. The color purple denotes a positive response.

The oxidase test is conducted based on synthesizing the cytochrome oxidase enzyme. This enzyme oxidizes the colorless substrate tetramethyl phenylenediamine dihydrochloride to create a darkpurple molecule when oxygen is present in the air. The purple color denotes a positive response.

Bacteria were cultured on a slant and then transferred into 250 ml Erlenmeyer flasks with 30 ml of starch broth. The flasks were incubated at 37°C and 230 rpm for 24 hours. After incubation, the cultures were centrifuged at 10,000 rpm for 10 minutes. Cells were disrupted using liquid nitrogen on a porcelain dish. TE buffer with lysozyme (20 mg/ml) was added to 500 μl a tube containing bacteria and incubated at 37°C for 30 minutes. Proteinase K and 10% SDS (w/v) were then added and incubated at 55°C for 30 minutes. DNA was precipitated with ethanol, chilled at –20°C for 30 minutes, and centrifuged (10,000 rpm for 10 minutes) to form a pellet. The pellet was washed with ethanol and dissolved in TE buffer. To remove RNA from DNA, the sample was treated with 1 ml of RNase solution (20 g/ml) at 37°C for 1 hour. The DNA was extracted with phenol and chloroform, precipitated, and tested for quality and quantity.

In a 100 μ1 reaction, genomic DNA was amplified using HotStar^® Master Mix (2×) (QIAGEN, Germany). Primers were designed using a conserved region of 16S rDNA from various bacteria. The samples without the primers in the HotStar^® Master Mix served as a negative control. Two bacterial primers, 27F: 5’-AGAGTTTGATCMTGGCTCAG-3’ and 1492R: 5’-TACGGYTACCTTGTTACGACTT-3’, were used in polymerase chain reaction (PCR) to amplify the 16S rDNA of the examined bacteria. The DNA sequence was then compared to the GenBank database using the BLAST tool at the National Center for Biotechnology Information (NCBI).

The samples were incubated for two to forty-eight hours. A variety of sterile media was used to fill 250 ml Erlenmeyer flasks. Two ml of preculture was injected into 50 ml flasks. The flasks were kept in a 37°C incubator with a 230 rpm rotation. Bacterial growth and α-amylase production were assessed following each growth phase.

Temperature was found to play a role in enzyme synthesis and growth. The amylase broth medium in the 250 ml Erlenmeyer flasks was supplemented with approximately 2 ml of the selected bacterial isolate. The flasks were then incubated for 12 h with shaking at 230 rpm and temperatures ranging from 25 to 60°C. At the end of the incubation period, the same methods were used to evaluate the growth and amylase activity of the inoculated bacteria.

The medium was prepared with a pH range from 3 to 9. 250 ml Erlenmeyer flasks were filled with 50 ml sterile medium, and 2 ml of preculture was added. After determining the necessary time, the flasks were placed in an incubator with a speed of 230 rpm and a temperature of 45°C.

The activity of α-amylase was measured at pH 6.9 and 25°C. 0.5 ml of the enzyme solution, diluted in 0.1 M phosphate buffer pH 6.5, was incubated with 0.5 ml of 1% soluble starch prepared in 0.1 M phosphate buffer. DNS (3,5-dinitrosalicylic acid) was used to determine the amount of the released reducing sugars. One unit of α-amylase activity was defined as the amount of enzyme that liberates 1 μmole of reducing sugar (maltose equivalents) per minute at 25°C and pH 6.9 (Salem et al. 2020).

The Bradford technique (Bradford 1976) was used to measure the total protein contents in the cell-free supernatant and purified samples. A calibration curve was performed using bovine serum albumin (BSA) as the reference.

The selected bacteria were allowed to grow in the medium while being treated with all the optimal conditions found in the literature for producing α-amylase. The culture medium was then centrifuged for 20 min at 4°C and 5,000 rpm. The cell-free filtrate was used as a crude enzyme solution to purify the α-amylase enzyme. The initial α-amylase activity and the protein concentration were measured as described.

The obtained supernatant was then treated with solid ammonium sulphate (80% saturation w/v) while being gently stirred at 4°C for 30 min. After that, it was centrifuged for 30 min at 8,000 rpm and 4°C. α-Amylase activity and protein concentration were measured as described previously.

The purification process was used to remove any remaining ammonium sulfate and to eliminate peptides and proteins with small molecular weights. The resulting residue was dialyzed overnight against 2 l of pH 8.5 Tris-HCl 20 mM buffer in a cellophane bag with a 20 kDa molecular mass cutoff. The concentrated cell free supernatant from dialyzing the original sample is now ready for the following purification steps.

The concentrated dialyzed solution was chromatographed on a Sephacryl^™ S-200 column (30 cm × 1.5 cm; Merck KGaA, Germany). The elution was performed at a flow rate of 30 ml/h followed by measuring the absorption at 280 nM and the α-amylase activity under standard conditions. The active fractions were collected, pooled, and analyzed by SDS-PAGE.

Analytical polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate (SDS-PAGE) was performed as described previously (Laemmli 1970). Standard protein markers (low molecular weight 14–60 kDa) were used as standards.

To study the effect of the pH of the culture medium on α-amylase production, the selected amylolytic strain was grown in 250 ml shake flasks with 50 ml starch nitrate medium for 48 h at 37°C with different pH levels ranging from 5 to 9. After centrifugation, the α-amylase activity was then measured at pH 6.9 and 25°C. The effect of temperature on amylase production was determined by growing the selected amylolytic strain in 250 ml shaking flasks with 50 ml starch nitrate medium for 48 h at different temperatures ranging from 25 to 50°C. The α-amylase activity was measured at pH 6.9 and 25°C after centrifugation.

To check the pH stability, the enzyme was incubated with buffers 100 mM: glycine-HCl (pH 4), sodium acetate (pH 5–6), phosphate (pH 7), Tris-HCl (pH 8) and glycine-NaOH (pH 9–12) (in ratio of 1:1) at 25°C for 1 h and assayed under standard assay conditions (pH 6.9 and 25°C). The effect of temperature on α-amylase stability was determined by incubating the enzyme at different temperatures (25 to 100°C) for 30 min. The residual activity was determined after centrifugation under standard conditions (pH 6.9 and 25°C).

Before the reaction started, the substrate, α-amylase, was introduced to a borate buffer at pH 6.0 and 50°C for one hour at various concentrations. The α-amylase activity assay was then performed under standard conditions.

To determine the optimal enzyme concentration, the reaction mixture was prepared with varying concentrations of pure enzyme. The purified enzyme was added to the reaction mixture at concentrations ranging from 0.25 to 3 U/ml and incubated for 1 hour at 50°C and pH 6. Subsequently, the α-amylase activity of the mixture was measured.

The bread was made with the following ingredients: 250 g of wheat flour, dry yeast, 3% sugar, 2% salt, 1.25% vegetable oil, and water. After being dissolved in distilled water, 0.5 U/kg and 1.0 U/kg of purified α-amylase were added to the bread mixture. It took 10 min to mix the ingredients. After spending 45 minutes at 50°C, the finished dough was cooked for 45 minutes at 200°C. After cooling for two hours at ambient temperature, the loaves were then cut into slices.