*3.5. Secondary Screening of α-Amylase-Producing Bacteria and Measurement of Enzyme Activity*

The α-amylase enzyme-producing bacteria, which had produced a stronger halo in the primary screening stage, were selected for secondary screening and enzyme activity measurement. At this stage, bacteria were cultured in nutrient broth and incubated in a shaker incubator at 30 ◦C and 100 rpm. After 24 h, the culture medium containing the grown bacteria was centrifuged at 12,000 rpm for 20 min, and the supernatant was used as a solution containing the α-amylase enzyme in the next stages of the experiment [23]. The Bernfeld method was used to measure alpha-amylase enzyme activity [24]. For this purpose, 400 µL of phosphate buffer solution, 100 µL of α-amylase enzyme extract, and

500 µL of 1% starch were incubated at 60 ◦C for 20 min. Then, 1 mL of DNS solution was added to the test tube and incubated for 5 min in a boiling water bath. After cooling, 1 mL of distilled water was added to it. After stirring the contents of the absorption tube, the sample was read at a wavelength of 540 nm.

## *3.6. Identification of Potential α-Amylase Enzyme-Producing Bacteria*

In this study, the identification of potential bacteria producing the α-amylase enzyme was carried out by examining phenotypic and biochemical characteristics as well as using 16S rRNA gene analysis. In order to extract the bacterial DNA contents, the boiling method was used [25,26]. Polymerase chain reaction (PCR) was applied to amplify a 1500-bp fragment of the 16S rRNA gene using the extracted DNA of potential α-amylase-producing bacteria (HR13, HR16, HR15, and HR14) by means of forward (50 - AGAGTTTGATCCTGGCTCAG-30 ) and reverse (50 -AAGGAGGTGATCCAGCC-30 ) primers at a final volume of 50 µL, containing 2 µL of extracted bacterial strain DNA, 0.5 µL of forward primer, 0.5 µL of reverse primer, 25 µL of amplicon master mix solution, and 22 µL of distilled water. The thermal cycling was carried out as follows: an initial denaturation at 95 ◦C for 1 min, followed by 30 cycles of denaturation at 95 ◦C for 20 s, annealing at 63 ◦C for 30 s, extension at 72 ◦C for 1 min, and the final extension at 72 ◦C for 5 min.

After checking the quality and quantity of PCR products on 1% agarose gel electrophoresis, they were sent to FAZA Pajooh Co. for double-sided sequencing. The sequences were compared with the 16S rRNA gene sequences of bacteria registered in the NCBI database using the BLAST tool available on the NCBI website. Then, the closest strain was selected based on the S rRNA16 gene sequence and biochemical tests [27,28].

In order to analyze the phylogenetic relationships, the target sequences were assessed and compared in accordance with the NCBI gene bank. In the end, using MEGA4 software and the Neighbor-Joining (NJ) algorithm with bootstrapping, 1000 repetitions of the phylogenetic tree were drawn.

#### *3.7. Effect of pH and Temperature on α-Amylase Activity*

The α-amylase enzyme activity was evaluated at a temperature range of 20–90 ◦C in a 20 mM phosphate buffer (pH 7.4). In order to examine the enzyme activity at any temperature, both the substrate (1% starch) and the enzyme solution (buffer and crude enzyme extract) must reach equilibrium at that temperature before measuring enzyme activity. The relative enzyme activity was measured at different pH values of 2–12 at room temperature. For this purpose, a mixed buffer (containing 25 mM tris-base, glycine, sodium phosphate, and sodium acetate) was prepared and adjusted using NaOH and HCl solutions at different pH values from 2 to 12. In this experiment, a 1% starch solution was also prepared as a substrate, and the enzyme activity was evaluated in the above buffer at different pH values.

## *3.8. Effects of pH and Temperature on Enzyme Stability*

To measure the temperature stability of the enzyme, first, the crude enzyme extracts of both bacteria were placed at 80 and 90 ◦C for different time intervals (5, 10, 20, 30, 40, 50, and 60 min). Then, 500 µL of the substrate was added to each of the samples, and the resulting mixture was incubated in the assay conditions. Finally, α-amylase activity was stopped using dinitrosalicylic acid reagent and the absorbance of the reaction mixture was read at a wavelength of 540 nm.

In order to measure the pH stability of the enzyme, first, the crude enzyme extracts of both bacteria were exposed to pH values of 8 and 9 for different times (5, 10, 20, 30, 40, 50, and 60 min). Then, 500 µL of the substrate was added to each of the samples, and the resulting mixture was incubated in the assay conditions. Finally, α-amylase activity was measured using the dinitrosalicylic acid reagent and then reading the absorbance of the reaction mixture at a wavelength of 540 nm.
