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

Lina S. Alhazmi

Lina S. Alhazmi

Department of Biological Sciences, College of Science, University of JeddahJeddah, Saudi Arabia
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Alhazmi, Lina S.
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Wafa A. Alshehri

Wafa A. Alshehri

Department of Biological Sciences, College of Science, University of JeddahJeddah, Saudi Arabia
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Alshehri, Wafa A.
  
Mar 26, 2025
Purification and Biochemical Characterization of α-Amylase from Newly Isolated Bacillus Cereus Strain and its Application as an Additive in Breadmaking's Cover Image
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Article Category: ORIGINAL PAPER
Published Online: Mar 26, 2025
Page range: 48 - 59
Received: Oct 02, 2024
Accepted: Dec 27, 2024
DOI: https://doi.org/10.33073/pjm-2025-004
Keywords
© 2025 Lina S. Alhazmi et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

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

Fig. 2.

Isolate WL showing positive starch hydrolysis test in presence of iodine.
Isolate WL showing positive starch hydrolysis test in presence of iodine.

Fig. 3.

The phylogenetic tree of Bacillus cereus WL with the accession number PP463909.1 and its close relatives based on the 16S rRNA sequence. The tree was constructed using the Neighbor-Joining method in MEGA software v11. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches.
The phylogenetic tree of Bacillus cereus WL with the accession number PP463909.1 and its close relatives based on the 16S rRNA sequence. The tree was constructed using the Neighbor-Joining method in MEGA software v11. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches.

Fig. 4.

Effect of incubation time on the growth and the α-amylase activity from Bacillus cereus WL.
Effect of incubation time on the growth and the α-amylase activity from Bacillus cereus WL.

Fig. 5.

Chromatography of Bacillus cereus WL on Sephacryl™ S-200.
Chromatography of Bacillus cereus WL on Sephacryl™ S-200.

Fig. 6.

The SDS-PAGE (12%) analysis of the Bacillus cereus WL. Lane 1 – 20 μg of partially purified amylase obtained after Sephacryl™ S-200 chromatography; Lane 2 – Molecular mass markers.
The SDS-PAGE (12%) analysis of the Bacillus cereus WL. Lane 1 – 20 μg of partially purified amylase obtained after Sephacryl™ S-200 chromatography; Lane 2 – Molecular mass markers.

Fig. 7.

Effect of temperature on the activity and the stability of the purified Bacillus cereus WL. α-Amylase was measured under stander conditions as described in Material and Methods section.
Effect of temperature on the activity and the stability of the purified Bacillus cereus WL. α-Amylase was measured under stander conditions as described in Material and Methods section.

Fig. 8.

Effect of pH on the activity and the stability of the purified Bacillus cereus WL. α-Amylase was measured under standard conditions as described in Material and Methods section.
Effect of pH on the activity and the stability of the purified Bacillus cereus WL. α-Amylase was measured under standard conditions as described in Material and Methods section.

Fig. 9.

Lineweaver-Burk plot (1/Relative activity (v) vs. 1/Concentration of substrate (S)) for α-amylase activity.
Lineweaver-Burk plot (1/Relative activity (v) vs. 1/Concentration of substrate (S)) for α-amylase activity.

Fig. 10.

Effect of the addition of α-amylase on the volume of bread. a – bread without enzyme; b – bread with 1 U/kg enzyme; c – bread with 2 U/kg enzyme.
Effect of the addition of α-amylase on the volume of bread. a – bread without enzyme; b – bread with 1 U/kg enzyme; c – bread with 2 U/kg enzyme.

Gram staining, morphological and physiological characterization of the isolates having a zone of clearance with iodine solution_

Sample Gram stain Morphology under light microscope Colony color Colony morphology Growth on α-amylase agar
M1 + Bacillus white filamentous ++++
M2 + Bacillus white irregular +++
WL + Bacillus white irregular ++++
M12 + Bacillus creamy circular ++
M13 + Bacillus creamy circular ++++
L5 − Bacillus white circular ++
L6 + Bacillus white circular ++
L8 + Bacillus creamy irregular +++
L15 + Bacillus creamy circular ++

Flow sheet of the Bacillus cereus WL purification_

Purification steps Total activity (U) Total proteina (mg) Specific activity (U/mg) Activity recovery (%) Purification factor
Crude enzyme 180 0.817 220 − 1
Participate by amm. sulfate (80%) 90 0.225 400 50 1.8
Sephacryl S-200 45 0.055 818.18 25 3.7

Screening of α-amylase activity (U/ml) using starch nitrate medium and selecting of WL as a potent isolate for further study_

Isolate code α-amylase activity (U/ml)
M1 511.08 ± 0.12
M2 572.10 ± 0.14
WL 579.12 ± 0.18
M12 509.98 ± 0.04
M13 499.99 ± 0.02
L5 568.12 ± 0.18
L6 539.08 ± 0.12
L8 568.12 ± 0.12
L15 539.08 ± 0.14

Physiological characters of the selected bacterial Bacillus cereus WL_

Character WL Strain
Temperature range 35–60°C
Optimum temperature 45°C
pH range 4–8
Optimal pH 6
Catalase +
Oxidase +
Language:
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