Enhanced production and biochemical characterization of a thermostable amylase from thermophilic bacterium Geobacillus icigianus BITSNS038

Figures & data

Figure 1. Growth kinetic profile for amylase production and cell biomass by strain G. icigianus BITSNS038. Results represented as Mean ± standard deviation of triplicates.

Figure 2. (A) Comparative graph showing experimental and predicted values of biomass formation of G. icigianus (B) Plot ln(X) vs. t showing variation of biomass formation in batch culture during the exponential phase of bacterial growth curve (C) Comparative graph showing experimental and predicted values of product formation (amylase activity) by G. icigianus (D) Regression analysis for product formation at t = 0, i.e. beginning of exponential phase

Table 1. Optimization condition achieved for each parameter after applying OFAT.

Factors	Optimum condition achieved
Incubation period	18–24 h
pH	7.0
Amount of inducer (starch)	7.5 g/L
Amount of tryptone	3 g/L
Carbon source	Starch soluble

Figure 3. (A) Effect of different pH on amylase production. (B) Effect of different carbon sources on amylase production. (C) Effect of different concentrations of starch as carbon source on amylase production. (D) Effect of different concentrations of tryptone as nitrogen source on amylase production. Results represented as Mean ± standard deviation of triplicates. Statistical data represented as Punnet chart where ns represents no significant difference.

Figure 4. (A) Effect of different temperatures on amylase activity. Statistical data represented as Punnet chart where ns represents no significant difference (B) Effect of pH on amylase activity (C) Study on thermal stability of amylase at 60°C and 70°C. Results represented as Mean ± standard deviation of triplicates (D) Plot of ln (A_t/A₀) vs. t for the determination of deactivation rate constant (K_d) (E) Lineweaver-Burk plot for the determination of K_m and V_max (F) Determination of α and β amylase in crude supernatant. Results represented as Mean ± standard deviation of triplicates.

Table 2. Kinetic and thermostability parameters evaluated experimentally.

Kinetic parameter	Value
Biomass production
Maximum specific growth rate (μmax)	0.041/h
Maximum biomass obtained (Xm) (reported here in terms of CFU/mL)	9.99 × 10^3
F-value	137.77
P-value	2.54 × 10^−6
R^2	0.984
Amylase production
Initial product (P0)	0.936 U/mL
Maximum product formed (Pm)	2.983 U/mL
Maximum specific product formation rate (μPm)	0.122/h
F value	13.04398
P-value	0.006865
R^2	0.945
Km	2.17 mg/mL
Vmax	4.16 U/mL
Thermostability parameter	Value
Deactivation rate constant (kd)	0.001
Half-life (t1/2)	11 h 55 min

Figure 5. %Hydrolysis of corn starch. Results represented as Mean ± standard deviation of triplicates.

Figure 6. Agar well diffusion assay revealing the lack of antibacterial activity of amylase enzyme on (A) E. coli (B) P. aeruginosa (C) B. subtilis and (D) S. aureus. Wells E and C contained 100 µL of partially purified and crude amylase enzyme, respectively.

Figure 7. Agar well diffusion assay depicting no antifungal activity of amylase on (A) Sclerotium sp. and (B) Rhizoctonia sp.

Figure 8. Antibiofilm activity of partially purified and crude amylase enzyme against two strains of EPS producing Bacillus sp. designated as (A) BITSNS010 (B) BITSNS018.

Figure 9. Molecular weight analysis of amylase enzyme using SDS-PAGE.

Table 3. Partial purification of amylase from G. icigianus (BITSNS038).

Step	Total protein content (µg/mL)	Total amylase activity (U/mL)	Specific activity (U/mg)	Fold purification
Crude amylase	25	3.30	0.132	1
Ammonium sulfate	19	5.26	0.277	2.1
Ultrafiltration (10 kDa)	14	4.95	0.353	2.67

Table 4. The comparison of the present research reports with the present work on amylase along with their applications.

Sl No.	Microbes	Temperature optima, thermostability & pH	Form	Applications	References
1.	A. pallidus BTPS-2	70°C, 40°C–80°C, pH 7.0	Purified	Starch liquefaction	Timilsina et al., 2021 [75]
2.	A. ayderensis FMB1	55°C, 50°C–60°C		Detergent & food industry	Matpan Bekler et al., 2021 [76]
3.	B. subtilis Y25	45°C, 30°C–50°C, pH 8.0	Purified	Deizing and starch hydrolysis in textile industry, detergents	Aladejana et al., 2020 [77]
4.	Geobacillussp.GS33	60°C, 40°C–90°C	Purified	Several industrial applications	Burhanoğlu et al., 2020 [78]
5.	B. licheniformis So-B3	70°C, 20°C–90°C, pH 5.0	Purified	Juice clarification in fruit juice industry, starch hydrolysis	Fincan et al., 2021 [13]
6.	G. icigianus	70°C and 30°C–80 °C, pH 7.0	Crude and partially purified	Antibiofilm agent and starch saccharification	Present Work

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