Statistical optimization of pectinases from thermophilic Aspergillus fumigatus BT-4 employing response surface methodology through submerged fermentation using agricultural wastes

Background: In this study, thermophilic pectinase-producing strains were isolated. Among all the isolates, strain No. 4 was identified as Aspergillus fumigatus BT-4 based on its morphology and 18 S rDNA analysis. This strain was employed to screen various fermentation media to enhance pectinase production. Pectinases are crucial enzymes with significant industrial applications, particularly in the food and textile industries. Identifying efficient pectinase producers and optimizing their production processes are essential for improving industrial applications.

Results: Maximum pectinase production was observed using 1% grapefruit peel in M5 media. Shake flask kinetics demonstrated the highest values of specific rate constant (qp), specific growth rate (µ), product yield coefficient (Y_p/x), volumetric rate of product formation (Q_p), and biomass formation (Q_x) after 72 h of incubation. Furthermore, Optimization of fermentation components via Response Surface Methodology (RSM) improved pectinase production by 50%, showcasing the effectiveness of factorial and central composite designs in fine-tuning parameters. The use of agricultural waste (grapefruit peel) significantly reduced production costs, offering an economically viable substrate alternative. The pectinase enzyme was purified through ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography, resulting in a 2.3-fold purification. The molecular weight of the purified enzyme was determined to be 48 kDa. Enzyme kinetics, determined using a Lineweaver-Burk plot at various pectin concentrations, showed a V_max of 32.7 UmL^{- 1} and a K_m of 0.3 mg mL^{- 1}. Thermodynamic parameters, including activation energy (Ea), enthalpy (ΔH), and entropy (ΔS), were measured at 41.74 kJmol^{- 1}, 39.53 kJmol^{- 1}, and 46.9 kJmol^{- 1}, respectively.

Conclusions: The study successfully isolated and identified Aspergillus fumigatus BT-4 as a potent thermophilic pectinase producer. Optimization of the fermentation process using 1% grapefruit peel in M5 media significantly enhanced pectinase production. Using grapefruit peel as an agricultural waste in pectinase production reduces costs by eliminating the need for expensive raw materials and utilizing a low-cost, sustainable, and locally available substrate. This approach also minimizes waste disposal expenses, making the process more economical. The enzyme was effectively purified, and its kinetic and thermodynamic properties were thoroughly characterized, revealing its potential for industrial applications. The comprehensive analysis of production kinetics and optimization strategies provides a robust foundation for scaling up pectinase production, contributing to more efficient and cost-effective industrial processes.