Stable protease from Bacillus licheniformis-MA1 strain: statistical production optimization, kinetic and thermodynamic characterization, and application in silver recovery from used X-ray films.

Abstract

Background: Alkaline proteases are useful enzymes for various industrial applications as bio-additives in detergents and in the recovery of silver from used X-ray films. Therefore, many strategies were used to increase enzyme production and reduce production costs by using microbial cultures, using agro-industrial waste, and improving growth conditions via statistical methods. The enzyme kinetics and thermodynamics were studied as well as its ability to recover silver was also evaluated.

Results: An alkaline protease suitable for industrial applications was produced by Bacillus licheniformis strain-MA1. The ability of B. licheniformis strain-MA1 to produce protease was optimized using multi-factorial designs (Plackett-Burman and Box-Behnken). Optimization process improved enzyme production by 9.6-fold over that obtained from the original medium. Highest alkaline protease production was reached after 72 h at pH 7.0, 35 °C, and 150 rpm. The protease was maximally active at 50 °C and pH 9.0 with high thermal and pH stability. The protease showed high catalytic efficiency and high affinity toward substrate with low activation energy (Ea). In addition, the thermodynamic parameters of protease enzyme (enthalpy, free energy, and entropy) were also investigated and showed its superior thermal stability. At 70 °C the thermal deactivation constant (kd) was 4.75-fold higher than that at 50 °C. The higher t_0.5, D-values, and activation energy for thermal denaturation (Ed) of the protease indicated its higher thermal stability and thus its potential application in industrial processes. The compatibility of the protease with laundry detergents at 40 °C was higher than at 50 °C. In the presence of EDTA, the protease enzyme retained 93.6% of its activity. Furthermore, the crude enzyme successfully hydrolyzed the gelatin layer from X-ray films waste after 1 h enabling recycling and reuse.

Conclusions: Stable alkaline protease from B. licheniformis strain-MA1 was suitable for some industrial aspects as a bio-additive in detergents and capable of recovering silver from used X-ray.