A Durable Biocatalyst Constructed by Immobilization of Recombinant Laccase-Like Multicopper Oxidase from Bacillus Mojavensis onto Hazelnut Shell Hydrochar: Its Characterization and Potential for Use in the Degradation of Sulfamethoxazole and Diclofenac

Abstract

The present study investigates the immobilization of recombinant LMCO from Bacillus mojavensis TH308 (BmLMCO) on hazelnut shell hydrochar (HSH) and the use of the thus constructed biocatalyst (HSH-BmLMCO) to degrade sulfamethoxazole and diclofenac. Immobilization of BmLMCO with 120 U/mg protein-specific activity was achieved with 77.3 f 2.7 % yield and 39.4 f 1.3 % activity loss under the optimal conditions (pH 6, 24 h, and 10 degrees C). FTIR analyses indicated that the hydroxyl, carbonyl, and carboxyl groups played a role in adsorption, while FESEM analyses showed that the enzyme clustered in the pores of FSH. Similar to the free enzyme, HSHBmLMCO exhibited maximum activity at 80 degrees C and pH 8. However, immobilization enhanced the thermal stability. The half-life of HSH-BmLMCO at 30, 40, 50, 60, and 70 degrees C was calculated to be about 648, 612, 496, 418, and 359 min, respectively. The addition of Cu2+ to the reaction mixture caused a 39.52 % enhancement in the activity of HSH-BmLMCO. Tween 20, Triton X-100, and SDS increased the laccase activity by 25.8 %, 26.4 %, and 7.0 %, respectively, while PMSF and beta-mercaptoethanol decreased the oxidative activity of the biocatalyst by about 35 %. The Michaelis constant (Km) values of the HSH-BmLMCO against 2,6-DMP, SGZ, and ABTS were 1.18, 0.52, and 0.21 mM, respectively. HSH-BmLMCO retained 53.4 f 3.5 % and 91.6 f 1.5 % of its activity when stored at 4 degrees C for 48 days. Its residual activity after 10 consecutive catalytic cycles was 91.6 f 1.5 %. The biocatalyst degraded sulfamethoxazole and diclofenac pharmaceuticals by about 85 % and 80 % within 30 min, respectively. Furthermore, no toxic effects of the degradation products on the aquatic model organism Daphnia magna were observed. So, to sum up, the study provides an enormous biocatalyst for practical bioremediation applications. To our knowledge, this is the first report describing the biocatalyst constructed with B. mojavensis LMCO and hazelnut shell hydrochars and demonstrating its potential remediation application.