Study On Heterologous Expression Of CotA Protein And Its Catalytic Properties

Laccases are multicopper oxidases that catalyze the oxidation of a variety of aromatic compounds.Because of their wide substrate range,laccases have great potential in various applications,such as pulp bleaching,dye decolorization and phenol sensing.In recent years,it was found that the CotA protein,which was a structural component of Bacillus subtilis endospore coat,had laccase catalytic activity.CotA laccase exhibited high thermal stability and high alkaline tolerance compared with laccase from other sources,and CotA protein was not easy to lose catalytic activity.These properties render CotA laccase important research significance and great potential for application.The CotA protein interacts tightly with other proteins in coat layer of the endospore.Therefore,it is difficult to isolate and purify CotA protein,leading to that the yield of CotA protein is low.The CotA laccase had been expressed in Escherichia coli,but the recombinant CotA often form inactive inclusion bodies.To enhance the productivity of CotA isolation and purification,the cot A gene from B.subtilis was secretory expressed in Pichia pastoris.The open reading frame of B.subtilis cot A gene was cloned and the cot A gene was 1542 bp encoding a 513 amino acid protein.The expression plasmid of p PICZαB-cot A was constructed.By gene transformation and screeing,the P.pastoris with the ability to express CotA protein was obtained.The laccase activity of the culture supernatant was 891.2 U/L.The CotA protein was purified to homogeneity by a simple two-step chromatographic method.The laccase activity and enzymatic properties of CotA protein were studied.Temperature and pH were important factors that influence the activity and stability of CotA laccase.The optimal enzymatic activity was found at pH 4.6,6.6 and 6.8 for ABTS,SGZ and 2,6-DMP oxidation,respectively.CotA protein was unstable under acid condition.However,the enzyme activity increased to 348.7% and 561.9% of its initial activity at pH 7.0 and pH 9.0 after 7 days of incubation.These results indicated that the CotA laccase possessed alkaliphilic property.The maximal enzyme activity was observed at 80 ℃ with SGZ as a substrate.The thermal denaturation profile indicated that the laccase was significantly thermostable at 60℃ and 70℃.The CotA laccase retained more than 72% and 57% of its initial activity after the incubation for 10 h.In this study,the indigo carmine was used as a model dye to investigate the decolorization ability of CotA laccase,and the decolorization conditions were optimized by response surface methods.Under the optimal decolorization condition,pH 8.6,60℃ and indigo carmine of 41.5 mg/m L,the dyecolorization rate was 91.4% within 45 min.The indigo carmine was degraded to a molecule with m/z 216.07 [M-Na]-which could be 4-amino-3-carboxybenzenesulfonic acid sodium salt.In order to immobilize the CotA protein for its reuse,a novel support material was developed.The hollow microspheres from Ganoderma lucidum spore were obtained and it could be used as a container to load Fe3O4 nanoparticles for preparing magnetic microspheres.The loaded amount of CotA on the microspheres was 75 mg/g and the activity recovery of the immobilized CotA was 81%.CotA immobilization in the magnetic spore microsphere led to a stabilizing effect towards heat denaturation,and the magnetic microspheres loaded with CotA could be easily and quickly recovered by an external magnetic field.The immobilized CotA was used for indigo carmine decolorization.After 1 h decolorization,99% of the indigo carmine had been removed by 10 mg microspheres.In addition,the immobilized CotA retained 75% of its activities after 10 consecutive cycles.The immobilized CotA presented good decolorization capability and reusability.The CotA protein showed bilirubin oxidase activity.The optimal pH and temperature for the CotA bilirubin oxidase were 8.0 and 60℃,respectively.The CotA bilirubin oxidase was more stable in the pH 7.0 than in alkaline and acidic solutions.The remarkable property of CotA bilirubin oxidase was its ability to tolerate high temperature.The half-life of CotA bilirubin oxidase was approximately 7 h at 90℃.CotA protein catalyzed the oxidation of bilirubin concomitantly with the reduction of oxygen molecule to water.The direct bioelectrocatalytic reduction of O2 was achieved on CotA modified graphite electrode,which suggested that the CotA had a potential in preparation of cathode in enzymatic biofuel cells.The direct bioelectrocatalytic reduction of H2O2 was achieved on CotA modified graphite electrode under anaerobic condition.These results indicated that the H 2O2 could be reduced to water by the catalysis of CotA,which was helpful to elucidate the mechanism of oxygen reduction by the CotA multi-copper oxidases.Based on the bioelectrocatalytic reduction,an amperometric biosensor for H2O2 was designed.The linear range of the H2O2 biosensor was from 0.05 to 4.75 mmol/L,with a detection limit of 3.1 μmol/L.The amperometric biosensor for H2O2 by CotA-modified electrode is a novel application for CotA protein.