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Extracellular Thermostable Laccase-Like Enzymes from Bacillus licheniformis Strains: Production, Purification and Characterization

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Abstract

This study presents the exploration of laccase-like enzyme produced by thermophilic bacterial strains present in the Tattapani hotspring located in Chhattisgarh, India. Two bacterial isolates namely TPNR1 and TPNR6 were found to be positive for laccase-like enzyme production on screening with guaiacol as a substrate. The biochemical and 16S rRNA gene sequence analysis indicated that the isolates have similarity with Bacillus licheniformis strains and, thus, named as B. licheniformis TPNR1 and B. licheniformis TPNR6. The activity of crude enzymes was estimated using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) as a substrate and measured as 3.1 U/mL and 7.1 U/mL for TPNR1 and TPNR6, respectively. The SDS-PAGE of purified enzymes showed that the enzymes are monomers with molecular weight of 44 (TPNR1) and 38 (TPNR6) kDa. The native-PAGE technique followed by activity staining using ABTS confirms the presence of purified active enzyme with laccase-like activity in both cases. Kinetic studies displayed that catalytic efficiency of the enzymes was higher for the substrate ABTS than 2,6-dimethoxyphenol for both enzymes. The maximum enzyme activity was observed at 50°C for both enzymes while the optimal pH for TPNR1 and TPNR6 was 5.0 and 6.0, respectively. The TPNR1 exhibited half-life of 4 h at 70°C and was stable at 60°C for 180 min with a residual activity of 79%. Similarly, the TPNR6 possessed half-life of 3.1 h at 70°C and retained 88% of its activity at 60°C for 180 min. In addition, the catalytic efficiency of the enzymes was tested by decolourization of toxic dyes which showed that the both enzymes are highly potential to degrade them.

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Funding

We would like to convey our sincere thanks to National institute of Technology Raipur for providing financial assistance (NITRR/SEED GRANT/2016-17/003) to perform this research.

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Correspondence to D. Vasanth.

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The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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Sharma, V., Upadhyay, L.S. & Vasanth, D. Extracellular Thermostable Laccase-Like Enzymes from Bacillus licheniformis Strains: Production, Purification and Characterization. Appl Biochem Microbiol 56, 420–432 (2020). https://doi.org/10.1134/S0003683820040146

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