Production of highly thermo-tolerant laccase from novel thermophilic bacterium Bacillus sp. PC-3 and its application in functionalization of chitosan film.

In this study, a novel thermophilic bacterial strain was isolated from Tattapani hot spring located in the Chhattisgarh state of India. The laccase was produced via submerged fermentation and purified by ammonium sulfate precipitation and anion exchange chromatography up to 13.7 fold. The 16S rRNA gene sequence and biochemical analysis revealed that the bacterial isolate is Bacillus sp. strain PC-3. The activity of extracellular crude laccase was determined to be 11.2 U/mL using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate. The SDS-PAGE revealed that the enzyme consists of single subunit with molecular size of 36 kDa. The laccase exhibited the maximum enzyme activity at temperature of 60°C and pH 7. Moreover, the laccase retained 99.1% of its original activity for 180 min and exhibited half-life of 3.75 h at 60°C. Similarly, the laccase retained 95% activity at pH 7 for 240 min and displayed significant activity at wider pH range. In addition, the laccase was used for functionalization of chitosan film and characterized for antioxidant and antimicrobial activity. Interestingly, the functionalized chitosan film showed the improved antioxidant and antimicrobial activity.