Cloning, purification and characterization of a novel thermostable recombinant tannase from Galactobacillus...

The exorbitant production costs associated with natural tannases pose a significant challenge to their widespread industrial utilization. Microbial expression systems provide a cost-effective method for enzyme production. In this study, a putative gene encoding the subtype B tannase (Gt-Tan) was cloned from Galactobacillus timonensis and expressed heterologously in Escherichia coli BL21 (DE3) cells. The Gt-Tan was purified using metal affinity chromatography and exhibited a monomeric structure with a molecular weight of 55 kDa. Gt-Tan showed optimal activity at a temperature of 50 ℃ and a pH of 6.0. It was also quite thermostable, with approximately 68.3 % and 54.7 % of its maximal activity retained after incubation at 45 ℃ for 2 h and 40 ℃ for 48 h respectively. Addition of Mn²⁺, Zn²⁺, Al³⁺, urea, n-butanol, and dimethylsulfoxide at a low concentration slightly enhanced the activity of Gt-Tan, whereas Cu²⁺, Fe³⁺, Fe²⁺, Co²⁺, SDS, cetyltrimethylammonium bromide, DTT, Tween 80, and β-mercaptoethanol significantly inhibited its activity. K_m and k_cat/K_m values were estimated to be 0.83 mM and 19.7 s¹ mM¹ for methyl gallate, 0.67 mM and 65.4 s¹ mM¹ for propyl gallate, and 0.22 mM and 240.8 s¹ mM¹ for tannic acid. These results enhanced our understanding of tannase and provided potential sources for applications in the chemical, feed, and food industries.