Characterization of cold-tolerant trehalose-6-phosphate synthase from the deep-sea bacterium <i>Microbacterium sediminis</i> YLB-01

A gene encoding the enzyme trehalose-6-phosphate synthase (TPS), which is part of the TPS trehalose synthesis pathway, was cloned from the deep-sea psychrotolerant bacterium <i>Microbacterium sediminis</i> YLB-01 and expressed in <i>Escherichia coli</i> BL21. The exogenously expressed TPS exhibited highest similarity (80.93% identity) to <i>Microbacterium</i> sp. TPS. The purified recombinant TPS was cold-tolerant, with low thermostability. The optimum temperature for TPS activity was 40°C, and the enzyme retained 72.6% of its maximal activity at 4°C. The optimum pH was 7.5. TPS activity was cation-dependent, with Mg²⁺, Co²⁺, or Ba²⁺ being essential for maximum activity. The kinetic constants of the recombinant TPS reaction rates confirmed that it was cold-tolerant. Molecular dynamics analysis showed that TPS was more flexible (0.8741Å) at 4°C than 1GZ5, its homolog in the mesophilic bacterium <i>E. coli</i>, and superposition of the 3D enzyme structures supported this. Enzymatic properties of recombinant trehalose-6-phosphate synthase (TPS).