Molecular and Biochemical Characterization of Salt-Tolerant Trehalose-6-Phosphate Hydrolases Identified by Screening and Sequencing Salt-Tolerant Clones From the Metagenomic Library of the Gastrointestinal Tract

The exploration and utilization of microbial salt-tolerant enzymatic and genetic resources are of great significance in the field of biotechnology and for the research of the adaptation of microorganisms to extreme environments. The presence of new salt-tolerant genes and enzymes in the microbial metagenomic library of the gastrointestinal tract has been confirmed through metagenomic technology. This paper aimed to identify and characterize enzymes that confer salt tolerance in the gastrointestinal tract microbe. By screening the fecal metagenomic library, 48 salt-tolerant clones were detected, of which 10 salt-tolerant clones exhibited stronger tolerance to 7% (wt/vol) NaCl and stability in different concentrations of NaCl [5%-9% (wt/vol)]. High-throughput sequencing and biological information analysis showed that 91 potential genes encoded proteins and enzymes that were widely involved in salt tolerance. Furthermore, two trehalose-6-phosphate hydrolase genes, namely, tre_P2 and tre_P3, were successfully cloned and expressed in Escherichia coli BL21 (DE3). By virtue of the substrate of p-nitrophenyl-α-D-glucopyranoside (pNPG) which can be specifically hydrolyzed by trehalose-6-phosphate hydrolase to produce glucose and p-nitrophenol, the two enzymes can act optimally at pH 7.5 and 30°C. Steady-state kinetics with pNPG showed that the K _M and K _cat values were 15.63 mM and 10.04 s^-1 for rTRE_P2 and 12.51 mM and 10.71 s^-1 for rTRE_P3, respectively. Characterization of enzymatic properties demonstrated that rTRE_P2 and rTRE_P3 were salt-tolerant. The enzymatic activity increased with increasing NaCl concentration, and the maximum activities of rTRE_P2 and rTRE_P3 were obtained at 4 and 3 M NaCl, respectively. The activities of rTRE_P2 increased by approximately 43-fold even after 24 h of incubation with 5 M NaCl. This study is the first to report the identification as well as molecular and biochemical characterization of salt-tolerant trehalose-6-phosphate hydrolase from the metagenomic library of the gastrointestinal tract. Results indicate the existence of numerous salt-tolerant genes and enzymes in gastrointestinal microbes and provide new insights into the salt-tolerant mechanisms in the gastrointestinal environment.