The Study Of Salt Tolerance Enzymes,Genes And Trehalose-6-Phosphate Hydrolase From The Gastrointestinal Tract Metagenome

Salt-tolerant enzymes and genes play an important role in the growth and metabolism of microorganisms in high-salt environments.And they are widely used in biotechnology fields such as seafood processing and washing.Hyperosmosis gastrointestinal cavity?equivalent to 0.3 mol/L?and dietary changes can lead to the production of osmotic pressure in the gastrointestinal tract.Therefore,gastrointestinal tract microorganisms may contain a wide variety of salt tolerance enzymatic and genetic resources.In this paper,the metagenome of the Nycticebus pygmaeusis and Bos frontalis gastrointestinal microbiome was studied.Screening and sequencing of salt-tolerant clones from constructed metagenomic libraries,analysis of potential salt-tolerant enzymes and genes,heterologous expression and identification of trehalose-6-phosphate hydrolase were performed to explore salt-tolerant enzymes and genes in the metagenome of gastrointestinal microorganism.It lays a theoretical foundation for exploring the salt-tolerance mechanism of gastrointestinal microorganisms and for the development and utilization of salt-tolerant enzymes and genes in this environment.The findings are summarized as follows:1.Screening,sequencing and analysis of salt-tolerant clones in metagenomic library of gastrointestinal microorganismsWith 7%NaCl?w/v?as the screening factor,4?1A,3-1,5₁₁ and 5₁₅?and 6?21₉A,24₇H,16₂E,16₂D,16₈H and 1₂G?clones with stronger salt-resistant ability were obtained and used to high-throughput sequencing and bioinformatics analysis from the N.pygmaeusis and B.frontalis gastrointestinal metagenome,respectively.A total of 1285 genes and enzymes were obtained from salt-tolerant clones,of which 91 were potential salt-tolerant genes and enzymes.2.Functional annotation of genes derived from salt-tolerant clonesGO analysis showed that most genes are associated with biological processes including metabolic and cellular processes.Based on the annotation against the COG database,these genes are mainly associated with carbohydrate transport and metabolism,transcription,replication,recombination and repair,inorganic ion transport and metabolism,defense mechanism,mobilome,prophages,and transposons.The KEGG analysis of the salt tolerant clones showed presumed components of carbohydrate metabolism,membrane transport,drug resistance,and nucleotide metabolism which perhaps implicated diverse metabolic pathways related to salt tolerance.3.Heterologous expression,purification and identification of 6-phosphate trehalose hydrolaseThe trehalose-6-phosphate hydrolases obtained by sequencing analysis were selected for further heterologous expression and purification.Two recombinant trehalose-6-phosphate hydrolases,namely,rTRE P2 and rTRE P3,were successfully obtained.The two enzymes can act optimally at pH 7.5 and 30°C.Both rTRE_P2 and rTRE_P3 are typical salt-resistant enzymes whose enzyme activity increases with the increasing NaCl concentrations.The activity also increased after 1 h of incubation with the involvement of 0.5–5 mol/L NaCl,and this trend remained after 24 h.The activity of rTRE_P2 increased by approximately 43-fold even after 24 h of incubation with 5 mol/L NaCl.Both activities were completely inhibited by 1 mmol/L PbCl₂,CTAB,AgNO₃,CuSO₄,HgCl₂ and ZnSO₄.rTRE_P2 and rTRE_P3 can cleave p-Nitrophenyl-?-D-glucopyranoside?pNPG?and trehalose-6-phosphate effectively.Steady-state kinetics with pNPG showed that the Km and Kcat values were 15.63mmol/L and 10.04 s^-11 for rTRE_P2 and 12.51 mmol/L and 10.71s^-11 for rTRE_P3,respectively.