Trehalose Synthase Produced By A Strain Of Polar Bacteria Pseudomonas Putida S1

Trehalose is a nonreducing disaccharide of two glucopyranosyl residues bonded with 1,1-α- glucoside linkage. As an important stress protectant in living organisms, trehalose has unspecific protective effects to life, biomembranes and biomolecules. Owing to its unique structure and biological functions, trehalose could be widely used in food industry, pharmaceutical industry, cosmetic industry and agriculture etc. Trehalose synthase could catalyze the conversion of maltose into trehalose by intramolecular transglucosylation, which is one of the most important treha1ose biosynthesis pathways and one of the enzymatic production methods of trehalose with industrial values. Trehalose synthase was mainly studied in thermophiles and mesophiles, but seldom in cold-adapted microorganisms. With unique geographical and climatic features such as extremely low-temperature, high salinity, and so on, the polar region is an important source of cold-adapted microbes. A trehalose synthase-producing strain of cold-adapted bacteria was screened from polar bacteria and its microbiology, molecular biology and enzymology were studied in this paper. The aims of the paper are to get novel trehalose synthase-producing strain with own intellectual property rights from new sources and established the theoretical and technological foundations for using this strain in industrial production of trehalose, and also to provide molecular biology and enzymology information of trehalose synthase from various kinds of microorganisms.A trehaolse synthase- producing strain of antarctic oceanic bacteria, S1, was screened from 83 strains of polar soil bacteria and 20 strains of polar oceanic bacteria. This strain of bacteria could produce trehalose from maltose with a high conversion rate of 70~76% when crude enzymes were used and 80~82% when partial purified enzymes were used. With its cell morphology, culture characteristics, physiological and biochemical characteristics,16S rDNA sequence analysis and phylogenetic characteristics, the strain S1 was identified as Pseudomonas putida S1 in accordance with the polyphasic taxonomy. Its growth characteristics showed that P. putida S1 was a strain of psychrotolerants and halotolerants and its trehalose synthase was an alkaline cold-adapted enzyme.To treat P. putida S1 with heat shock (30 min at 25℃or 30℃) or salt stress ( by adding 0.5 ~ 0.6 mol / L of NaCl ) after cultivation at 20℃for 20 h could promote its trehalose synthase production by 70-76% and 90-92% respectively. But cold shock treatment ( 30 min at 5℃, 0℃and -17℃) did not promote trehalose synthase production. Heat Shock and salt stress has no obvious collaborative effects.TreS,The gene encoding trehalose synthase,was cloned successfully from P. putida S1 with PCR method。The nucleotide sequence analysis of the gene revealed that treS has a 2067bp open reading frame, which encodes a protein of 688 amino acid residue with the molecular weight of about 75.7 KD. The results of Blast in Genbank showed that the homology of trehalose synthase from P. putida S1 and those from other strains of Pseudomonas was more than 70%. However, the homology of trehalose synthase from P. putida S1 and some other reported trehalose synthase, such as those from Pimelobacter sp. R48 and the Thermus aquaticus, was below 30%. The recombinant E.coli M15/pQE30T-TS, which could overexpress treS from P. putida S1, was constructed by inserting treS gene into vector pQE30T and then transformed it into E. coli M15. The activity and special activity of trehalsoe synthase in the cell lysate of IPTG-induced E.coli M15/pQE30T-TS were 18.1u/ml and 5.36 u/mg protein, about 50-fold and 120-fold higher than the lysate of the parent strain Pseudomonas putida S1 respectively.The recombinant trehalose synthase was purified from E.coli M15/pQE30T-TS by affinity chromatography on Ni-NTA column. The optimum temperature for enzymatic reaction was 25℃and the enzyme was stable below 35℃. The recombinant trehalose synthase showed a maximum activity at pH 8.5 and was stable at a pH range of 7.5–9.0. The addition of 1mmol/L of Hg2+, Cu2+, Fe3+ and Zn2+ inhibited the enzyme activity at various degrees and Tris-HCl strongly inhibited the enzyme activity. The Km and Vmax values of the purified recombinant trehalose synthase were estimated to be 10.2mmol/L and 27.0μmol/mg protein/min for maltose as substrate, and 87.5mmol/L and 26.0μmol/mg protein/min for trehalose. These results revealed that the enzyme had a higher affinity for maltose.The trehalose synthase from P. putida showed differences from other reported trehalose synthase in nucleotide sequence, amino acid sequence and enzymological properties. These reveled that the trehalose synthase from P. putida was a novel enzyme. The nucleotide sequence of its gene was submitted to and accepted by Genbank and the Accession Number was EU310374.