Construction And Expression Of MTSase And MTHase Fusion Strains

Trehalose is a non-reducing disaccharide derived from two molecules of glucose by?,?-1,1-glycosidic linkages,it has good non-specific protective effect on the organism.It is widely used in pharmaceuticals,foodstuffs and cosmetics,which has a prospect vision of application.At present,the most common method for the production of trehalose is extraction,but the yield too low to meet the current market requirements.In this paper,maltooligosyl trehalose synthase?MTSase?and maltooligosyl trehalose hydrolase?MTHase?were studied,by inserting different binding peptides as well as different fusion sequences,MTSase,MTHase fusion enzyme strains were constructed and their expression were analyzed.It was proved that the strain of Arthrobacter oxydans contained trehalose synthesis related enzymes MTSase and MTHase after fermentation validation.The mtsase gene sequence was 2328 bp length,it encoded 775 amino acids and the mthase gene was 1770 bp length and encoded 589 amino acids.NCBI data showed that the similarity with Arthrobacter phenanthrenivorans Sphe3 was 87%.The target gene mtsase and mthase were obtained by PCR and ligated by the expression vector pET-22b to achieve the expression of mtsase and mthase in Escherichia coli.The activities of recombinant MTSase and MTHase were 17.85U/mL and 15.16 U/mL,respectively.The two enzymes was incubate in 4%amylose as the substrate for 24 h?40°C,pH 7.0?and the conversion of trehalose was 45.87%.In the present study,?GGGGS?₂ and?EAAAK?₃ were selected as the linker peptide,and mtsase-mthase and mthase-mtsase were designed to achieve the connection.Four strains of MTSase and MTHase fusion strains were successfully constructed.The fusion protein was detected by SDS-PAGE and the molecular weight was about 150 KDa.Nevertheless,four fusion proteins showed as biofunctional enzyme,which can catalyze the production of trehalose.Among them,the fusion enzyme SP2H has a high activity of 11.53 U/mL.The fermentation medium M9-ZJ-LM-X was obtained by optimizing the fermentation medium,and the enzymatic properties of the fusion enzyme were studied and the optimal reaction temperature was 50?,pH 6.5.The activity of the fusion enzyme was 24.13 U/mL after 20 h of incubation in 4%amylose?50°C,pH6.5?,the yield of trehalose was 43.43%,which was 2 times higher than that of the LB condition and 5 times higher than original strain,compared with the mixed enzyme system,although it did not improve the conversion rate,the reaction time shortened by nearly 4 h.