Research On Cloning,Expression And Immobilization Of Trehalose Synthase

Trehalose has been widely used in food,medicine,and agriculture industries in recent years because of its good stability and non-specific protective effect on biological macromolecules.At present,industrial methods for producing trehalose mainly include chemical synthesis,microbial fermentation,microbial extraction,enzyme conversion and genetic transformation.The use of trehalose synthase to produce trehalose in a one-step process has the advantages of short process flow,easy to control and cheap raw materials.Therefore,trehalose synthase has an excellent development and application prospect in industrial large-scale production.Based on the above background,this study selected a trehalose synthase gene from Pyrophilus torridus at 60 ? and pH0.7,using two different expression systems: Escherichia coli and Pichia pastoris.Cloning and expression were performed,and a fast,simple,and efficient method for the immobilization of trehalose synthase was explored.The main research work is as follows:1.According to E.coli codon bias,the trehalose synthase gene(GenBank: AE017261)was optimized and a fragment of trehalose synthase(TreS)gene with a length of 1677 bp was synthesized.After double digests in EcoR I and Xho I restriction sites designed at both ends,the gene fragment was ligated into pET-28a(+)vector and the resulting plasmid pET-28a(+)-TreS was transformed into E.coli BL21 for expression.The molecular weight of the recombinant protein induced by the engineered bacteria was about 65 kDa,the optimum inducer IPTG concentration was 0.1 mmol/L,the optimal induction temperature was 20 ?,and the optimum induction time was 12 h.Thin layer chromatography(TLC)results of the reaction between crude enzyme solution and maltose or trehalose showed that the recombinant strain had the catalytic activity of catalyzing maltose to trehalose;after purification of the crude enzyme solution,high-performance liquid chromatography(HPLC)results further demonstrate that the purified enzyme also had the ability to catalyze maltose to trehalose.2.To study the effects of different expression systems on the expression level of the enzyme,the Gibson method was used to connect the trehalose synthase fragment into the EcoR I site of the plasmid pPICZ?A to construct the expression vector pPICZ?A-TreS,and then homologously recombined into the Pichia pastoris GS115 genome.In this study,methanol-induced strains were selected with high concentration of methanol.TLC results showed trehalose synthase activity was detected both intracellularly and extracellularly in the recombinant Pichia pastoris.3.Using chitosan as the carrier,the trehalose synthase expressed and purified in E.coli BL21 was immobilized.The results of single-factor tests showed that the optimum trehalose synthase addition amount was 32 mg/g(chitosan)and the optimum adsorption time was 2.5 h;the rate of residual enzyme activity is 64.64% after 9 instances of repeated experiments.Compared with the free enzyme,the thermal stability of the immobilized enzyme did not change significantly,but the pH stability is superior.The reaction process indicates that the maximum trehalose yield of this immobilized enzyme is 61.4% at the point of 2 hours throughout the reaction.