Study On Expression Of Trehalose Synthase Genes From Pseudomonas Putida P06in E.coli

Trehalose is one kind of non-reducing saccharide, which has very importantbiological significance, protecting biological macromolecule from extreme conditions,such as dry, high temperature, low temperature, radiation and high permeability. For thisreason, it has great application value and the market prospect.In recent years, thedomestic study of trehalose mainly concentrated in the yeast extraction and microbialfermentation, and just started in the field of molecular biology. But with more and moretrehalose synthase genes made public, it will be a hot spot that the research on trehalosein molecular biology.In this thesis, the trehalose synthase genes of pseudomonas putida was cloned andwas expressed efficiently in the vector, then the large target protein was acquired, whichavoid the control of metabolic modulation mechanism to trehalose synthase genes. Thestable recombinant was obtained and enzyme quantity and enzyme activity wasimproved significantly, lay a foundation for the future industrialized production of theformation of trehalose from maltose by trehalose synthase.In this study, with pseudomonas putida P06genomic DNA as template, by PCR,double-enzyme cleavage method, vectors connection, and introduced into EcoliBL21(ED3), the trehalose synthase genes were successfully cloned. the analysis oftrehalose synthase gene sequences and the phylogenetic tree of trehalose synthaseconstructed, has proved the homology and the conservatism of the trehalose synthasegene from pseudomonas putida. After IPTG induction of recombinant, the target proteinwas successfully expressed. The enzyme activity has reached11.84U/mL crude enzymesolution and297.6U/g dry cell weight, significantly higher than the original strain. Andafter subculture for six generation, the plasmid loss rate was20%, indicating that thegenetically engineered bacteria had genetic stability.Inducing conditions in shaking culture and key parameters of enzyme reaction hasbeen optimized and the results showed as follow: the induction temperature is25℃,inducer dosage was0.6mmol/L，the induction began at OD600of0.8; In the crudeenzyme reaction system, the optimum pH was7.5, reaction temperature35℃, reactiontime was2h, and the substrate concentration was30%. Then trehalose content ofsamples was detected by HPLC and the enzyme activity reached to318.12U/mL crudeenzyme solution. High density culture in5L fermenter was performed and the culture conditionswere explored. Fed-batch of glucose and nitrogen sources extended log phase andincreased the bacteria yield. Other conditions was as follows: fermentation time was21h, dissolved oxygen maintained above30%, and dry cell weight reached a maximumvalue of25.64g/L at21h. Then after the culture lasted for12h, the induction wasperformed for6h, so that the cell dry weight reached20.4g/L and the enzymaticactivity was159U/mL crude enzyme solution.