The Study On Improving The Characteristics Of Fusion Enzyme By Molecular Modification

Trehalose is one of non reducing disaccharide,which is composed of two pyranosyl glucose monomers linked by 1,1 glycoside bond.Its chemical name is?-D-glucopyranosyl-?-D-glucopyranosyl??-D-glucopyranosyl-?-d-glucopyranoside?,which is called the sugar of life.The chemical properties of trehalose are very stable,and it has anti dehydration function in the organism.Therefore,under the stress conditions of drought,cold,high salinity and alkali,trehalose can protect the biofilm and protein to reduce the damage,and has a very good non-specific protective effect on the organism.The market application of trehalose is hindered because of its complex preparation process and high production cost.In this study,?-amylase and trehalose synthetase from Bacillus cereus were used as the starting points.By replacing the trehalose producing bacteria and changing the connection mode of the fusion enzyme,?-amylase trehalose synthetase fusion enzyme was constructed,and some enzymatic properties of the fusion enzyme were preliminarily explored.This research mainly includes the following aspects:.?1?Construction and expression of bifunctional fusion enzyme of?-amylase and trehalose synthetase from different sources:the genes of trehalose synthetase from Pseudomonas stutzeri,thermobaculum terrene,Pseudomonas putide P06 were cloned by PCR,and?-amylase and trehalose fusion enzyme E.coli BL21?pET-28a-bap3ts Ps?,E.coli BL21?pET-28a-bap3ts Tt?and E.coli BL21?pET-28a-bap3ts Pp?were constructed by?EAAAK?3.After the recombinant strain was induced and expressed,SDS-PAGE showed that all the four fusion proteins had been expressed.In the catalytic 6%amylose,four recombinant fusion enzymes showed dual functional activity.The conversion rates of the three fusion enzymes were 26.587%,23.473%and 23.7654%respectively after reacting with 6%amylose at 45?for 12 hours.The optimal reaction temperature was 45?,the optimal p H was 6.5,7.5 and 7.5,respectively.EDTA and CO²⁺inhibited the activity of fusion protease,while Mn²⁺and Ca²⁺did not significantly affect the activity of all fusion protease.?2?Construction of?-amylase trehalose synthetase bifunctional fusion enzyme by anti protease degradation linker:In this experiment,?PT?₈P linker was designed,pET-28a-bap1tsps was used as plasmids,restriction endonuclease Not?and Hind?were used to insert the linker between?-amylase and trehalose synthetase by T4ligase,and E.coli BL21?pET-28a-bap4tsps?,a fusion enzyme strain of?-amylase and trehalose synthetase,was constructed,SDS-PAGE showed that the fusion protein had been expressed.The optimum reaction conditions of the fusion enzyme are:temperature 45?,p H 6.5,under which the conversion rate of 6%amylose in 12hours is 86%.The analysis of circular dichroism spectrum data by cdnn software shows that the fusion enzyme replacing the connecting peptide and the original fusion enzyme Compared with E.coli BL21?pET-28a-bap3ts Ps?,the spatial structure of Ecoli BL21?pet-28a-bap4ts Ps?did not change significantly,but compared with?-amylase and trehalose synthetase,the two fusion enzymes changed significantly in reverse parallel.?3?Construction of?-amylase trehalose synthetase fusion enzyme by insertion fusion:The trehalose synthetase gene was inserted into the 446th amino acid of?-amylase gene,namely threonine and proline,and the fusion enzyme of?-amylase and trehalose synthetase was constructed and expressed in E.coli.Although SDS-PAGE showed that the fusion protein had been expressed,the fusion enzyme showed only?-amylase activity,while trehalose synthetase did not.