| Sucrose Isomerase?SIase?can translocate?-1,2-glycosidic bond in sucrose molecules to form?-1,6-glycosidic bond or?-1,1-glycosidic bond,thereby obtaining isomaltulose or trehalulose,which is also called isomaltulose synthase or trehalose synthase depending on its main product.Isomaltulose and sucrose are similar in physical properties such as sweetness and solubility.Compared with sucrose,isomaltulose has the characteristics of anti-caries,prevention from diabetes,anti-fatigue,easy to absorp,etc.The preparation of isomaltulose by bio-enzymatic can get high reaction rate,in mild reaction conditions,and easy to separation,which is consistent with the requirements of green environmental protection.In this study,based on the recombinant B.choshinensis/pNCMO2-SI constructed before,the culture mediums on the level of shake flask and 3-L fermenter were optimized,and the promoters were further screened to increase the enzyme yield.Simultaneously,the sucrose isomerase gene was expressed in the host strain Bacillus subtilis WSH11,and the two expression systems were compared.The main results are listed as follows:?1?The optimal culture conditions of recombinant B.choshinensis/pNCMO2-SI were determined by shake flask optimization:glucose 10 g·L-1,polypeptone 8.5 g·L-1,beef extract8.5 g·L-1,NH4H2PO4 2 g·L-1,after 48 h of culture,the extracellular supernatant activity was98.4 U·mL-1.The 3-L fermenter culture conditions were further optimized,and the initial carbon source was 10 g·L-11 glucose.The initial nitrogen source was 15 g·L-11 polypeptone,5 g·L-11 beef extract.Fermentation temperature was 30?.After 35 h of culture,the activity reached 275 U·mL-1.?2?In order to increase the expression level of SIase,the effects of different promoters(promoters Papr,Pnpr,Pamymy derived from Bacillus subtilis and promoter Pxylyl derived from Bacillus megaterium)on the expression of SIase by recombinant strains were investigated.The optimal promoter was Paprpr in shake flask fermentation.The corresponding recombinant BCpNapr-SI was fermented in TM medium for 48 h,and the extracellular supernatant activity was 85.1 U·mL-1,followed by Pnprpr and Pamy.The corresponding recombinant strains were BCpNnpr-SI and BCpNamy-SI,respectively,and the corresponding extracellular supernatant activities were 80 U·mL-11 and 73 U·mL-1.The extracellular supernatant activity of recombinant strain BCpNxyl-SI which was mediated by the xylose promoter Pxylyl was proved to be 38 U·mL-11 by shake flask fermentation for 48 h.?3?The fermentation process of different promoters were optimized in shake flask,and the recombinant strains BCpNapr-SI,BCpNnpr-SI,BCpNamy-SI and BCpNxyl-SI were cultivated under optimal conditions for 48 h in shake flask,the extracellular supernatant activities were proved to be 137.0 U·mL-1,126.3 U·mL-1,110.6 U·mL-11 and 47 U·mL-1,respectively,which were 1.61 folds,1.58 folds,1.52 folds and 1.23 folds of the unoptimized shake flask levels.Based on the studies above,different recombinant strains was evaluated by3-L fermenter level.The activity of extracellular supernatant activity was 485.5 U·mL-1when recombinant strain BCpNapr-SI mediated the promoter Paprpr was cultivated for 52 h,which was 1.76 folds of that of the original promoter.?4?The SIase gene was ligated with the vector pHY300PLK-CGT and vector pBSMuL-LacZ,respectively,and the recombinant plasmids pHY300PLK-SI and pBSMuL-SI were constructed and transformed into B.subtilis WSH11 to obtain recombinant strains BSpHY-SI and BSpBS-SI.The extracellular supernatant activities were determined to be 7.4U·mL-1and 10.7 U·mL-11 after 48 h of fermentation in TB medium.The culture conditions of the recombinant BSpBS-SI were optimized at the shake flask level.The results of optimal culture conditions were listed as below:bean powder 16 g·L-1,fish meal peptone 8 g·L-1,glycerol 8 g·L-1,Mg2+1 mM.Under this condition,the enzyme activity was 20.4 U·mL-11 after48 h of constant temperature fermentation at 33?.Based on the above conditions,the recombinant strain BSpBS-SI was cultured in 3-L fermenter.The feed medium was glucose 150g·L-1,bean powder 100 g·L-1,fish meal peptone 50 g·L-1,dissolved oxygen and rotating speed were coupled to ensure the dissolved oxygen DO of 30%.When the recombinant strain BSpBS-SI was cultivated for 75 h in 3-L fermenter,the cell growth concentration OD600reached 115,and the extracellular supernatant activity reached 209.5 U·mL-1. |
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