The Construction And Application Of Recombinant Bacillus Subtilis Displaying Trehalose Synthase On The Spore Surface

Surface display is a molecular biological technology for foreign immobilization of polypeptides and proteins on the surfaces of phages,cells or spores.In addition,the genome sequencing of Bacillus subtilis has been completed,which is a food-safety strain.The spore generated by Bacillus subtilis is also highly safe and more convenient for the construction of spore surface display system.Trehalose has the biological functions of protecting macromolecular organisms in extreme environment.At present,the methods of trehalose mass production are enzymatic,including double enzymatic and single enzymatic methods.Single enzymatic method is that maltose is isomerized into trehalose under the action of trehalose synthase(TreS).The process of this method is simple and easy to operate,and it has attracted the attention of scientists all over the world.In view of the above content,this paper carried out the following research work:(1)11 kinds of spore capsid proteins were selected and displayed Enhanced Green Fluorescent Protein(EGFP)on the surface of B.subtilis WB800n,respectively.Western Blot and Immunofluorescence analysis showed that EGFP was successfully displayed on the spore surface of recombinant spores of 11 strains,respectively.The relationship between the fluorescence intensity of EGFP displayed by different capsid proteins was as follows:CotX>CotY>CotF>CotC>CotE>CotG>CotV>CotB>CotM>CotA>CotW.(2)According to the fluorescence intensity displayed by different spore capsid proteins,CotX,CotY,CotF,CotC,CotE and CotG were selected as the molecular carriers to construct the recombinant Bacillus subtilis displaying trehalose synthase TreS on the spore surface.The enzyme activities on the surface of the dry heavy spore of the 6recombinant strains were measured as 3687.64 U/g(cotX),1791.16 U/g(cotY),862.18U/g(cotF),886.11 U/g(cotC),621.96 U/g(cotE),484.51 U/g(cotG),which were basically consistent with the measured of green fluorescence intensity.(3)Using both CotC and CotG as molecular carriers,recombinant Bacillus subtilis was constructed to display TreS on the spore surface.By confocal laser microscopy,Western Blot and enzyme activity analysis,it was proved that the TreS was successfully displayed on the spore surface of Bacillus subtilis.After 96 h shaking flask fermentation in TB medium,the enzyme activity on the surface of dry heavy spore of recombinant bacteria displaying TreS was determined to be 1511.6 U/g.The molecular numbers of TreS displayed by CotC and CotG respectively and together on single spore were2.84×10~9,1.38×10~9,4.65×10~9by Dot Blot analysis.By optimizing the TB medium,the total number of fermentation bacteria was 1.66×10~9 cfu/mL,the number of spores was1.50×10~9 cfu/mL,and the spore production rate was up to 90.36%.(4)The spore germination genes sleB and cwlJ were knocked out,so as to control the germination of recombinant spores displaying TreS effectively in the transformation system.Through the transformation of recombinant B.subtilis WB800n(?sleB,?cwlJ)/cotC-treS-cotG-treS spores to maltose under different conditions,it was proved that the TreS displayed on the spore surface had good thermal stability and pH tolerance,and could maintain high enzyme activity after four cycles of transformation.