| ?-galactosidase??-Gal?is a kind of enzyme which is widely used in food industry.In recent years,the application and development of?-Gal are mainly restricted by the factors such as low stability,poor tolerance of organic solvents,low reuse rate and so on.These factors limit the further application of?-Gal in food industry.The surface display technique of?-Gal on spores of Bacillus subtilis is a kind of immobilization technique of immobilized?-Gal on surface protein of spores.Because of the high stability and resistance of Bacillus subtilis spores,spores can become an excellent carrier for immobilization.However,the low activity of?-Gal on spores has always restricted the development of the biocatalyst.In this study,three different display mechanisms were used to display thermostable?-Gal from Bacillus thermophilus on the surface of Bacillus subtilis spores.Optimization of spore display mechanism,screening the most suitable bio-enzyme preparation for food industry.Five surface display systems of?-Gal spores,based on the principle of cohesin-dockerin interaction,were successfully constructed by selecting cohesin modules and dockerin modules of type?,type?and type?from different anaerobe:?-Gal-Doc?-1/CotG-Coh?-1,?-Gal-Doc?-2/CotG-Coh?-2,?-Gal-Doc?-3/CotG-Coh?-3,?-Gal-Doc?-1/CotG-Coh?-1,?-Gal-Doc?-1/CotG-Coh?-1.The enzyme activities showed were 1.9,1.86,1.97,1.71,1.64 U/mg?dry weight?,respectively.At the same time,direct fusion display system,CotG-?-Gal,1.83 U/mg spore?dry weight?and?-Gal spore surface display system without anchored protein by Pcry1Aa promoter,Pcry1Aa-?-Gal,2.48 U/mg spores?dry weight?.Immunofluorescence microscopy and Western blot analysis confirmed that the above-mentioned?-Gal spore surface display system was successfully constructed.?-Gal-Doc?-3/CotG-Coh?-3 and?-Gal-Doc?-1/CotG-Coh?-1,CotG-?-Gal and Pcry1Aa-?-Gal are three kinds of?-Gal spore surface display systems based on different display mechanisms.The number of?-Gal molecules fixed on the surface of each group of spores was determined by semi-quantitative dot blotting.The number of?-Gal molecules on the single spore surface of Pcry1Aa-?-Gal was 1.3×105,and that of CotG-?-Gal was 4.3×104,The values of?-Gal-Doc?-3/CotG-Coh?-3 and?-Gal-Doc?-1/CotG-Coh?-1 were 3.0×104 and 2.8×104,respectively.The difference between display amount and apparent activity corresponds to the space distance between?-Gal and spores are caused by different display mechanisms,indicating that steric hindrance is the key factor affecting enzyme activity.Studies on the enzymatic properties of recombinant spores?-Gal-Doc?-3/CotG-Coh?-3,?-Gal-Doc?-1/CotG-Coh?-1,CotG-?-Gal,Pcry1Aa-?-Gal and free?-Gal?dissociated from the surface display system of non-anchored protein spores?.The optimum pH and temperature for showing?-Gal on different spores were 6.0 and 75?respectively.The thermalstable:CotG-?-Gal>?-Gal-Doc?-3/CotG-Coh?-3 and?-Gal-Doc?-1/CotG-Coh?-1>Pcry1Aa-?-Gal;Anti-solvent property:CotG-?-Gal>?-Gal-DocI-3/CotG-CohI-3 and?-Gal-Doc?-1/CotG-Coh?-1>Pcry1Aa-?-Gal;After 6 cycles of recycling,the enzyme activity of all kinds of recombinant spores did not lose much,and the residual enzyme activity was kept at about 60%;The Km values of?-Gal-Doc?-3/CotG-Coh?-3 and?-Gal-Doc?-1/CotG-Coh?-1 were 3.83 mM and 3.71 mM,CotG-?-Gal4.43 mM,Pcry1Aa-?-Gal 5.71 mM,respectively.The results showed that the increase of the steric barrier between?-Gal and coat protein CotG was beneficial to the improvement of substrate affinity and the efficiency of enzyme activity display.However,the enlargement of space distance leads to the excessive exposure of?-Gal to the external environment,resulting in a weaker stability than the direct fusion type.In conclusion,compared with?-Gal-Doc?-1/CotG-Coh?-1 and Pcry1Aa-?-Gal,direct fusion display type CotG-?-Gal and scaffolding protein display type?-Gal-Doc?-3/CotG-Coh?-3 have high enzyme activity and excellent enzymatic properties,which are very suitable for the field of food industry catalysis. |
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