Bioinformatics Analysis,Molecular Modification And Immobilization Of ?-glucosidase

Beta-glucosidase is a cellulase that has function in the metabolic reactions of cellulose,which can decompose the cellobiose into glucose that is easier to be used by microorganisms.It can also be used to hydrolyze the aroma precursor material of fruit and tea,and thus has functions of incense.Beta-glucosidase has a very important application value in the fields of food,household chemicals,renewable resources and other fields.However,there are still many shortcomings in practical applications,such as the high cost,the low stability,the poor adaptability and so on.These factors have limited the application of ?-glucosidase.Therefore,the transformation of ?-glucosidase has a very important significance.In this study,the(3-glucosidase gene derived from Thermobifida fusca has been cloned and the mEsprit-Bglc-His plasmid has been successfully constructed.Moreover,some of the enzymatic properties of ?-glucosidase have been studied.The results show that the molecular weight of the enzyme is 55 kDa,the optimum temperature is 60?,the optimum pH is 7.0,the optimum reaction time is 30 min,and the Km of the substrate p-NPG is 0.19 mmol/L.The thermal stability,pH stability and storage stability of the enzyme are not well.To improve the stability of this beta-glucosidase,bioinformnatics analysis has been used in this study.The conserved region sequence has been found by homology comparison,and two conserved regions are selected.The structure of the sequence between the two conserved regions has been analyzed by four different sources of ?-glucosidase.The results show that the content and position of a-helix and proline in this sequence are different.Substitution of this sequence is likely to introduce higher levels of alpha-helix and proline,which increases stability.In this study,Soil metagenomic section library has been established to introduce a sequence to replace the original sequence,between the two conserved regions.Although the new?-glucosidase gene has not been obtained by screening,this study provides a new idea for the efficient development and utilization of microbial gene resources,which provides reference value for the research of related fields.In this study,(3-glucosidase has been immobilized on decanedioic acid-covered magnetic Fe3O4 nanoparticles(DMNPs)to enhance the properties of the enzyme.The optimal pH of the immobilized enzymes is 7.0,the optimum temperature is 40?.In addition,the immobilized enzymes have increased reusability,thermal stability,pH stability,storage stability,and organic solvent stability are observed when compared with the free enzymes.The immobilized enzymes have 60%residual activity after 10 cycles of reuse.The Km of the substrate p-NPG is 0.28 mmol/L,which is slightly increased compared with the free enzymes.Moreover,the reuse times are satisfactory even crude plant materials(e.g.,rice husk and corn cob)are used as substrates,which should be valuable for decomposition of natural organic materials and full utilization of bioresources.