Heterologous Expression And Characterization Of ?-xylosidase In Aspergillus Oryzae NSAR1

Ginsenoside Rd has obvious neuroprotection,anti-inflammatory,anti-aging,antioxidant and other activities.At present,Rd are mainly extracted from Panax ginseng,Panax notoginseng and Panax quinquefolius.But the high cost limits the scope of industrial application.However,the production of recombinant microbial enzyme has the advantage of lower cost.This thesis clone the ?-xylosidase( ?-anxyl)gene from A.niger NG1306 strain and construct an expression vector.And then introduce it into the A.oryzae NSAR1 strain,aiming to take advantage of the heterologous expression of closely related species to explore expression and characterization of ?-xylosidase in A.oryzae.In this thesis,firstly,genetically stable A.oryzae pyr G auxotrophic strain was constructed by the ultraviolet radiation mutagenesis method using A.oryzae RIB40 strain as the starting strain.Secondly,the ?-anxyl gene was transformed into A.oryzae by protoplast method and the engineering strains was induced by starch to produce enzyme.Then the activity of target protein against synthetic substrate p-nitrophenyl- ?-D-pyranoside(p NPX)and natural substrate ginsenoside Rb₃ was detected by enzyme-plate assay and TLC.Finally,the target protein was purified by the combined method of(NH₄)₂SO₄ step-by-step precipitation and ultrafiltration for enzymatic characterization.The results of this paper are as follows:(1)No pyr G mutant strain was screened by the ultraviolet radiation mutagenesis method.(2)The recombinant plasmid p TAex3- ?-anxyl CHis was constructed by cloning ?-anxyl gene.(3)The p TAex3- ?-anxyl CHis plasmid was successfully transformed into A.oryzae NSAR1.The ?-xylosidase secreted by the engineered strain had strong activity on p NPX and could converted ginsenoside Rb₃ to Rd.(4)The purified ?-xylosidase protein was about 110 k Da.(5)The specific activity of ?-xylosidase is 147 U/mg,and the K_m,V_max and K_cat values using p NPX as the substrate under the optimal conditions(p H 4.0,60°C)are:4.86 m M,373?mol·min^-1·mg^-1,10.25 s^-1,respectively.It can tolerate 20%ethanol.The ?-xylosidase was heterologously expressed by A.oryzae NSAR1 showed activity against both p NPX and ginsenoside Rb₃.Compared with literature reports,the specific activity of the enzyme was greatly improved.This study broadens the scope of heterologous expression of exogenous genes in A.oryzae,and provides a basis for the industrial application of ?-xylosidase.