Expression Of Wheat Fungal Cell Wall Hydrolase And Its Effect On The Growth As Well As Toxin Production Of Aspergillus Flavus

In the process of storage,wheat is easily polluted by storage fungi such as Aspergillus flavus,which causes damage and loss of wheat quality.The accumulation of aflatoxin,a metabolite produced by Aspergillus flavus in the process of reproduction also causes food safety problems.Therefore,it is of great significance to study the growth and production of aflatoxin in wheat storage.In this study,the whole genome of Yangmai 15 was extracted.The genes of fungal cell wall hydrolase ?-1,3-glucanase,Chitinase and endo-1,4-?-mannosidase were amplified by PCR,and the coding sequences was 924 bp,894 bp and 1203 bp.After linked with pGAPZ?A and pPIC9 K,they were transferred into Pichia pastoris X-33 and GS115 for constitutive expression and inducible expression,respectively,and their biochemical and antifungal properties were characterized herein.The molecular weight of recombinant ?-1,3-glucanase is approximately 33 kDa.?-1,3-glucanase displays optimal activity at pH 6.5,remaining relatively high at pH 5.5–8.0.The optimal reaction temperature of ?-1,3-glucanase is 50 °C,retaining approximately 84% residual activity after heat treated at 50 °C for 1 h.The steady-state kinetic parameters of ?-1,3-glucanase against laminarin was determined and the Km and Vmax were 1.32 ± 0.20 mg/mL and 96.4 ± 4.4 U/mg protein,respectively.The specific activity of ?-1,3-glucanase against laminarin was 13.6 U/mg under the reaction condition of pH 6.5 and 50 ?.The molecular weight of recombinant Chitinase is approximately 36 kDa,which was higher than its deduced molecular weight owing to glycosylation.Chitinase displays optimal activity at pH 5.5.The optimal reaction temperature of Chitinase is 45 °C,retaining approximately 93 % residual activity after heat treated at 50 °C for 30 min.The specific activity of Chitinase against colloidal chitin was 29.5 U/mg under the reaction condition of pH 5.5 and 45 ?.The 3D structure modeling of chitinase indicated that it contains a chitin-binding domain at the N-terminus and a catalytic domain at the C-terminus.The molecular weight of recombinant Chitinase without binding domain is approximately 26 kDa.Chitinase without binding domain displays optimal activity at pH 5.0 and the optimal reaction temperature is 45 °C,retaining approximately 61 % residual activity after heat treated at 50 °C for 30 min.The specific activity of Chitinase without binding domain was 18.61 U/mg under the optimum reaction conditions.The molecular weight of recombinant endo-1,4-?-mannosidase is approximately 43 kDa,it shows the highest activity at pH 4.0 and 45 ?.After heat treated at 45 ? for 30 min,endo-1,4-?-mannosidase still retains about 86 % residual activity.The specific activity of endo-1,4-?-mannosidase against galactomannan was 12.13 U/mg under the reaction condition of pH 4.0 and 45 ?.The mainly hydrolysate of konjac glucomannan by endo-1,4-?-mannosidase was mannobiose,while the hydrolysate of galactomannan was mannobiose,mannotriose and mannotetraose.After 24 hours of hydrolysis,the amount of reducing sugar was 33 ?g/mL and 13 ?g/mL respectively.The recombinant Glu-GS115,Chi-GS115 and Man-GS115 were optimized in shake flask.The results showed that the optimum methanol induction concentration of the recombinant strain was 1 %,2 % and 1.5 % respectively.The best induction time was 96 h,120 h and 72 h.The inhibitory effect of purified ?-1,3-glucanase,chitinase and endo-1,4-?-mannosidase against the seven fungi commonly associated with wheat kernel was assessed in vitro.Three kind of fungal cell wall hydrolase exerted differential inhibitory effects on hyphal growth of Fusarium graminearum,Alternaria sp.,A.glaucus,A.flavus,A.niger,and Penicillium sp.Spore formation and mycelial morphology of fungal were significantly affected by fungal cell wall hydrolase.Different concentrations of fungal cell wall hydrolase had different effects on spore germination and mycelial growth of A.flavus.When the concentration of ?-1,3-glucanase reached 104 ?g/mL,the diameter of colony decreased by 19.35 %,and the growth of peripheral mycelia and spore germination were slightly inhibited.When the concentration of chitinase reached 116 ?g/mL,the growth of mycelia was more prosperous,the spore germination was completely inhibited.When the endo-1,4-?-mannosidase reached 111?g/mL,the diameter of colony increased by 16 %,the growth of mycelium was more prosperous,the spore germination was completely inhibited.The inhibition rates of ?-1,3-glucanase,chitinase and endo-1,4-?-mannosidase to aflatoxin production by A.flavus were 33.27 %,67.56 % and 33.17 %,respectively.