Tannase Production From Aspergillus Niger Bysolid-state Fermentation Of Tea Stalks And Tannase Immobilization On Magnetic Nanoparticles

Tannin acyl hydrolase,commonly referred as tannase,is an attractive biocatalysts for biodegradation of tannins.Tannase has extensive application prospect in various industries,especially in beverage and pharmaceutical industries.In the present work,tannase production by solid state fermentation and tannase immobilization on carboxyl functioned magnetic Fe₃O₄nanoparticles?CMNPs?were investigated.First of all,tannase production by non-heat-treated solid state fermentation using tea stalks as fermentation substrate was explored.Next,high density polyurethane sponge,a flexible polyurethane foam,was used as a novel inert carrier for tannase production under a modified solid state fermentation system.Moreover,using this system,the additional nitrogen and carbon sources concentration were optimized by response surface methodology using Box and Behnken factorial design to determine the optimum conditions for tannase accumulation.Furthermore,the process parameters of tannase immobilization on carboxyl functioned magnetic nanoparticles?CMNPs?were studied.And the physical properties of the tannase-CMNPs were investigated as well.Finally,chemical properties of the immobilized tannase and free tannase were evaluated simultaneously.?1?Tannase production from Aspergillus niger by non-heat-treated solid state fermentation using tea stalks as culture substrate was studied.Compared with tannase production of heat-treated group,the production of non-heat-treated solid state fermentation increased by3.2-fold,and supplementation with tannic acid was confirmed to have no influence on tannase production.Meanwhile,extra addition of inorganic salts,carbon and nitrogen sources had synergistic effect on tannase formation.At the same time,supplementing sucrose obtained higher tannase yield than other carbon sources,but decreased enzyme productivity at the initial stage of fermentation.Also,additional ammonium chloride supplementation played an important role in tannase synthesis process.It not only increased tannase production,but also shortened the fermentation time dramatically.Moreover,being supplemented with 7%?w/w?sucrose and ammonium chloride can obtain highest tannase harvest?23.6 U/gds?.?2?High density polyurethane sponge was used as the inert carrier to adsorb tea stalks infusion to produce tannase as a modified solid state fermentation system.And there was no distinct decrease on tannase production when the polyurethane sponge particles were recycled for 5 cycles.It was shown that the maximum tannase activity were obtained when Aspergillus niger was incubated for 96 h,the side length of polyurethane sponge cubes were 0.2 cm,tea stalks extract content 95%,initial pH 4.0,inoculum size 6.4×10⁷ spores/gds,incubation temperature 30 ^oC,and tannic acid and ammonium sulfate were used as the additional carbon sources and nitrogen sources,respectively.However,the peak of tannase productivity was obtained when the side length of polyurethane sponge cubes was 0.2 cm,tea stalks extract content 90%,initial pH 5.0,supplementation with tannic acid and ammonium sulfate,inoculum size 6.4×10⁷ spores/gds,incubated at 30 ^oC,for 96 h.Furthermore,biomass accumulation were remarkable promoted by additional glucose and yeast extract.Undesirably,the yeast extract made a forceful suppress effect on tannase production.?3?On the base of single-factor experiment,tannic acid and glucose were determined as the carbon sources.Meanwhile,ammonium sulfate and yeast extract were affirmed as the nitrogen sources.Combination of carbon and nitrogen sources,namely the additive amount of tannic acid,glucose,ammonium sulfate and yeast extract were studied using the Box-Behnken Design methodology.The response surface results indicated that the maximum tannase activity was15.43 U/gds when the additive amount of tannic acid,glucose,ammonium sulfate and yeast extract was 7.49%,8.11%,9.26%and 2.25%,respectively.Maximum yield of tannase was obtained when MSSF was carried out at 30°C,90%of tea stalks extract,additional 7.49%tannic acid,8.11%glucose,9.26%ammonium sulfate and 2.25%yeast extract,initial pH 5.0,inoculum size 6.4×10⁷ spores/gds,after 120 h of incubation.The highest tannase activity of optimized condition was 6.36 times over the initial condition.?4?Tannase was immobilized onto CMNPs?about 10 nm?by crosslinking with glutaraldehyde.The best efficiency of enzyme immobilization was carried out at 31°C,3.5%glutaraldehyde,1.75 U/mL free tannase,220 rpm,pH 7,after 26 h of immobilization with 20 mg of CMNPs.The physical properties of the tannase-CMNPs were systematic investigated.The results of Fourier transform infrared?FTIR?and thermogravimetric analysis?TGA?indicated that the free tannase was successfully combined with the CMNPs.Analysis result of X-ray diffraction?XRD?showed that the crystal structure of CMNPs had no obvious changed after enzyme immobilization process.Tannase-CMNPs was observed by scanning electron microscope?SEM?and transmission electron microscopy?TEM?,the electron microscope photographs showed that the tannase-CMNPs was irregular three dimensional structure.According to the vibrating sample magnetometer?VSM?analysis results,both the CMNPs and tannase-CMNPs were superparamagnetic nanoparticles,however,the saturation magnetisation value of tannase-CMNPs only half of CMNPs.Zeta potentiometric analysis indicated that tannase-CMNPs has very good stability in aqueous phase.The average grain diameter of tannase-CMNPs?362 nm?was significantly higher than the CMNPs when both they were suspended in deionized water.?5?Chemical properties of the immobilized tannase and free tannase were investigated simultaneously.The results indicated that the optimal reaction temperature of immobilized tannase and free tannase were found to be 50°C similarly.However,the optimal reaction pH were 6.0 and 7.0,respectively.Comparing with the free enzyme,the immobilized tannase exhibited better stability to temperature,pH,and storage.Immobilized tannase activity was stimulated by 1 mM Fe²⁺,5 mM Mg²⁺,and 10 mM Mn²⁺,on the contrary,Ca²⁺,Zn²⁺,Cu²⁺,and10 mM Al³⁺showed significant inhibiting effect.SDS,EDTA,urea,tween 80,and Triton X-100were also decreased immobilized enzyme activity.Then,the kinetic parameters of free and immobilized tannase were determined.K_m values of the free and immobilized tannase was 0.29mM and 0.45 mM,respectively.V_max values changed from 0.12 to 0.26?M·min^-1 after immobilization.After 6 cycles for catalytic hydrolysis of propyl gallate?PG?,the immobilized tannase still maintained more than 60%of its initial activity.