Preparation Of Magnetic Chitosan Nanoparticles And Their Study As Immobilized Carriers For Saccharomyces Cerevisiae

Magnetic polymer microspheres are usually composed of the magnetic cores(Fe₃O₄)to ensure a strong magnetic response and a polymeric shell to provide favorable functional groups and features for various applications in cell separation,drug delivery systems, enzyme/cell immobilization and so on.Immobilization provides a favorable approach to maintain their inherent biological activity and to easy recovery for their recycled use.Thus,it is very important to design the novel carriers and choose appropriate method in the process of immobilization.(R)-mandelic acid((R)-MA)is a useful compound as a raw material or synthetic intermediate for the preparation of pharmaceutical compounds such as antibiotics(e.g.penicillium-type or cephalosporin-type)or sympathetic nerve affecting drugs(e.g. ephedrin).Therefore,the preparation of chiral mandelic acid is a challenging target.Biotransformation of phenylglyoxylic acid(PGA)to (R)-MA will be well studied by Saccharomyces cerevisiae as catalyst because of its high efficiency,high stereoselectivity,mild reaction conditions and good economic and social benefits.Therefore,in this paper,the work focused on how to design the good immobilization carders,achieve immobilization cell/enzyme and to prepare(R)-MA using Saccharomyces cerevisiae as biocatalyst.Two kinds of novel magnetic chitosan microspheres were achieved by the covalent binding of chitosan(CTS)or alpha-ketoglutaric acid chitosan (KCTS)onto the surface of Fe₃O₄ nanoparticles.CTS and the two magnetic chitosan microspheres were used as carriers to immobilize Saccharomyces cerevisiae alcohol dehydrogenase(SCAD)and Saccharomyces cerevisiae cells,Asymmetric microbial reduction of PGA to(R)-MA with the suggested immobilized SCAD or cells was also studied.The main research can be summarized as following.Magnetic Fe₃O₄/CTS nanoparticles were prepared by the covalent binding of CTS onto the surface of Fe₃O₄ nanoparticles which were prepared by hydrothermal method using H₂O₂ as an oxidizer.The functionalized magnetic Fe₃O₄/KCTS nanoparticles have been prepared by the covalent binding of KCTS onto the surface of Fe₃O₄ magnetic nanoparticles via carbodiimide activation.The products were characterized by IR,XRD,TEM,TGA,VSM.Results showed that the Fe₃O₄ magnetic nanoparticles have the spinel structure and were 22.9 nm in size.The magnetic Fe₃O₄/CTS nanoparticles and Fe₃O₄/KCTS nanoparticles were monodisperse and regular sphere with a mean diameter of 35nm and 26 nm,The saturated magnetization of composite nanoparticles were 21.5 emu·g^-1,24.8emu·g^-1,respectively.All the nanoparticles showed the characteristics of superparamagnetism and may be applied to the magnetic-field assisted drug delivery systems, cell/enzyme immobilization and many other industrial processes.A new route for preparing PGA was studied by acetophenone as the starting material after oximation by isopropyl nitrite,then hydrolysis by hydrochloric acid and sodium nitrite.Calculated on the basis of acetophenone,the final yield is 57.5%.The suitable experimental conditions were also discussed and the structure of PGA were demonstrated by IR and ¹HNMR.The content of PGA in ferments could be analyzed quickly and conveniently by colorimetry.In the aqueous solution of sodium hydroxide,PGA can coordinate with 2,4-dinitrophenylhydrazine to form a complex which has maximum absorption at 458 nm.The linear regressive equation established Y=13.512X-0.01212,R=0.9994.Thus the content of PGA can be quantitatively determined and can be used as an effective means to monitor the process of(R)-MA production by microorganism bioreduction.Immobilized of SCAD into the magnetic Fe₃O₄/KCTS nanoparticles was studied using absorption method.After absorption 60 min,the absorption rate of SCAD immobilized on the Fe₃O₄/KCTS can reach 24.87%at 25℃.And some properties of magnetic immobilized and native SCAD such as optimum temperature,optimum pH and stabilities to heat and operational stability were studied and compared.Because of its magnetic responsibility,it was simple and convenient to retrieve magnetic SCAD. The adsorption of SCAD protein on the magnetic Fe₃O₄/CTS nanoparticles was studied in a batch adsorption system.The adsorption capacities and rates of SCAD protein onto the magnetic Fe₃O₄/CTS nanoparticles were evaluated.The adsorption isothermal data could be well interpreted by the Freundlich isotherm model.The kinetic experimental data properly correlated with the Lagergren first-order kinetic model,which indicated that the reaction is the adsorption control step.The apparent adsorption activation energy was 27.62 kJ·mol^-1and the first-order constant for SCMD protein was 0.01254 min^-1at 293 K. Adsorption enthalpies of SCAD onto magnetic Fe₃O₄/CTS nanoparticles were calculated,and the results indicated that the adsorption was an endothermal process.The covalently immobilized of SCAD to magnetic Fe₃O₄ nanoparticles via glutaraldehyde coupling reaction was studied.The amino functional group on the magnetic Fe₃O₄/CTS particles surface and the amino group of the dehydrogenase were coupled with glutaraldehyde. The immobilization process did not affect the size and structure of magnetic nanoparticles.Furthermore,the immobilized SCAD enhanced thermal stability and good durability in the repeated use after recovered by magnetic separations.Bioreduction PGA to(R)-MA by SCAD belongs to the double substrate enzyme biocatalysis response with NADH as coenzyme,which follows a sequentially ordered mechanism.The reaction mechanism is: Coenzyme A(NADH)enters the enzyme's coenzyme combining site, after forming EA,substrate PGA enters the enzyme's substrate combining site again,then forms three Yuan active complexes(EAB),After completing the redox reaction,the EAB turns to the EPQ complexes, EPQ complexes release product P((R)-MA)and return to original state form coenzyme Q(NAD⁺).Following the sequentially ordered mechanism and the King-Altman method,the kinetics of the reaction was simply studied without considering the effect of other components.Result fits in Michaelis-Menton theory. v=(V_max[S])/(K_m+[S])The enzyme kinetic constants K_m and V_maxof the magnetic Fe₃O₄/CTS particles immobilized SCAD and the Fe₃O₄/KCTS nano-particles immobilized SCAD were also studied using Lineweaver-Burk pair of reciprocal method.The asymmetric microbial reduction of PGA to(R)-MA with immobilized Saccharomyces cerevisiae cells on globar chitosan was studied.The immobilization conditions and characterization of the immobilized cells were carried out.The optimum substrate concentration, pH and reaction temperature were found to be 10 mmol·L^-1,6.5,30℃, respectively,for the reaction under which the convesion of PGA and optical purity of the(R)-MA were as high as 67.86%and 98.05%e.e.The immobilized cells showed good operation and storage stability.At the same time,Saccharomyces cerevisiae cells were immobilized by using magnetic Fe₃O₄/CTS nanoparticles as carriers.Then the immobilized cells were utilized as biocatalysts to catalyze the reduction of PGA to(R)-MA.It was found that the conversion of PGA and optical purity of the(R)-MA were 29.3%and 86.69%e.e.under the optimum conditions.The optimum conditions were identified as 10 mmol·L^-1of substrate concentration at pH 6.0 and 30℃for 11 h.Being stored at 4℃for a week,the activity of immobilized cells was reduced to 3.6%from 29.3%.Immobilized cells showed better acitivity than free cells.