Hydrolysis Activity Of Merrifield-chitosan-resin Complexed With Metal Ions For Phosphate Bond And Peptide Bond

Hydrolysis of phosphate bond and peptide bond has an important theoretical significanceand application value in the field of food processing, biopharming, and so on. To design anartificial enzyme with high stability and catalytic activity is an important direction in futurecatalysis research. Chitosan is the second largest renewable resources with the reputation ofuniversal polysaccharide, in which great deals of active groups are contained and can bemodified into various functional materials.In this study, merrifield chitson resin-supported cyclen (MCRC) was prepared byattaching1,4,7,10-tetraazacyclododecane (cyclen) to merrifield chitson resin (MCR). Thencoordinated with Mg(II), Cu(II), Zn(II), Fe(II), Fe(III), Ce(III), Ce(IV) to be differentimmobilized artificial enzymes, respectively. Their physicochemical characteristics andhydrolysis activity were investigated and the conclusions were as follows:1）The hydrolysis activity toward phosphomonoester bond and peptide bond wereevaluated by investigating the hydrolysis of PNPP Na2and Bovine Serum Albumin (BSA),which turned out to be that MCRC-Ce(III) was superior to other solid artificial enzymes.SEM, FTIR, DSC and XRD were applied to describe the surface detail and structure ofMCRC-Ce(III), revealing that the spherical MCRC-Ce(III) had a smooth surface and theCe-cyclen skeleton was successfully attached to the chitosan resin. The average diameter ofMCRC-Ce(III) is500μm, which ensures a high mobility and specific surface area inheterogenous system to accelerate the intense interaction between substrates and active sites.2） The hydrolysis activity of MCRC-Ce(III) toward phosphomonoester bond andphosphodiester bond were evaluated on PNPP Na2and pBR322plasmid DNA. The resultsindicated that PNPP Na2can be hydrolyzed by MCRC-Ce(III) at physiological pH, and theapparent rate constant was8.39×10-2M·min-1, which is807times as much as its ownhydrolysis rate on the same condition. The catalytic ability could maintain97.8%of theoriginal after being reused for6times. In addition, MCRC-Ce(III) was found to promote cleavage of pBR322plasmid DNA from supercoiled form to the open circular form andlinearized form under physiological conditions.3）The proteolytic activity of MCRC-Ce(III) was evaluated on BSA, myoglobin (Mb)and lysozyme (LZM). The results demonstrated that under phyisiological conditions, BSA andMb could be hydrolyzed more than88%and83%during24h and12h at60℃respectively,while LZM can be hydrolyzed up to97%during10h at50℃. The pseudo-first-order kineticconstant (Kobs) of hydrolyzing BSA, Mb and LZM were7.05,16.70and36.80×10-2M·h-1,while the Vmaxwere17.56,22.46and37.60×10-2M·h-1, respectively. The high hydrolysisactivity of MCRC-Ce(III) demanded for the cooperation of different parts, and the kineticscomply with Michaelis-Menten mechanism. MCRC-Ce(III) had a strong stability that morethan80%of the original catalytic ability maintained to BSA after being reused for6times,and90%to Mb. The optimum pH for the proteolytic activity of MCRC-Ce(III) was6.0, andhigh reaction rate was obtained within the scope of pH5.0~9.0, consistent with the pH rangein conventional food.In conclusion, as a new type of immobilized artificial metalloenzyme, MCRC-Ce(III)could hydrolyze phosphate bond and peptide bond efficiently, and could break the limitationof natural enzymes on pH and temperature, revealing a broad large-scale application prospect.