Using Thermoresponsive Suppotrs To Adsorb And Release Enzymes Repeatedly

Catalysts can be simply classified into two types: homogeneous andheterogeneous. Homogeneous catalysts usually show excellent activity （new worldrecord of turnover number:4,550,000） and selectivity but troublesome separation andrecycling leading to large wastes against green chemistry. Although heterogeneouscatalysts have the merit of easy recovery, in many cases they are less active than theirhomogeneous counterparts. Ideally, the advantages of homogeneous andheterogeneous catalysts should be combined.Based on the progress in artificial molecular chaperone, this paper proposes anew strategy. When the reaction is on, active species catalyze the reactionhomogeneously and there is almost no interaction between the active species and thesupport. When the reaction is off, the species are adsorbed on the support by heatingin a heterogeneous state. After recovery, the species are released from the support bycooling, and go into next round of reaction. Herein, we describe a model systememploying thermoresponsive Au@PNIPAM nanoparticles as supports that can adsorband release enzymes （papain, catalase） repeatedly.Gold nanoparticles are prepared by citrate-mediated reduction of HAuCl4. Then,ligand-exchange reactions are performed by mixing citrate-protected goldnanoparticles colloids with thiol-terminated PNIPAM which is prepared by RAFTpolymerization. Characterization by XPS, NMR, IR, TGA and UV-Vis confirms theformation of Au@PNIPAM. DLS and TEM show its thermo-responsiveness.The adsorption rate of papain increases from11.7%at25°C to90.0%at50°C,which is ascribed to the transition of PNIPAM from hydrophilic to hydrophobic state.The papain adsorbed at50°C can be released after the system is cooled to25°C. Theadsorption-release process can be cycled at least three times.When catalyzing the casein hydrolysis at25°C,“papain+Au@PNIPAM” systemshows93.4%relative activity. After the reaction, the reaction mixture is heated to50°C. Then, papain/Au@PNIPAM is recovered by centrifugation and transferred into fresh buffer. There is80.7%papain released from Au@PNIPAM in RUN2, with83.1%relative activity. In RUN3,62.0%papain is released, with62.6%relativeactivity.Then, we apply this strategy in the catalase reaction. In RUN1,94.9%catalaseis not adsorbed on Au@PNIPAM, with the activity almost equal to that of freecatalase. More than90%catalase is adsorbed on Au@PNIPAM at50°C, while74.4%and49.5%catalase is released in RUN2（relative activity78.0%） and3（relativeactivity53.2%）, respectively.The Au core of Au@PNIPAM plays no role in adsorption-release process, and itis also too expensive to be widely used in industrial process. Therefore,Au@PNIPAM is superseded by SiO₂@PNIPAM. Unfortunately, SiO₂@PNIPAMdoes not fit for this strategy because it only adsorbs36%papain under optimumconditions.Besides, this paper employs mesoporous SBA-15as support to immobilizepapain. Michaelis constant shows the papain immobilized on carboxyl-modifiedSBA-15has a higher affinity for substrate casein than free enzyme. The ionic liquid[Simim+][Cl] is beneficial to improve the activity of the immobilized papain becausethe papain immobilized on ionic liquid-decorated SBA-15shows a higher specificactivity than that on SBA-15.