Effects Of Nano Particles Addition On Structure And Properties Of Lipase Cross-linked Enzyme Aggregates(CLEAs)

The research aimed to increase the activity of cross-linked enzyme aggregates (CLEAs). According to the good properties of nanoparticle, nano-TiO2, nano-MgO, nano-Ni, nano-Cu and nano-Fe3O4were introduced in the preparation of lipase (Candida sp.1619) CLEAs respectively. They were named as NTiO2-CLEAs, NMgO-CLEAs, NNi-CLEAs, NCu-CLEAs and NFe3O4-CLEAs). In order to know effects of different nanoparticles on activity, morphological structure, and performance, infrared spectrometric analyzer, scanning electron microscope, and fluorescence spectrometer were used to determine nano-CLEAs. In addition, NTiO2-CLEAs had the best performance in five nano-CLEAs, so the optimal conditions for NTiO2-CLEAs preparation and its application in the treatment of simulated wastewater with olive oil and soybean oil were studied. The experimental results follow as:(1)Relative to CLEAs without nanoparticle, the activity of CLEAs with nano-TiO2particle increased by15.2%at most, while activity of CLEAs with nano-MgO, nano-Ni, nano-Cu and nano-Fe3O4particles decreased by92.6%,54.6%,97.7%,63.3%and at most respectively. After optimization, the optimal conditions for NTiO2-CLEAs preparation were experimentally determined of lmg/ml nano-TiO2,five times the volume of enzyme solution of acetone,2.08%glutaraldehyde, cross-linking time of2h, cross-linking temperature of4℃, and centrifugation speed of6000rmin-1.(2)Relative to CLEAs without nanoparticle, CLEAs with nano-TiO2particle had smaller particle, more uniform particle size and more pore density; while CLEAs with nano-MgO, nano-Ni, nano-Cu and nano-Fe3O4particles had more no-uniform particle size and less pore density. Result determined by Fourier Transform Infrared Spectrometer showed that, infrared spectrum of NTiO2-CLEAs was similar to CLEAs without nanoparticle, while the infrared spectrum of CLEAs with nano-MgO, nano-Ni, nano-Cu and nano-Fe3O4particles showed great changes compared to CLEAs without nanoparticle. Result determined by fluorescence spectrum showed that relative to CLEAs without nanoparticle, nano-TiO2addition had greater effect on endogenous fluorescent spectrometry and three-dimensional fluorescence spectrum than other four nanoparticles.(3)NTiO2-CLEAs had greater pH and thermal stability than CLEAs without nanoparticle, but its storage stability and reusability were relatively lower than CLEAs without nanoparticle. Relative to CLEAs without nanoparticle, the kinetics of enzyme reaction Km of NTiO2-CLEAs decreased by24.8%and Vmax increased by56.7%, indicating that NTiO2-CLEAs had great affinity to substrate and reaction rate.(4) In the treatment of simulated olive oil wastewater experiment, the maximum olive oil conversion rate was95.1%under catalysis of NTiO2-CLEAs but it was only78.9%under catalysis of CLEAs without nanoparticle. The reaction conditions were olive oil volume was1ml,23.3mg of CLEAs, volume ratio of oil to water was1:9, temperature of37℃, and reaction time of12h. In the treatment of simulated soybean oil wastewater experiment, soybean oil conversion rate under catalysis of NTiO2-CLEAs increased by18.9%than CLEAs without nanoparticle.In addition, NTiO2-CLEAs had greater tolerance for Na+and Ca2+than CLEAs without nanoparticle.In summary, it is possible to increase the activity of CLEAs and improve their properties by addition of nanoparticles, but the detailed influence mechanism need further study.