The Improved Bio-imprinted Lipase And Its Application In The Resolution Of1-phenylethanol

Bio-imprinting is a novel strategy to enhance lipase activity andenantioselectivity, which can trap the active conformation of enzymes. The synthesisof enantiomerically pure compounds or separation of racemic mixtures is in highdemand in pharmaceuticals and agrochemical industries. Some single enantiomers hasbeen produced by this method.In our study, improved activity and enantioselectivity of PPL was prepared bybio-imprinting and some improvements were made to increase dispersivity ofenzymes. Meanwhile, these lipase samples were applied to resolving the1-phenylethanol, which is an importantly pharmaceutical precursor. The conclusionscan be drawn as follows:The preparation and characterization of bio-imprinted lipase: Bio-imprinting canenhance lipase activity and enantioselectivity in organic solvent. The template was thekey factor to determine the performance of bio-imprinted lipase. The ideal templatewas the combination of (+)-ethyl mandelate and (R)-phenylethanol; the esterificationactivity could be644.8U and the enantioselectivity E was beyond2000. The optimalpH was7.2. The porous structure was the reason why the bio-imprinted lipase has soexcellent properties.Resolution of1-phenylethanol by bio-imprinted lipase: In the resolution of1-phenylethanol, the ideal solvent was isopropyl ether and the optimal temperaturewas35oC. The ratio of substrates (1-phenylethanol:vinyl acetate) was1:3and the bestconcentration of catalyst was62.5mg/ml. The conversion was43.2%by bio-imprintedlipase, of which the ultimate conversion is50%. What's more, the activity of chitosancoated bio-imprinted lipase was improved1.4-fold compared with bio-imprintedlipase. However the activity of bio-imprinted lipase nanogel was decreased andneeded to be improved. Besides, all these lipase samples exhibited excellentenantioselectivity and reusability.The preparation and characterization of chitosan-TPP coated bio-imprintedlipase: Combining the chitosan nanopartical with bio-imprinting technology, thechitosan-TPP coated bio-imprinted lipase was obtained. The optimal preparationconditions were as follows: The mass concentration of chitosan was0.75%; the ratio of mass concentration of chitosan to TPP was1:1and the volume ratio was1:1.5; thebest cross-linking time was10min and the temperature was15oC. The results showedthat the chitosan-TPP coated bio-imprinted lipase was spherical and the diameter wasabout50nm. Besides, the secondary structure of chitosan-TPP coated lipase waschanged obviously.The preparation and characterization of bio-imprinted lipase nanogel:Combining the nanogel with bio-imprinting, the bio-imprinted lipase nanogel wasobtained. The optimal preparation conditions were as follows: The optimalconcentration of NAS was7.5mg/ml and the pH was8. The bio-imprinted lipasenanogel was spherical regularly as chitosan coated lipase and the diameter was about30~40nm. In addition, most of the bio-imprinted lipase nanogel was distributedsingly.