Preparation Of Enzyme-inorganic Crystal Hybrid Nanoflowers And Its Appilcation In Biocatalysis

Enzymatic catalysis is an effective and green catalytic method in organic synthesis,but its difficulty in separation from reaction mixture has been a drawback for its development.The immobilization methods have the superiority in above mentioned aspects,but traditionally immobilized methods have the disadvantages of reduced activity,which have been a drawback in industrial application.While there was an easy and effective immobilized method that the preparation of enzyme hybrid nanoflowers?hNFs?.The enzymatic active sites in hNFs are limited in a specific space by the inorganic support,that is,the hNFs have the confined spaces.The hNFs could be applied in organic synthesis by utilizing the advantages of confined catalysis based on the preparation of hNFs.In the hNFs,the inorganic metal salt could function as both support and co-catalyst.In this work,the hNFs confining papain as biocatalyst has been prepared using co-crystallization and used in promiscuous catalysis.The papain-Cu₂?PO₄?₃ hNFs was firstly prepared through mixing CuSO₄ aqueous solution with papain in phosphate saline?PBS?at room temperature.The resulting hNFs was characterized by SEM and its activity was verified through a hydrolysis reaction with N-benzoyl-DL-arginine amide as substrate.The load rate of hNFs was 94.5%,and under optimal condition,this nano-biocatalyst demonstrated a 15-fold hydrolytic activity compared with papain of free form,along with better thermal-stability.A series of reaction factors?reaction temperature,time,and solvent?have been investigated for Knoevenagel condensation reaction with hNFs as catalyst.At optimal conditions,the product yield of hNFs catalytic reaction was 1.25 fold higher than that of free enzyme with benzaldehyde and acetylacetone as substrates.The results showed that the hNFs had the 87.8%of initial catalytic activity after 72 h,which show that the stability is improved immensely compared with free enzyme.Further,the substrate scope has been extended and it was found that most of results show that the catalytic activity of hNFs was better than that of free enzyme.Moreover,it was found that unsaturated aromatic aldehydes bearing strong electron-withdrawing substituents gave higher yields than those containing strong electron-donating substituents under same reaction conditions.We speculated that the copper ion may play a role in decreasing the electron density on the unsaturated aldehydes to promote the forming of carbon-carbon bond.Because of the toxicity of copper ion and its extensive detrimental effects on humans and the environment,biocompatible calcium ion was chosen to replace it and was used to prepare hNFs to catalyze the synthesis of drug.Six kinds of enzymes including papain,bromelain,trypsin,Lipase from Porcine Pancreas?PPL?,Lipase from Thermomyces Lanuginosus?TLL?and Lipase B from Candida Antarctica?CALB?were chosen to prepare the enzyme@Ca₃?PO₄?₂ hybrid nanoflowers?hNFs?.The loading efficiencies of the obtained hNFs were all about 95%and show better activity and thermal stability compared with corresponding free enzyme.Moreover,the used enzyme-inorganic hybrid nanoflowers?hNFs?were dissolved by adding phosphoric acid solution and heated for minutes to remove the denatured enzyme proteins and recover the primary calcium phosphate and secondary calcium phosphate.By weight-checking,it was found that the recovery rate of Ca₃?PO₄?₂ were all above95%,even up to the 99%.Furthermore,the reblossoming hNFs prepared using the recovered phosphate salts all gave the consistent catalytic activities.Enzyme was confined in specific space named confined space.Enzyme and substrate could integrate better and promote the reaction rate.Meanwhile,the Ca₃?PO₄?₂-hNFs has been prepared using TLL as organic component and it has been applied in the synthesis of Clindamycin Palmitate.In addition,its hydrolytic activity was 8.2 times of free enzyme,along with better thermal stability.In the preparation of TLL-hNFs,calcium phosphate from animal bones was also explored to use as inorganic component.The resulting hNFs was named natural hNFs and its hydrolytic activity was the 8.9 times of free enzyme.When the PE was used as solvent,the yield was69.6%catalyzed by natural hNFs and 79.1%catalyzed by free enzyme.The catalytic efficiency of free enzyme was better than hNFs,however,the natural hNFs could be reused for cycle catalysis and the catalytic activity still remain above 90%of initial activity even after 10 cycle times?12 h every cycle?.The yield of vitamin A palmitate could achieve 90.39%catalyzed by hNFs,while the yield was only 61.31%catalyzed by free enzyme.What's more,the hNFs could be reused for cycle catalysis and the catalytic activity still remain 73.2%of initial activity even after 10 cycle times?12 h every cycle?.In summary,enzyme@Cu₃?PO₄?₂ hNFs and enzyem@Ca₃?PO₄?₂ hNFs were prepared successfully using co-crystallization technology.The stability and activity was improved simultaneously compared with free enzyme.Some problems such as heavy metal pollution and difficult recovery of support during the immobilization could be eased by our green and dual-cycle immobilization method depending on the calcium phosphate crystallization,dissolution and recrystallization.The hNFs could also be applied in organic synthesis such as palmitate drug synthesis,which would further enrich and push the development of enzyme immobilization.