Construction Of A Novel Magnetically Responsive Immobilized Enzyme And Its Enzymatic Biotransformation For The Preparation Of Glycyrrhetinic Acid

In this paper,an unreported glucuronidase（Aspergillus bertholletiae KAE8374379.1）was screened based on genomic databases NCBI.The glucuronidase was immobilized using MOFs and magnetic cross-linked enzyme aggregates.Its stability,reusability and by-product tolerance were investigated.1.Two kinds of glucuronidases from Aspergillus bertholletiae（KAE8374379.1）and Pseudomonas aeruginosa（KFY40603.1）were screened by homologous sequence comparison based on the NCBI database.The expression vectors pet28A-ABGUS and PET28A-KFY were constructed.Their catalytic activity,mode and products of hydrolysis were probed.Glucuronidase（Ab GUS）from Aspergillus bertholletiae was found to that it has high hydrolytic activity towards glycyrrhetinic acid（GA）,with the conversion ratio 91.4%.The products were verified by mass spectrometry and it was found that the product of the hydrolysis process was highly specific.What’s more,there was hardly no by-product glycyrrhetinic acid monoglucuronide（GAMG）.2.To solve the poor stability of free enzymes during the reaction and its non-reusability,we prepared a magnetically responsive zirconium-based MOFs specific carrier（Fe₃O₄@Ui O66）,which cleverly integrated glucuronidase purification and immobilization.Fe₃O₄@Ui O66has good magnetic response,regular shape and high specificity.The immobilizedβ-glucuronidase exhibited a wider range of p H adaptability,temperature stability and storage stability.The relative activity of the immobilized enzymes remained approximately 80%after 8 cycles.More importantly,it was innovatively discovered that the magnetic nanoparticles could be regenerated using dithiothreitol.After a simple treatment,the immobilization capacity of the regenerated MOFs remained at more than 90%for the first 3 cycles.In addition,a significant increase in the tolerance of the enzyme to the by-product glucuronide was found after immobilization.3.Magnetic cross-linked enzyme aggregates（MCLEAs）were successfully constructed by introducing Fe₃O₄ modified with surface carboxyl groups.Before cross-linking using glutaraldehyde,carbodiimide was used to enhance the interaction between glucuronidase and carboxyl-functionalised Fe₃O₄,significantly enhanced the recovery of enzymatic activity.Compared to free enzymes,MCLEAs had higher acid/alkali and thermal stability,retaining 63.3%of catalytic activity after6 cycles.More importantly,we likewise found an improved by-product tolerance of MCLEAs,with the catalytic activity in the 10 m M glucuronide system being 221.5%of that of the free enzyme.Raman spectroscopy results showed that the ordered structure ofβ-glucuronidase increased from 43.9%to 50.6%after immobilization,which resulted in more hydrogen bonding and thus increased enzyme rigidity,explaining the increased stability and tolerance.