Study On The Preparation Of Immobilized Penicillin Acylase Onto Magnetic Nanocrystalline Cellulose And The Synthesis Of Cefaclor

Cefaclor is a second generation of semi synthetic cephalosporin antibiotics,and it can interference the synthesis of bacterial cell wall by deactivating the transpeptidase.Thus,cefaclor has a strong bactericidal effect on many kinds of gram-positive or gram-negative bacteria and is widely used in clinical practice.The traditional chemical method for cefaclor synthesis has the disadvantages of complicated steps,large consumption of organic solvents,the waste of the synthesis process exhibit negative impact on the environment and human.Nowadays,the enzymic synthesis of cefaclor has attracted more and more attention due to its advantages such as high efficiency,mild reaction conditions and less contamination.Penicillin acylase,which can efficiently catalyze the condensation of?-lactam nucleus and acyl side chain,has been proved to be applied in the synthesis of various?-lactam antibiotics.In this paper,tri?hydroxymethyl?phosphine?THP?as a new cross-linking agent,was first used to immobilization of penicillin acylase on a new material,magnetic cellulose nanocrystalline.The prepared immobilized penicillin acylase could be separated and recovered conveniently and exhibit high enzyme loading,activity recovery,catalytic activity as well as good stability.Moreover,the reaction conditions of the PA-THP-MNCC-catalyzed synthesis of cefaclor were systematically optimized.In order to further impove the reaction efficiency,the DES-containing systems were applied as the reaction media for the synthesis of cefaclor.The main results are as follows:Immobilized penicillin acylase PA-THP-MNCC and PA-GA-MNCC were prepared by using THP and glutaraldehyde?GA?,respectively.Under the optimal preparation conditions,the enzyme loading and the activity recovery of PA-THP-MNCC were 172.3 mg?enzyme?/g?carrier?,77.6%,respectively.The enzyme loading and activity recovery of PA-GA-MNCC were 148.4 mg?enzyme?/g?carrier?,48.7%,respectively.The results showed that using THP as the cross-linking agent to prepare immobilized enzyme had better immobilization effect.X-ray diffraction?XRD?,scanning electron microscopy?SEM?and Fourier transform infrared spectroscopy?FTIR?et.al were used to characterize the physicochemical properties of MNCC and the relative immobilized enzyme.The result of PA-THP-MNCC,PA-GA-MNCC and PA were compared,and it revealed that the content of?-helix in secondary structure of PA-THP-MNCC increased by 26.6%and 19.83%respectively,and the content of coil in secondary structure of PA-THP-MNCC decreased by 24.26%and 16.25%,respectively indicating the increasing of structural rigidity and stability of PA-THP-MNCC.Then,the enzyme properties of free PA,PA-GA-MNCC and PA-THP-MNCC were studied.The optimum temperature and pH of them were 40,40,50oC and 9,9.5,10,repectively.Besides,the PA-THP-MNCC showed superior thermal and pH stability as well as solvent tolerance comparing with its free PA or PA-GA-MNCC.Moreover,after 20 days,PA-THP-MNCC had remained 93%activity compared with initial activity,which significantly higher than that of free PA and PA-GA-MNCC.The kinetic study showed that PA-THP-MNCC has better substrate affinity and catalytic efficiency than PA or PA-GA-MNCC.These results demonstrated that the PA-THP-MNCC had higher stability and activity.PA-THP-MNCC was used to the synthesis of cafaclor.The effects of key conditions such as temperature and substrate molar ratios on the enzymatic reaction were explored.The optimal temperature,p H,substrate molar ratio of 7-ACCA and PGME,7-ACCA concentration and enzyme dosage were 20oC,6.5,4.0,30 mmol/L and 3 U/mL,respectively.Under the optimal conditions,the yield of cafaclor was 84.3%,where the synthesis/hydrolysis?S/H?ratio was 1.24.In order to futher improve the yield of cafaclor,DES-containing systems were applied as the reaction media.The optimal reaction conditions for the enzymatic synthesis carried out in the DES-based co-solvent system?ChCl:Glycol-H₂O,7:3?were as follows:the optimum substrate molar ratio?7-ACCA:PGME?,temperature,pH,and enzyme dosage were 4.5,20oC,6.5,4 U/m L,while the concentration of 7-ACCA was 30 mmol/L.The yield of cafaclor and S/H were 92.1%and 1.85,respectively.In this paper,the penicillin acylase was successfully immobilized on the magnetic nanocrystalline cellulose by a new crosslinker THP.PA-THP-MNCC exhibited good stability and catalytic activity,and was used in the synthesis of cefaclor in both aqueous phase and DES-containing systems.It provides a theoretical guidance for the use of new crosslinking agent THP and the synthesis of cefaclor by enzymatic method.