| Ketoprofen is an important member of the nonsteroidal anti-inflammatory 2-(3-benzoylphenyl)propionic acid drugs, and currently its racemate is sold in the world. Pharmacological studies have shown that(S)- ketoprofen can be used for the treatment of various arthritis, while(R)- ketoprofen is mainly used to relieve pain. Among various methods for preparation of enantiomeric pure ketoprofen, the enzymatic approach has drawn more and more attention due to its advantages such as high efficiency, high enantioselectivity, greeness and mild reaction conditions. In many cases, however, the industrial applicationof enzymatic production of enantiopure ketoprofenis limited because of the high cost of the applied enzyme. Therefore, it is of great importance to obtain cheap and available enzyme preparations.In this paper, a cheap and available domestic lipase(Pseudomonas Cepacia lipase,PCL)was screened and it can effectively catalyze the asymmetrichydrolysis of ketoprofen ethyl ester(KE). However, the free PCL has a number of limitationspreventing its further applicationssuch as easy inactivation, difficulty to recycle and separate. Therefore, the immobilization of PCL is used toovercome these drawbacks.This study not only conforms to the development trend of chiral drugs, will also have a significant economic and social benefits.Cellulosenanocrystal(CNC) have attracted increasing researchers’ attention in recent years because of their high surface-to-volume ratio, high biocompatibility, cheap and renewable characteristics. Recently, our group had prepared a novel low-cost magnetic CNC(MCNC) enzyme carrier via responding improvement of magnetism and chemical modification. This novel MCNC carrier has been proved to be an effective carrier for immobilization. Research shows that papain immobilized through this carrier has high catalytic activity and stability. Moreover, it can be easily separated from the reaction system under magnetic field.However, the universality of this novel carrier with low cost and easy separation is needed to be explored.In the present study, we firstly immobilized PCL onto the novel MCNC by precipitation-cross-linking process and had successfully obtained a magnetic nanoscale enzyme preparations with high activity, high stability(named PCL@MCNC). The effects of different preparation conditions on the activity recovery of PCL@MCNC were examined. The results revealed that the highest activity recovery was obtained under the precipitation time of 1.0 h, support/enzyme mass ratio of 10:1, crosslinker concentration of 40 m M, and cross-linking time of 4.0 h. Under these optimum parameters, the protein loading of PCL@MCNC wad as high as 82.2 mg/g MCNCand the activity recovery of PCL@MCNC reached 95.9%.Secondly, the microstructure and enzymology properties of PCL@MCNC wereinvestigatedsystematically. The aggregation stateof enzyme on the carrier, the internal secondary structure and surface morphologyof PCL@MCNC were analyzed using FTIR, SEM and AFM. The results showed that: 1) the content of alpha helix increased followed by the increased rigidity and stability of secondary structure after immobilization; 2) the structure of the PCL@MCNC is relatively compact after immobilization. Meanwhile, the width of PCL@MCNC had significantly increased(20.0 nm vs 75.8 nm) and the height of it had slightly induced. There were some network gaps in PCL@MCNC which could help to promote the smooth progress of enzymatic reaction to a certain extent. The results of enzymology properties revealed that the optimal p H of PCL@MCNC and free enzyme were 6.0 and 6.5, respectively; the optimal temperature of PCL@MCNC was 40 oC, and free enzyme was 35 oC. In addition, the PCL@MCNCwere superior to the free enzyme in terms of thermal stability, organicsolvent tolerance and p H tolerance;Moreover, after 25 days, PCL@MCNC had remained 92.3% activity compared with initial activity, much higher than the free enzyme(73.7%). It was found that the catalytic efficiency(Vmax/Km) of PCL@MCNC was higher than that of free enzyme(60 10-2 vs 39 10-2 min-1). These resultsshowed that the PCL immobilized on the surface of MCNC had improved adaption of p H and temperature,higher stability and higher catalytic efficiency.A comparative study of PCL@MCNC and PCL was made to find out the influence of various related factors on the asymmetric hydrolysis of KE. Among them, the optimal reaction conditions catalyzed by PCL were as follows: the optimal buffer p H value, content of Triton X-100, substrate concentration and reaction temperature were found to be 6.0, 1.0%, 5 m M and 35 oC, respectively, under which the initial reaction rate, the product yield and the product e.e. were 25.5 mmol/(L.h), 36.1% and 85.3%, respectively. At the same time, the optimal conditions catalyzed by PCL@MCNC were as follows: the buffer p H value, content of Triton X-100, substrate concentration and reaction temperature were found to be 6.5, 1.5%, 6 m M and 40 oC, respectively. Under these optimum parameters, the initial reaction rate, the product yield and the product e.e. were 20.1 mmol/(L·h), 39.0% and 86.4%, respectively. Compared with the free enzyme, the product yield and the product e.e of asymmetric hydrolysiscatalyzed by PCL@MCNC were slightly enhanced.In the present study, we report firstly for the successful preparation, characterization and application of PCL@MCNC. In comparsion with the present immobilization technology, this preparation process exhibited more advantages such as simpleness, high enzyme loading, high enzyme activity, high stability of enzyme and rapid separation of enzymefrom the reaction medium. Meanwhile, PCL@MCNC could effectively catalyze asymmetric hydrolysis of KE to(R) – ketoprofen. This study is helpful to rich immobilization technology of enzyme. Additionally, it provides not only theeffective new wayof the preparation of(R) – ketoprofen, but also the established efficient biocatalytic system which may be widely used for asymmetric resolution of other racemic 2- aryl propionic acid drugs(such as ibuprofen, naproxen). |
PDF Full Text Request