Preparation And Properties Of Cross-linked Enzyme Aggregates Of Phenylalanine Ammonia Lyase On Silica Gel

Now，PAL has been widely applied in clinic and industry,but the stability is relativelypoor.In order to improve the stability of PAL in practical application,the PAL–CLEA swith a silica core and PAL-CLEAs with macroporous silica gel were prepared bycombining the cross-linked enzyme aggregates technology and Carrier immobilizedtechnology. In addtion, the properties were also investigated.1) the determination of the preparation condition PAL–CLEA s with a silica core andthe study of propertiessilica gel content、the concentration of cross-linking agent and cross-linking timewere optimized in the method of Single factor. The optimized conditions were as follow:silica gel content,100mg/mL; the concentration of cross-linking agent,0.2%;cross-linking time,3h.The temperature, pH, storage stability, acid-base stability, denaturant stability andreusability were discussed. The results were as follows: The optimum temperature and pHof CLEAs were at50℃and9.5respectively.In the condition of50℃and60℃,theresidual rate of enzyme activity was higher then the PAL-CLEAs。 And when the pH ofenvironment was higher then6, residual rate of enzyme activity reduced seriously；theTolerance to denaturant was significantly higher then PAL-CLEAs. after2transformingbatches, There are an obvious decline in enzyme activity; the residual rate of enzymeactivity was50%,when PAL–CLEA s with a silica core were stored in25℃.2) the determination of the preparation condition PAL-CLEAs with macroporoussilica gel and the study of properties.Firstly, The conditions for the preparation of aminated macroporous silica gel wereoptimized，the ratio of APTES and macroporous silica gel,2:1(v/w); temperature,110℃;time,10h. The content of amino-group was478.3μmol/g under optimized conditions.Secondly, The conditions for the preparation of MSG-PAL-CLEAs and SG-PAL-CLEAswere optimized and could be summarized as:0.05g MSG and1mL free PAL sulotion (pH8.8) were incubated at room temperature for2.5h, and then precipitated with80%-saturation ammonia sulfate for1h followed by cross-linking with1%glutaraldehydefor1h. The optimized condition for preparing SG-PAL-CLEAs was the activity recoveriesof MSG-PAL-CLEAs and SG-PAL-CLEAs were approximately45%.Finally，The temperature, pH, storage stability, acid-base stability, denaturant stabilityand reusability were discussed and the kinetic parameter were measured. The results were as follows: The optimum temperature and pH of CLEAs were at50℃and8.8,respectively. Compared withPAL-CLEAs, the Tolerance to denaturant、temperaturestability、reusability and storage stability were enhanced signally. MSG-PAL-CLEAsretained about79%of it initial activities after four transforming batches,but PAL-CLEAsretained about49%after two transforming batches. At25℃,PAL-CLEAs retained about26%of it initial activities after29d, MSG-PAL-CLEAs retained about50%.