以大孔硅胶为载体的苯丙氨酸解氨酶交联酶聚体的制备及性质研究
Preparation and properties of cross-linked enzyme aggregates of phenylalanine ammonia lyase in the aminated macroporous silica gel
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摘要: 通过交联酶聚体与氨基化硅胶(活化硅胶)的共价交联,制备了以氨基化硅胶、无氨基化硅胶为载体的苯丙氨酸解氨酶交联酶聚体MSG(SG)-PAL-CLEAs、SG-PAL-CLEAs。优化了硅胶的氨基化条件以及MSG-PAL-CLEAs的制备条件,并且对游离酶,苯丙氨酸解氨酶交联酶聚体(PAL-CLEAs),MSG-PAL-CLEAs和SG-PAL-CLEAs的稳定性进行了比较。优化的硅胶氨基化条件:3-氨丙基三乙氧基硅烷(APTES)/硅胶2∶1(v/w),氨基化温度110℃,氨基化时间10h,在此条件下所得硅胶键合氨基数目是480μmol/g硅胶。优化的MSG-PAL-CLEAs制备条件:0.05g硅胶/mL酶液,吸附时间2.5h,吸附pH8.8,沉淀剂为硫酸铵,饱和度80%,交联剂戊二醛的浓度为1%,交联时间1h。SG-PALCLEAs的制备条件和MSG-PAL-CLEAs的制备条件大体相似,不同的是沉淀剂硫酸铵的饱和度为40%,交联剂戊二醛的终浓度为1.5%。研究结果表明,制备得到的MSG-PAL-CLEAs和SG-PAL-CLEAs的酶活回收率均为45%左右。与传统的PAL-CLEAs相比,MSG-PAL-CLEAs的重复使用性和储藏稳定性均有了显著提高,MSG-PAL-CLEAs转化4个批次后,酶活保留79%,而PAL-CLEAs转化2个批次酶活仅保留49%。25℃,PAL-CLEAs储存29d酶活保留26%,而MSG-PAL-CLEAs的酶活保留仍高达50%。Abstract: The cross- linked enzyme aggregates of phenylalanine ammonia lyase ( PAL) in aminated ( activited) macroporous silica gel ( MSG ( SG) - PAL- CLEAs) were prepared by the cross- linked enzyme aggregates technology.The conditions for the preparation of aminated macroporous silica gel and MSG ( SG) - PAL- CLEAs were optimized, respectively.Moreover, stability of free PAL, PAL-CLEAs, MSG-PAL-CLEAs and SG-PAL-CLEAs was evaluated and compared.The optimized conditions for preparing amino- modified macroporous silica gel ( MSG) were as follows: the ratio of APTES and macroporous silica gel, 2∶1 ( v/w) ; temperature, 110℃; time, 10h.The content of amino-group was 480μmol /g under optimized conditions.The optimum conditions for preparing MSG- PAL-CLEAs could be summarized as: 0.05g MSG and 1mL free PAL sulotion ( pH8.8) were incubated at room temperature for 2.5h, and then precipitated with 80%- saturation ammonia sulfate for 1h followed by crosslinking with 1% glutaraldehyde for 1h.The optimized condition for preparing SG- PAL- CLEAs was similar with the MSG- PAL- CLEAs except that the saturation of ammonia sulfate was 40% and the terminal concentration of glutaraldehyde was 1.5%.The results showed that the activity recoveries of MSG- PAL- CLEAs and SG- PALCLEAs were approximately 45%.Moreover, compared with PAL- CLEAs, the reusability and storage stability of MSG- PAL- CLEAs and SG- PAL- CLEAs were improved.MSG- PAL- CLEAs retained about 79% of it initial activities after four transforming batches, but PAL- CLEAs retained about 49% after two transforming batches.At25℃, PAL-CLEAs retained about 26% of it initial activities after 29d, MSG-PAL-CLEAs retained about 50%.