壳聚糖微球固定化重组TEM-116型ESBL的研究  

Study on immobilization of recombinant TEM-116 ESBL with chitosan microspheres

作　　者：徐荣[1] 韩冬青[1] 王震[1] 楼永良[1] 陈秀枢[1] 

机构地区：[1]温州医学院微生态学研究所,温州325000

出　　处：《中国抗生素杂志》2011年第1期64-69,共6页Chinese Journal of Antibiotics

基　　金：国家高科技发展"863"计划(No.2001AA215051和2003AA215072)

摘　　要：目的利用固定化酶技术,使重组TEM-116型超广谱β-内酰胺酶(ESBL)在清除环境中残留抗生素的应用中可重复使用,并改善其稳定性。方法以戊二醛为交联剂将重组TEM-116型ESBL固定于壳聚糖微球载体,并采用单因素轮换法和响应曲面法对固定化条件进行优化。结果 4℃条件下,当戊二醛浓度0.54%、交联pH5.4、给酶量0.58IU/g壳聚糖微球、固定化pH6.2时,固定化酶的酶活和回收率分别可达到0.49IU/g和84%(0.49IU/0.58IU)。与游离酶相比,固定化酶的酸碱稳定性和可重复利用性得到显著提高,在pH5.8～7.4范围内酶活性保持在73%～100%的水平,经7次重复性使用,每次1h,酶活力保留高达80%。结论通过固定化酶技术,以重组TEM-116型ESBL为代表的耐药酶能够开发为具有广泛应用前景的环保制剂,以消除外环境中残留的抗生素。Objective To improve the stability of recombinant TEM-116 ESBL and make it reusable in clearing the environmental residual antibiotics through immobilization. Methods Recombinant TEM-116 ESBL was immobilized on chitosan microspheres by glutaraldehyde-crosslinking method, then the immobilizing conditions were optimized by means of one-factor-at-a-time experimentation and response surface methodology. Results The activity and the recovery rate of the immobilized TEM-116 ESBL were up to 0.49IU/g and 84%（0.49IU/0.58IU） respectively under conditions as follows： glutaraldehyde concentration 0.54%, crosslinking pH 5.4, ratio of TEM- 116 ESBL to chitosan microspheres 0.58IU/g, immobilizing pH 6.2, at 4℃. The acid-base stability and the reusability of the immobilized TEM-116 ESBL were better than those of free enzymes. The activity of the immobilized enzyme could retain 73% to 100% in the range ofpH 5.8 to 7.4, and it could still retain 80% after 7 times repeated use （1 hour per time）. Conclusion Through immobilization, the drug-resistant enzymes represented by recombinant TEM-116 ESBL have bright future to be developed as protective agents in clearing the environmental residual antibiotics.

关 键 词：壳聚糖 固定化酶 超广谱Β-内酰胺酶 

分 类 号：Q55[生物学—生物化学]