【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。更多还原

【Abstract】 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 of pH 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.更多还原