吸附法纯化青霉素酰化酶

Studies on the purification of the penicillin acylase by the adsorption method

目的纯化发酵液中的青霉素酰化酶。方法采用吸附纯化法。结果按 1 2 % (W /V)的比例将皂土加到含青霉素酰化酶的发酵上清液中 ,可将 98%的酶吸附 ,而同时吸附的杂蛋白质仅占发酵上清液总蛋白质的 14 5 %左右。吸附过程受pH影响较大 ,在 pH 6 5～ 7 5时 ,吸附效果最佳。使用不同种类的缓冲液洗涤酶 -皂土复合物 ,发现缓冲液的种类对酶的洗涤影响不大。使用含5 0 %以上的PEG和 8 0 %的NaCl的磷酸缓冲液可以将酶全部洗脱。结论此法可在常温下操作 ,酶活力收率较高 。

Objective To investigate the purification method of the penicillin acylase. Methods The purification of the penicillin acylase was carried out by the adsorption method. Results The bentonite as an absorbent 1 2%( W/V ) was added into the supernatant of fermentation broth of Bacillus subtilis 97 1 for adsorption of the penicillin acylase. It was found that the 98% activity of the penicillin acylase and 14 5% protein in the supernatant were absorbed. The adsorption of enzyme was obviously affected by different pH of the supernatant, at 6 5～7 5 pH and the effect of adsorption was the best. Various kinds of buffers were used to wash the adsorbent enzyme complex; the amount of the washed out enzyme was not obviously changed by different kinds of buffers. When the phosphate buffer containing the 5% PEG and 8% NaCl was used to elute the complex, 100% of the enzyme activity absorbed on the complex was eluted. Conclusions This method is easy of and suitable for being carried out at room temperature, especially the recovery rate of the enzyme activity is very high. So it can be used for the isolation and purification of the penicillin acylase from the fermentation broth.