羟基阿特拉津脱乙胺基水解酶(AtzB)典型酶学性质的研究

Typical characterization of hydroxyatrazine N-ethylaminohydrolase (AtzB)

采用Ni-NTA亲和层析法对基因重组菌中的羟基阿特拉津脱乙胺基水解酶(AtzB)进行分离纯化,并对该酶的典型酶学性质进行研究。纯化处理后,AtzB的纯度提高15倍,酶活回收率高达15.2%。酶学性质研究表明,AtzB的最适反应温度为40℃;最适反应pH为9.0。在最适反应条件下,AtzB对底物最大反应速率Vmax为3.17μmol.L-1.min-1,米氏常数Km为0.45 mmol.L-1。反应缓冲液中Cu(Ⅱ)对酶活力有相对较强的抑制作用(P<0.05),Zn(Ⅱ)对酶活力影响相对较小(P<0.05),而Co(Ⅱ)、Cr(Ⅲ)、Ca(Ⅱ)和Ni(Ⅱ)则对酶活力基本没有影响。盐度对AtzB活力影响较大,当缓冲液中NaCl浓度达到1 mol.L-1时,AtzB完全失活。

The purification of Hydroxyatrazine N-Ethylaminohydrolase was achieved by combination method of Ni-NTA affinity chromatography, and the properties were subsequently measured. AtzB was purified 15-fold to apparent homogeneity with 15.2% overall recovery. The properties of purified AtzB showed that the optimum temperature and pH was 40 ℃ and 9.0, respectively. Kinetic studies of the AtzB showed that the Vmax and Km were 3.17 pmol. L-1 min-1 and 0.45 mmol. L-1, respectively. The activity of pudfied AtzB was strongly inhibited by Cu（ll） （P〈0.05）, and moderately inhibited by Zn（ll） （P〈0.05）, However, Co （11）, Cr （111）, Ca （11） and Ni （11） have little effect on the activity of purified AtzB. In addition, the influence of NaCI on enzyme activity is strong, when its concentration reached 1 tool . L-1, the purified AtzB was a reflection of complete inactivity.