粪产碱杆菌ZWS11菌株对烟嘧磺隆的酶促降解特性

Enzymatic degradation characteristics of nicosulfuron by Alcaligenes faecalis ZWS11

为明确粪产碱杆菌Alcaligenes faecalis ZWS11菌株对烟嘧磺隆的酶促降解特性,采用丙酮沉淀、超声波破碎和高速离心等方法,提取制备了ZWS11菌株的胞外和胞内粗酶液以及菌体碎片悬浮液,并分别测定了其酶活力。结果表明：当V（菌体发酵液）：V（丙酮）=1：3时提取到的胞外粗酶液具有较高的酶活力;胞外粗酶液、胞内粗酶液和菌体碎片悬浮液对24.9μmol/L烟嘧磺隆的平均降解率分别为87.4%、16.9%和17.4%,胞外粗酶液的降解能力与胞内粗酶液或菌体碎片悬浮液相比差异显著（P〈0.05）,由此确定对烟嘧磺隆起降解作用的酶属于胞外酶。最适降解条件研究表明：该降解酶的酶促反应适宜温度为35℃,较适p H值为6.0,反应时间为30 min,在此条件下其对烟嘧磺隆的降解率在80%以上。此外,该降解酶在35~70℃、p H值4.0~9.0范围内均能够保持较高的酶活力,对烟嘧磺隆的降解率均在60%以上,表明该降解酶具有较好的热稳定性和酸碱稳定性。加入不同浓度十二烷基硫酸钠（SDS）和苯甲基磺酰氟（PMSF）,对该降解酶的活力表现出了不同程度的抑制作用。研究结果可为烟嘧磺隆降解酶制剂的规模化生产及被烟嘧磺隆污染土壤的生物修复提供科学依据。

In order to study enzymatic degradation characteristics of nicosulfuron by Alcaligenes faecalis ZWS11, extracellular, intracellular crude enzymes and cell debris were extracted from strain ZWS11 by methods of acetone precipitation, ultrasonic fragmentation and centrifuge separation. The enzyme activity of the extraction from ZWS11 was systematic studied under laboratory conditions. The results showed that the enzyme activity was high when the ratio of fermentation broth and acetone was1：3（V/V）. At the presence of extracellular, intracellular crude enzymes and cell debris, the average degradation rates of nicosulfuron（24.9 μmol/L） were 87.4%, 16.9% and 17.4%, respectively. The degradation capability was significantly different between the extracellular enzymes and intracellular crude enzymes or cell debris（P〈0.05）, which suggested that it was the extracellular enzymes that catalyzed the degradation of nicosulfuron. The optimal condition for enzymatic degradation of nicosulfuron were as follows： 30 min of reaction time, p H 6.0 and 35 ℃; more than 80% of nicosulfuron was degraded by extracellular enzymes under this condition. Moreover, high degradation rates（〉60%）were achieved when the p H ranged from 4.0 to 9.0, and temperature ranged from 35 ℃ to 70 ℃, which demonstrated that the degrading enzymes had a broad p H tolerance and a good thermal stability. The activity of degrading enzymes could be inhibited in the presence of different concentrations of SDS and PMSF. The results provided scientific basis for further studies on the large-scale production of nicosulfuron-degrading enzymes and the bioremediation of nicosulfuron-contaminated soil.