阴沟肠杆菌w10j15中拟除虫菊酯类杀虫剂降解酶的酶学性质研究
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摘要
本文采用HP6890气相色谱仪测定了联苯菊酯，甲氰菊酯和氯氰菊酯在水体中的添加回收率，3种菊酯在水体中都分别设计了1 mg／L，10mg／L，100mg／L 3个不同浓度的添加回收试验。结果表明，3种菊酯在水样中的添加回收率在95.47％至103.21％之间，变异系数在2.21％-4.72％之间。
从农药长期污染的土样中分离到一株阴沟肠杆菌w10j15，该菌株可以利用拟除虫菊酯类杀虫剂作为唯一碳源和能源进行生长。经研究表明(王兆守，2003)，阴沟肠杆菌w10j15的生长最适p H为7.0，最适温度为30℃。该菌株培养72h，对50mg／L联苯菊酯、甲氰菊酯和氯氰菊酯的降解率分别为52.57％、50.13％和54.31％。在富集培养基培养72h后，可产生最高的联苯菊酯降解酶活性，达到约8U／mL(U为每分钟所降解的农药mg数)。
W10j15中提取的胞内酶降解联苯菊酯、甲氰菊酯和氯氰菊酯的最适反应p H均为7.5，最适温度均为40℃。该酶对联苯菊酯的米氏常数Km=440.58nmol／mL，最大反应速率Vmax=50.25nmol／min：对甲氰菊酯的米氏常数Km=332.23nmol／mL，最大反应速率Vmax=48.08nmol／min；对氯氰菊酯的米氏常数为Km=372.00nmol／mL，最大反应速率Vmax=63.29nmol／min。该酶与底物作用20min，对浓度分别为50mg／L联苯菊酯、甲氰菊酯、氯氰菊酯的降解率分别为75％、58％、78％。该酶通过硫酸铵沉淀、凝胶过滤、离子交换柱层析等多步纯化；就联苯菊酯而言，降解酶比活力达到125.7U／mg，纯化了6.37倍，回收率为91.7％。经SDS—聚丙烯酰胺凝胶电泳测定，蛋白染色呈现一条主带，该降解酶的分子量约为65K D。等电聚焦测得的酶的等电点约为6.8。冻干后的酶保存于4℃冰箱中一个月后，对联苯菊酯的降解活力剩余70％。
In this study, the HP6890 Gas Chromatography (GC) was used to measure the recovery rates of bifenthrin, mecthrin and cypermethrin in water. The concentration of bifenthrin, mecthrin and cypermethrin were set at 1mg/ L, 10 mg/ L, 100 mg/ L respectively with water. The recovery rates of 3 pesticides were between 95. 47% and 103. 21%, and the coefficient ranged from 2. 21% to 4. 72%.
From the soil contaminated by pesticides, an E. cloacae strain wlOjl5 was isolated. This microorganism could use bifenthrin, mecthrin and cypermethrin as its unique carbon source. The best growth conditions for wlOjl5 was pH=7. 0, at 30 C . The degradation rates to bifenthrin, mecthrin and cypermethrin were 52.57%, 50.13%, 54.31% respectively. After 72 hours, the microorganism had the highest enzyme product and the highest enzyme activity of roughly 8U/mL (I) refers to mg/min).
The enzyme has the highest degradation rate at pH=7. 5 and 40 C . Bifenthrin, mecthrin and cypermethrin could be degraded 75%, 58% and 78% respectively. The enzyme has
Km=440. 58nmol/mL and Vmax=50. 25nmol/min for bifenthrin;
Km=332.23nmol/mL and Vmax=48. 08nmol/min for mecthrin and
Km=372.00nmol/mL and Vmax=63. 29nmol/min for cypermethrino
As far as biphenthrin is concerned, through the procedure of the ammonium sulfate precipitation, the gel gum filter and the ion exchange, the enzyme attains the specific ratio of 125.7U/mg, the purified ratio of 6.37, and the recovery rate of 91.7%. With the SDS-PAGE electrophoresis, the protein presented as a dark line. Compared with the protein marker, the molecular weight of the enzyme is roughly 65 KD. The IEF-PAGE measurement showed pI of the enzyme was 6. 8. The enzyme has 70% of the total degradation activity after one month restoration at 4C.
从农药长期污染的土样中分离到一株阴沟肠杆菌w10j15，该菌株可以利用拟除虫菊酯类杀虫剂作为唯一碳源和能源进行生长。经研究表明(王兆守，2003)，阴沟肠杆菌w10j15的生长最适p H为7.0，最适温度为30℃。该菌株培养72h，对50mg／L联苯菊酯、甲氰菊酯和氯氰菊酯的降解率分别为52.57％、50.13％和54.31％。在富集培养基培养72h后，可产生最高的联苯菊酯降解酶活性，达到约8U／mL(U为每分钟所降解的农药mg数)。
W10j15中提取的胞内酶降解联苯菊酯、甲氰菊酯和氯氰菊酯的最适反应p H均为7.5，最适温度均为40℃。该酶对联苯菊酯的米氏常数Km=440.58nmol／mL，最大反应速率Vmax=50.25nmol／min：对甲氰菊酯的米氏常数Km=332.23nmol／mL，最大反应速率Vmax=48.08nmol／min；对氯氰菊酯的米氏常数为Km=372.00nmol／mL，最大反应速率Vmax=63.29nmol／min。该酶与底物作用20min，对浓度分别为50mg／L联苯菊酯、甲氰菊酯、氯氰菊酯的降解率分别为75％、58％、78％。该酶通过硫酸铵沉淀、凝胶过滤、离子交换柱层析等多步纯化；就联苯菊酯而言，降解酶比活力达到125.7U／mg，纯化了6.37倍，回收率为91.7％。经SDS—聚丙烯酰胺凝胶电泳测定，蛋白染色呈现一条主带，该降解酶的分子量约为65K D。等电聚焦测得的酶的等电点约为6.8。冻干后的酶保存于4℃冰箱中一个月后，对联苯菊酯的降解活力剩余70％。
In this study, the HP6890 Gas Chromatography (GC) was used to measure the recovery rates of bifenthrin, mecthrin and cypermethrin in water. The concentration of bifenthrin, mecthrin and cypermethrin were set at 1mg/ L, 10 mg/ L, 100 mg/ L respectively with water. The recovery rates of 3 pesticides were between 95. 47% and 103. 21%, and the coefficient ranged from 2. 21% to 4. 72%.
From the soil contaminated by pesticides, an E. cloacae strain wlOjl5 was isolated. This microorganism could use bifenthrin, mecthrin and cypermethrin as its unique carbon source. The best growth conditions for wlOjl5 was pH=7. 0, at 30 C . The degradation rates to bifenthrin, mecthrin and cypermethrin were 52.57%, 50.13%, 54.31% respectively. After 72 hours, the microorganism had the highest enzyme product and the highest enzyme activity of roughly 8U/mL (I) refers to mg/min).
The enzyme has the highest degradation rate at pH=7. 5 and 40 C . Bifenthrin, mecthrin and cypermethrin could be degraded 75%, 58% and 78% respectively. The enzyme has
Km=440. 58nmol/mL and Vmax=50. 25nmol/min for bifenthrin;
Km=332.23nmol/mL and Vmax=48. 08nmol/min for mecthrin and
Km=372.00nmol/mL and Vmax=63. 29nmol/min for cypermethrino
As far as biphenthrin is concerned, through the procedure of the ammonium sulfate precipitation, the gel gum filter and the ion exchange, the enzyme attains the specific ratio of 125.7U/mg, the purified ratio of 6.37, and the recovery rate of 91.7%. With the SDS-PAGE electrophoresis, the protein presented as a dark line. Compared with the protein marker, the molecular weight of the enzyme is roughly 65 KD. The IEF-PAGE measurement showed pI of the enzyme was 6. 8. The enzyme has 70% of the total degradation activity after one month restoration at 4C.
引文
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