Pseudoxanthomonas indica and application thereof in degrading chloronicotinyl insecticide imidacloprid

[0021] Example 1: Isolation, screening, identification and biological characteristics of biodegradable IMI strains

[0022] 1. Strain isolation

[0023] Collect soil from Xianlin area, Nanjing City, Jiangsu Province, add 2g of soil to 18mL sterile water containing 5 glass beads, shake for 10min, let it stand for 5min, take 1ml of the suspension and add it to 19ml of mineral salt medium containing 200mg / LIMI . The composition of the mineral salt medium is: 1.36g / LKH 2 PO 4 , 2.13 gL Na 2 HPO 4 , 0.5gL MgSO 4 ·7H 2 O and 10ml / L metal ion liquid, pH7.5. The composition of the metal ionic liquid is: 0.40gL CaCl 2 2H 2 O,0.30g / L H 3 BO 3 ,0.04gL CuSO 4 ·5H 2 O, 0.10g / L KI, 0.20g / L FeSO 4 ·7H 2 O, 0.40g / LMnSO 4 ·7H 2 O, 0.20g / LNaMoO 4 2H 2 O and 10.0mL / L concentrated hydrochloric acid. The samples were incubated for one month at 30°C in a shaker with a rotational speed of 220rpm. Take 100 μl sample and dilute to 10 -3 and 10 -4 Afterwards, LB plates were coate...

[0030] The Pseudoxanthomonas indica CGMCC6648 stored at -80°C was streaked on LB solid medium and cultured in a 30°C incubator. A single colony was picked and inoculated into a 100 mL Erlenmeyer flask containing 25 mL of LB liquid medium, and cultured at 30°C with shaking at 220 rpm for 24 hours. Inoculate 1% of the inoculum into a 500mL Erlenmeyer flask containing 100mL LB liquid medium, culture at 30°C for 12h with shaking at 220rpm, when OD 600 When the value reaches 5, take 10ml of the bacterial liquid and centrifuge at 8000rpm for 5min at 4°C to collect the bacterial cells. Phosphate buffered saline (0.2mol / LNa 2 HPO 4 / KH 2 PO 4 , pH7.5) After washing twice, suspend in 10mL IMI phosphate buffer containing 500mgL. Take 2ml of the above-mentioned transformation reaction solution, divide it into three 50ml centrifuge tubes, each tube contains 2ml, and shake and culture at 30°C and 220rpm for 48h. HPLC analysis ( image 3 ) IMI degradation and 5-hydroxy IMI generation...

[0032] Basically the same as example two, just added the glucose of 100mmol / L in the phosphate buffer solution, HPLC analysis ( Figure 4 ) results showed that IMI decreased by 0.66mmol / L, and 0.59mmol / L 5-hydroxy IMI was generated at the same time, and the degradation rate of IMI was 43.6%. The above results indicated that glucose can greatly promote the degradation of IMI and the generation of 5-hydroxy IMI.

[0033] Under the same culture and transformation conditions, Stenotrophomonas maltophilia CGMCC1.1788 was used to replace Pseudomonas CGMCC6648, HPLC analysis ( Figure 5 ) results showed that IMI decreased by 0.48mmol / L, and 0.40mmol / L 5-hydroxy IMI was generated at the same time, and the degradation rate of IMI was 28.2%. The above results indicated that Pseudoxanthomonas indica CGMCC6648 had much higher IMI degradation and hydroxylation abilities than Stenotrophomonas maltophilia CGMCC1.1788.