Related Enzymes’ Screening And Alcohol Dehydrogenase Expression Of Arthrobacter Sp. HW08 On Degrading Swainsonine

Locoweeds are the poisonous plants of Astragalus spp. and Oxytropis spp. They distribute in the northwest rangeland region of China widely. Their toxic constituent swainsonine can lead to animal poisoning disease with the characteristics of neurological and reproductive disorders, which resulting in serious harm to the development of grassland animal husbandry industry of China. However, there is a lack of effective control methods so far, microbial degradation of SW provides a new thought to solve the locoweed poisoning disease. Our laboratory had isolated a Arthrobacter sp. HW08 strain on degrading SW. In order to confirm the Arthrobacter sp. HW08’s related enzymes on degrading swainsonine, this study adopted differential proteomics research approach along with the liquid chromatography tandem mass spectrometry(LC-MS/MS) to Arthrobacter sp. HW08, screened degrading SW enzymes. Real-time quantitative PCR was used to verify genes’ relative expression. Then we cloned and expressed the NADP-dependent alcohol dehydrogenase A1R6C3, then detected the degrading SW capacity. The achieved results were as follow:(1) Lable-free quantitative differential proteomics was adopted to analysis on Arthrobacter sp. HW08 whether induced by SW or not. Through comparing the differences in protein expression, combined Gene Ontology and KEGG pathway analysis, we screened 8 enzymes which relate to SW degradation. They were: Sugar phosphate isomerase A1R5X7; Putative sugar phosphate isomerase/epimerase A1R5X8; NADP-dependent alcohol dehydrogenase A1R6C3; Uncharacterized protein A1R872; NAD(P) transhydrogenase subunit beta A1RBS8; Cold-shock DNA-binding protein family A0JY59; Acetyl-CoA acetyltransferase A0JZ95 and putative cold shock protein J7LTJ9.Gene relative expression of 8 proteins were validated on transcription level by real-time quantitative PCR(qRT-PCR). The results showed that the experimental group genes AAur-1890(3.41), AAur-1891(2.27), AAur-2040(2.12), Arth-2600(2.27), Arth-2986(3.73), and ARUE-c09510(11.4) significant increased in comparison to the control group(p < 0.05), which were consistent with the expression on protein level. However, AAur-2719(0.81) and AAur-4018(0.61) was not significant different to the control group.(2) Prokaryotic expression and purification of NADP-dependent alcohol dehydrogenase A1R6C3, the experiment detected the purified enzyme ability on degradation SW. As a result, the recombinant bacterial E.coli BL21-pET32a-AAur 2040 expressed a protein with the molecular weight 56 kDa, which was consistent with the expected size. Then we optimized the induction conditions of IPTG: when the concentration of IPTG induction was 1 mmol/L, the expression protein reached the highest level in 3 hours. The target protein was purified by affinity chromatography with His·tag and identified by Western blot correctly. Alcohol dehydrogenase which concentration was about 0.5 mg/mL can degrade about 18.52 μg of SW in 1 h, the degradation rate was 46.3%.