Study On Key Enzymes Of Pyrrolidine Pathway Degrading Nicotine By Pseudomonas ZZ-5

Nicotine is a natural alkaloid,which can cause damage to many tissues such as heart,brain,lung and blood vessel after entering the human body and is easy to cause many diseases such as lung cancer.China is a big country of tobacco production and consumption.The waste produced in the process of tobacco processing contains high concentration of nicotine,which poses a great threat to the environment and human health.Microbial treatment of this type of pollution is an efficient,reliable and non-secondary pollution green treatment,and the biodegradation method has mild reaction conditions,simple operation and low cost.It is an effective means to fully degrade nicotine.At the same time,2,5-dihydroxypyridine,the intermediate product of nicotine degradation by microbial enzymatic method,is an important precondition for pharmaceutical synthesis and has great potential for application.Therefore,it has great prospects to isolate and purify the key enzymes which can degrade nicotine efficiently,study their degradation mechanism and give full play to their degradation ability.In this paper,Pseudomonas sp.ZZ-5 was used as the experimental strain,and the purified 6-hydroxy-3-succinylpyridine Hydroxylase(HSPH_ZZ)was obtained by protein purification steps such as?NH₄?₂SO₄ precipitation,DEAE sepharose,Pheny1 sepharose and Superdex-200 gel filtration.SDS-PAGE showed a single band with a molecular weight of approximately 44.3 KDa.The protein was purified 17.6 times to give a yield of 13.4%and a specific activity of 5.1 U/mg.The N-terminal amino acid sequence of HSPHzz was M-S-G-H-L-R-V-I-V-I-V-G-G-P by protein N-terminal sequencing.The full length of HSPHzz gene was obtained by molecular cloning.The gene size was 1206 bp and contained401 amino acids.The sequence of HSPHzz amino acid was highly similar to that of HSPHs from Pseudomonas putida.HSPHzz gene was cloned into prokaryotic expression vector pET22b,and the recombinant plasmid pET22b-HSPHzz was obtained.HSPHzz was expressed and purified,and its enzymatic properties were studied.The molecular weight of recombinant HSPHzz was 45 KDa;the optimum temperature and pH were 30?and 8.5;the enzyme activity was not significantly affected by Mg²⁺,Ni²⁺,Ca²⁺,Mn²⁺,K⁺,Na⁺,Cu²⁺,Co²+,Zn²⁺and Fe²⁺;methanol,acetone,formaldehyde,dimethylformamide,chloroform,toluene and 1%?w/v?Triton X-100 significantly inhibited the enzyme activity;under the optimum conditions,the enzyme produced 2,5-DHP?85.3 mg/L?within 40 minutes and the conversion rate was 74.9%?w/w?.In order to improve the catalytic performance and reduce the cost of biocatalytic process,Immobead 150 was used to immobilize HSPHzz?ImmHSPHzz?and study its catalytic performance.The optimum pH of ImmHSPHzz was 9.0,the optimum temperature was 35?,and the optimum concentration of enzyme and substrate were 30 mg/L and 0.75 mM,respectively.Under the optimum conditions,2,5-DHP of 94.5 mg/L was produced after 30minutes,and the conversion rate was 85.4%.After 8 reaction cycles and 6 days storage,the initial enzyme activities of 51.3%and 75.0%were retained respectively.Compared with free HSPH_ZZ,ImmHSPHzz has a wider range of pH and temperature tolerance,higher conversion and storage stability.The key enzyme NOX was preliminarily analyzed.The purified recombinant NOX was cloned and expressed in Escherichia coli.The enzyme was an extracellular enzyme with a molecular weight of 55 KDa.Nicotine was used as a substrate to detect its activity.The enzyme degraded nicotine to produce pseudooxynicotine,which was initially identified as nicotine oxidase.A preliminary analysis of nicotine oxidase?NOX?was carried out,and purified recombinant NOX was obtained by cloning and expressing in Escherichia coli.The enzyme was an extracellular enzyme with a molecular weight of 55 KDa and nicotine as a substrate.Using nicotine as a substrate,the enzyme activity was detected by LC-MS analysis.The enzyme degraded nicotine to form pseudooxynicotine,and the enzyme was initially determined to be nicotine oxidase.