Cloning,Expression And Purification Of Cyclohexylamine Oxidase From Pseudomonas Plecoglossicida NyZ12

Cyclohexylamine(chemical formula C6H13N),also named as six hydrogen aniline or amino cyclohexane,is an important chemical intermediate.Cyclohexylamine is released into the environment in the process of production and usage.Experiments have confirmed that this compound is carcinogenic.As cyclohexylamine is widely used in the industrial production and it is significantly harmful to the human body,it is necessary to take certain measures to control and degrade industrial waste amine generated.The microbial degradation is low cost,low energy consumption.The elimination of cyclohexylamine and it’s residues in environment via biodegradation is one of the effective ways.Currently,only two pure cultures were reported that they could use cyclohexylamine as sole carbon and nitrogen sources to grow,One is a Gram-positive bacteria Brevibacterium oxydans IH-35 A which isolated by Iwaki H.The other is Pseudomonas plecoglossicida NyZ12 isolated by Zhou Ning-Yi research group in Wuhan Institute of Virology,Chinese Academy of scienses.Pseudomonas plecoglossicida NyZ12 is a newly discovered Gram-negative bacterium that has the ability to degrade cyclohexylamine and it has been confirmed that the cyclohexylamine metabolic pathway in Pseudomonas plecoglossicida NyZ12 is via cyclohexanone.In this study,the whole-genome of NyZ12 was sequenced.Through comparative analysis and alignment,five candidate genes which maybe encode cyclohexylamine oxidase were cloned and expressed under different promoters.We used a variety of experimental methods to determine the actual encoding gene,purified the enzyme and preliminarily detected the enzyme activity of cyclohexylamine oxidase.Main research contents are as follows:1.Firstly,studies on the ability of wild strains NyZ12 to degrade cyclohexylamine showed that it could degrade 10mmol/L cyclohexylamine within 20 hours.Set up the method that detected the concentrate of cyclohexylamine by spectrophotometry.And confirmed the cyclohexylamine oxidase was induced by substrate.2.Using bioinformatics tools to analyze the whole-genome sequencing and then find five candidate genes,amo2631,amo4207,amo5539,amo0425,amo4637,which maybe encode cyclohexylamine oxidase.These genes were cloned into plasmid pUC18 respectively and the result recombinant plasmids were transformed into E.coli DH5?.3.Using spectrophotometry detect the ability of the series of pUC18 engineered bacteria to degrade substrate cyclohexylamine,it proved only DH5?[pUC18-2631] has the ability to degrade cyclohexylamine,thus we preliminarily determined that amo2631 is a key gene that encoding cyclohexylamine oxidase in these five genes.4.By real-time fluorescence quantitative PCR to study the expression level of these five genes after substrate induction,and we found that the expression level of gene amo2631 significantly increased.This is consistent with transcriptome analysis results,and it’s the further evidence shoued that amo2631 is likely to encode the key enzyme-cyclohexylamine oxidase in NyZ12.5.In order to make the expression level of target genes high and get soluble proteins and easily purified,these five genes were cloned into plasmid pVLT33 primers with His tag desigened,and the result recombinant expression vecteors were transformed into E.coli DH5?.Gas chromatography was used to identify whether detect the series of pVLT33 engineered bacteria could degraded substrate cyclohexylamine and produce the products.The results showed that only intermediate cyclohexanone was detected in the supernatant of DH5?[pVLT33-2631],so it proved that amo2631 was the key gene coding cyclohexylamine oxidase in NyZ12.6.The expression of target genes in the series of pVLT33 were realized in engineered bacteria without the formation of inclusion bodies.Horseradish peroxidase were adopted to measure the crude amine oxidase enzyme activity.The results shows that the crude enzyme concentration of DH5?[pVLT33-2631] is 914.97 U/m L,so it futher proved that the amo2631 has the catalytic activity towards substrate cyclohexylamine.7.The protein amo2631 with six histidine tag were purfied from induced DH5? [pVLT33-2631] cell,and a large amount of purified pure enzyme were acquired by His Trap HP affinity chromatography.