Studies On The Redundancy Mechanism Of The Initial Step Of Cyclohexylamine Degradation In Pseudomonas Plecoglossicida NyZ12

As an important fine chemical intermediate,cyclohexylamine released into environment polluted the atmosphere and water.This compound also has certain carcinogenicity.Pseudomonas plecoglossicida NyZ12 was isolated using cyclohexylamine as the sole carbon source,nitrogen source and energy source.The molecular biological mechanism of cyclohexylamine degradation in NyZ12 has not been clarified so far.In the previous research on the degradation mechanism of cyclohexylamine in Pseudomonas NyZ12,amo2631 was cloned into the broad host expression vector pVLT33 and expressed in the host Pseudomonas PaW340,and finally the protein encoded by amo2631 was purified.During the transformation,the production of the product cyclohexanone was also successfully detected,and the function of amo2631 was verified.However,amo2631 was knocked out using strategies of scarless gene knockout,the mutant was still able to grow well with cyclohexylamine as the sole carbon and nitrogen source,and only the growth rate was affected,indicating that the strain has a metabolic redundancy mechanism for the initial metabolic step of cyclohexylamine,which indicated two or more genes involved in the metabolic process of cyclohexylamine to cyclohexanone.In this study,we intended to explore the redundant genes involving in the initial metabolic steps of cyclohexylamine,and fully reveal the molecular mechanism of cyclohexylamine catabolism in Pseudomonas NyZ12.In order to achieve the purpose,bioinformatics analysis,genomic library construction and transposon insertion mutantion library have been used to screen the genes related to cyclohexylamine degradation.Among them,the predicted genes orf671 and orfN4 were obtained by bioinformatics analysis,and the genes orf566,orf567,orfN2 and orf5356 were screened after constructing the Pseudomonas NyZ12 genomic library.The transposon insertion mutant library did not screen related genes.After the above predicted genes were cloned into pUC18 vector and transformed in DH5?,the whole cell transformation was performed to detect the degradation of the substrate cyclohexylamine by spectrophotometer.The degradation of cyclohexylamine in DH5a[pUC 18-orf566]and DH5?[pUC 18-orf671]was obvious.orf566 and orf671 were cloned into pET28b expression vector and transformed into BL21(DE3)strain,respectively.The proteins were purified after the induction.The transformation assay of cyclohexylamine was performed in the crude enzyme solution,and the product was detected by GC-MS.The metabolic intermediate cyclohexanone(rentation time 3.300 min)was successfully detected in the orf671 transformation product.It was proved orf671 was one of the participating genes in the initial metabolic step of cyclohexylamine,and its mechanism of action needs to be studied further in the later stage.Due to the large size of orfN4,it was not expressed effectively in pUC18 and pET28b.Therefore,gene knockout of orf1727 was carried out.NyZ12?5 did not lose the ability to grow in cyclohexylamine as the sole carbon and nitrogen source,but its growth rate was obvious slower than that of NyZ12?4,so it may be a gene related to cyclohexylamine metabolism,which needs further confirmation by subsequent research.In addition,cyclohexylamine oxidase from Acinetobacter YT-02 was cloned and heterologous expressed,and its enzymatic properties were studied.The enzyme molecule is a dimeric protein with a molecular weight of about 91 kDa.The optimum temperature is 50?.This enzyme is thermolabile and loses its activity after incubation for 30 min at 50?.The metal ions Mg2+.Co2+ and K+have a certain inhibitory effect on the enzyme activity.The kinetic parameters Km and Vmax were 0.25±0.02 mM and 4.3 ±0.083 pMmiN-1,respectively.