| Laccases(EC1.10.3.2)are a type of copper-containing polyphenol oxidases,which have a wide range of catalytic specificity for aromatic compounds,steroids and biopigments.Statistics showed that laccases could catalyze the oxidation of about 300 different types of substrates,and they had great application potential in furniture decoration,low-pollution textile,paper decolorization,daily washing food and beverage industries.In recent years,more and more attentions had been paid to the study of laccase.Laccases have a wide range of sources.They could be broadly divided into plant laccases,insect laccases,fungal laccases and bacterial laccases.Compared with other laccases,bacterial laccases have many advantages,such as wider source,shorter growth cycle,better thermal stability and more wider pH range adaptativily.Escherichia coli laccase,as an important bacterial laccases,had been reported in more and more literatures.Although some literatures had studied on E.coli laccase,the effects of its signal peptide,leading peptide and third domain on its function had not been reported.Although some studies had been reported on the structural differences between E.coli laccase and fungal laccase,the effects of these differences on their functions were still unclear.In this study,the structure and their fuctions of laccase CueO from E.coli was primarily reveated.Firstly,the heterologous expression of CueO in Pichia pastoris was realized,and the enzymatic characteristics of the expressed product were studied.In order to elucidate the effects of the signal peptide,lead peptide,third domain and the unique"cap" structure of different domains on its function,the mutants were constructed by partial deletion or substitution the Key amino acid of CueO,and the expression products of there mutants were analyzed.In order to further improve the expression of mutant genes,we optimized the mutant genes according to the codon preference of P.pastoris,and achieved its high heterologous expression.The main results of this study were as follows:(1)E.coli laccase CueO was cloned and expressed in P.pastoris,and the optimum substrate was ABTS,the optimum reaction pH was 3.0,the optimum reaction temperature was 450C,the optimum reaction time was 10 min,and the optimum Cu2+ion was 2.0 mM.After 72 hours of methanol induction in shake flask fermentation,the highest protein expression level was 81.26 ug/mL and the highest enzyme activity was 356.67 U/L.Among the seven common metal ions,except the Mn2+ions,the activity of CueO was inhibited by other metal ions.(2)The mutants were successfully constructed and fransformed into P.pastoris.I was found that the deletion of signal peptide(CueO-?Sp)increased the protein expression and enzyme activity of CueO,which were 250.29 ug/mL and 1248.36 U/L,respectively,3.08 times and 3.5 times higher than that of the wild type CueO.Although the deletion of the third domain increased the protein expression of CueO,it resulted in the inactivation of CueO,and the deletion of the leading peptide hindered the secretion of protein.Moreover,the deletion or replacement of the alpha-helix in the connecting segments between CueO domains would lead to the inactivation of CueO.(3)By referring to the codon preference of P.pastoris,the mutant CueO-?Sp,which removes signal peptide,was optimized.Two optimized genes were obtained and introduced into P.pastoris to realize heterologous expression.The protein expression and enzyme activity of the optimized gene were higher than those of the mutant before optimization.The highest protein expression was 450.19 ug/mL,and the highest enzyme activity was 2880.69 U/L,which were 1.8 and 2.3 times of CueO-?Sp before optimization,and 5.54 and 8.07 times of wild type CueO respectively. |
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