Abstract: Using Pichia pastoris X-33 as host,the expression level,optimum pH and optimum temperature of polyphenol oxidase(CueO)from Escherichia coli were measured at the shake flask level,and the effect of CueO's signal peptide,leader peptide,third domain,and the "cap" structure of the linker region on its function were investigated. The study demonstrated that the highest expression,highest enzyme activity,optimum pH and optimum temperature were 81.26 μg/mL,356.67 U/L,2.5 and 45℃ from CueO at methanol induction for 72 h,respectively. Meanwhile,results showed that compared with the wild type,the expression level and enzyme activity were increased to 3.08 times and 3.5 times,respectively,after the self-signal peptide of CueO was removed. The secretion of CueO was hindered,after the leading peptide was removed. Although the protein concentration was increased,but the enzyme activity was completely lost,after the third domain of CueO was removed. Thereafter,CueO's enzyme activity was lost,regardless of whether the "cap" structure of the linker region was deleted or replaced. More importantly,this result is completely different from the current literature reports. The study laid some theoretical foundation for the industrial application of CueO.