One pair of primers were designed and synthesized on the base of the cDNA sequence encoding Schizosaccharomyces pombe N-glycanase reported on the GenBank. The cDNA sequence encoding Peptide N-glycanase was cloned from the Schizosaccharomyces pombe by RT-PCR. And then the RT-PCR product was cloned into the expression vector pET-15b. The expression vector pET-15b(+)/Png1p was transformed into E. coli BL21(DE3). The results showed that the relative molecular weight of the enzyme was determined to be approximately 39 kD using SDS-PAGE. The expression products after induction and purification can catalyze the cleavage of N-linked oligosaccharides from glycoprotein coped with heat, but have no action on the native glycoprotein with the help of DTT. The percentage of deglycosylated RNase B treated with equate Png1p in different reaction temperature, pH, concentration of DTT and denatured temperature showed that the optimum temperature, the optimum pH is 30°C; the optimum concentration of DTT is 10 mmol/L and the optimum denatured temperature is 100°C.