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A proline specific endopeptidase gene pep was amplified from Aspergillus niger TCCC41013 by RT-PCR. This 1581-bp length pep gene encodes a signal peptide of 22 amino acid residues and a mature peptide of 504 amino acid residues. The pep gene exhibits over 99% identities at both the nucleotide and amino acid sequence levels with a gene previously isolated from A niger CBS 513.88. To achieve high level expression of the proline specific endopeptidase (PEP) in Bacillus subtilis, a pep gene variant termed pepm was generated by first PCR-amplifying the portion of the pep gene encoding the mature peptide and then site-directed mutagenesis to convert the amplified DNA fragment (5 CCC to 5 CCA, 5 CCC to 5 CCG and 4 ACT to 4 ACA, respectively). The pepm gene was subsequently cloned into the expression vector pWB980 to produce pWB980-pepm which was then introduced into B. subtilis WB600 for PEP overexpression. SDS-PAGE analysis showed that the molecular weight of the expressed product was about 55 kDa, consistent with the expected size of PEP. The enzyme activity of the recombinant PEP in fermentation supernatant was as high as 832.6 mU/ml. This recombinant enzyme could effectively reduce chill haze in beer.