Background:　Protein　glutaminase　specifically　deamidates　glutamine　residue　in　protein　and　therefore　significantly　improves　protein　solubility　and　colloidal　stability　of　protein　solution.　In　order　to　improve　its　preparation　efficiency,　we　exploited　the　possibility　for　its　secretory　expression　mediated　by　twin-arginine　translocation　(Tat)　pathway　in　Bacillus　licheniformis.　Results:　The　B.　licheniformis　genome-wide　twin-arginine　signal　peptides　were　analyzed.　Of　which,　eleven　candidates　were　cloned　for　construction　of　expression　vectors　to　mediate　the　expression　of　Chryseobacterium　proteolyticum　protein　glutaminase　(PGA).　The　signal　peptide　of　GlmU　was　confirmed　that　it　significantly　mediated　PGA　secretion　into　media　with　the　maximum　activity　of　0.16　U/ml　in　Bacillus　subtilis　WB600.　A　mutant　GlmU-R,　being　replaced　the　third　residue　aspartic　acid　of　GlmU　twin-arginine　signal　peptide　with　arginine　by　site-directed　mutagenesis,　mediated　the　improved　secretion　of　PGA　with　about　40%　increased　(0.23　U/ml).　In　B.　licheniformis　CBBD302,　GlmU-R　mediated　PGA　expression　in　active　form　with　the　maximum　yield　of　6.8　U/ml　in　a　25-1　bioreactor.　Conclusions:　PGA　can　be　produced　and　secreted　efficiently　in　active　form　via　Tat　pathway　of　B.　licheniformis,　an　alternative　expression　system　for　the　industrial-scale　production　of　PGA.　(C)　2019　Pontificia　Universidad　Catolica　de　Valparaiso.　Production　and　hosting　by　Elsevier　B.V.　All　rights　reserved.