title: Efficient Extracellular Expression of Metalloprotease for Z-Aspartame Synthesis.
abstract: Metalloprotease PT121 and its mutant Y114S (Tyr114 was substituted to Ser) are effective catalysts for the synthesis of Z-aspartame (Z-APM). This study presents the selection of a suitable signal peptide for improving expression and extracellular secretion of proteases PT121 and Y114S by Escherichia coli. Co-inducers containing IPTG and arabinose were used to promote protease production and cell growth. Under optimal conditions, the expression levels of PT121 and Y114S reached >500 mg/L, and the extracellular activity of PT121/Y114S accounted for 87/82% of the total activity of proteases. Surprisingly, purer protein was obtained in the supernatant, because arabinose reduced cell membrane permeability, avoiding cell lysis. Comparison of Z-APM synthesis and caseinolysis between proteases PT121 and Y114S showed that mutant Y114S presented remarkably higher activity of Z-APM synthesis and considerably lower activity of caseinolysis. The significant difference in substrate specificity renders these enzymes promising biocatalysts.
authors.affiliation: College of Biotechnology and Pharmaceutical Engineering and ‡School of Pharmaceutical Sciences, Nanjing Tech University , No. 30 Puzhu South Road, Nanjing 211816, China.
authors.forename: Fucheng
authors.initials: F
authors.lastname: Zhu
authors.affiliation: College of Biotechnology and Pharmaceutical Engineering and ‡School of Pharmaceutical Sciences, Nanjing Tech University , No. 30 Puzhu South Road, Nanjing 211816, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.affiliation: College of Biotechnology and Pharmaceutical Engineering and ‡School of Pharmaceutical Sciences, Nanjing Tech University , No. 30 Puzhu South Road, Nanjing 211816, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.affiliation: College of Biotechnology and Pharmaceutical Engineering and ‡School of Pharmaceutical Sciences, Nanjing Tech University , No. 30 Puzhu South Road, Nanjing 211816, China.
authors.forename: Bingfang
authors.identifier: http://orcid.org/0000-0002-4502-9531
authors.initials: B
authors.lastname: He
mesh_terms: D001089:Arabinose / Q000378:metabolism; D001218:Aspartame / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D001089:Arabinose; D045726:Metalloproteases; D001218:Aspartame
keywords: Escherichia coli, arabinose; Z-aspartame; extracellular expression; metalloprotease