title: Peptide synthesis in neat organic solvents with novel thermostable proteases.
abstract: Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli. The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80°C and 60°C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40-50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions.
authors.affiliation: Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Ana
authors.initials: A
authors.lastname: Toplak
authors.affiliation: Enzypep B.V., Urmonderbaan 22, Chemelot Campus, 6167 RD Geleen, The Netherlands.
authors.forename: Timo
authors.initials: T
authors.lastname: Nuijens
authors.affiliation: Enzypep B.V., Urmonderbaan 22, Chemelot Campus, 6167 RD Geleen, The Netherlands.
authors.forename: Peter J L M
authors.initials: PJ
authors.lastname: Quaedflieg
authors.affiliation: Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Bian
authors.initials: B
authors.lastname: Wu
authors.affiliation: Biotransformation and Biocatalysis, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Electronic address: d.b.janssen@rug.nl.
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
mesh_terms: D019840:2-Propanol; D000096:Acetone; D000097:Acetonitriles; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis; D011232:Chemical Precipitation; D060326:Chemistry Techniques, Synthetic; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics; D004126:Dimethylformamide; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D006358:Hot Temperature; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D012997:Solvents; D013824:Thermus / Q000201:enzymology* / Q000235:genetics; D020002:tert-Butyl Alcohol
chemical_list: D000097:Acetonitriles; D001426:Bacterial Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012997:Solvents; D000096:Acetone; D004126:Dimethylformamide; D010447:Peptide Hydrolases; D020002:tert-Butyl Alcohol; D019840:2-Propanol; C032159:acetonitrile
keywords: Organic solvent; Peptide synthesis; Protease; Subtilase; Thermophile; Thermostable