TY - JOUR A1 - Haeger, Gerrit A1 - Jolmes, Tristan A1 - Oyen, Sven A1 - Jaeger, Karl-Erich A1 - Bongaerts, Johannes A1 - Schörken, Ulrich A1 - Siegert, Petra T1 - Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis JF - Applied Microbiology and Biotechnology N2 - N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from Paraburkholderia monticola DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC–MS and NMR. KW - Chaperone KW - Biocatalysis KW - Aminoacylase KW - Acylation KW - Acyl-amino acids KW - Biosurfactants Y1 - 2024 U6 - https://doi.org/10.1007/s00253-023-12868-8 SN - 1432-0614 N1 - Corresponding author: Petra Siegert IS - 108 PB - Springer CY - Berlin ER - TY - JOUR A1 - Haeger, Gerrit A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - A convenient ninhydrin assay in 96-well format for amino acid-releasing enzymes using an air-stable reagent JF - Analytical Biochemistry N2 - An improved and convenient ninhydrin assay for aminoacylase activity measurements was developed using the commercial EZ Nin™ reagent. Alternative reagents from literature were also evaluated and compared. The addition of DMSO to the reagent enhanced the solubility of Ruhemann's purple (RP). Furthermore, we found that the use of a basic, aqueous buffer enhances stability of RP. An acidic protocol for the quantification of lysine was developed by addition of glacial acetic acid. The assay allows for parallel processing in a 96-well format with measurements microtiter plates. Y1 - 2022 U6 - https://doi.org/10.1016/j.ab.2022.114819 SN - 1096-0309 IS - 624 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Haeger, Gerrit A1 - Probst, Johanna A1 - Jaeger, Karl-Erich A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Novel aminoacylases from Streptomyces griseus DSM 40236 and their recombinant production in Streptomyces lividans JF - FEBS Open Bio N2 - Amino acid-based surfactants are valuable compounds for cosmetic formulations. The chemical synthesis of acyl-amino acids is conventionally performed by the Schotten-Baumann reaction using fatty acyl chlorides, but aminoacylases have also been investigated for use in biocatalytic synthesis with free fatty acids. Aminoacylases and their properties are diverse; they belong to different peptidase families and show differences in substrate specificity and biocatalytic potential. Bacterial aminoacylases capable of synthesis have been isolated from Burkholderia, Mycolicibacterium, and Streptomyces. Although several proteases and peptidases from S. griseus have been described, no aminoacylases from this species have been identified yet. In this study, we investigated two novel enzymes produced by S. griseus DSM 40236ᵀ . We identified and cloned the respective genes and recombinantly expressed an α-aminoacylase (EC 3.5.1.14), designated SgAA, and an ε-lysine acylase (EC 3.5.1.17), designated SgELA, in S. lividans TK23. The purified aminoacylase SgAA was biochemically characterized, focusing on its hydrolytic activity to determine temperature- and pH optima and stabilities. The aminoacylase could hydrolyze various acetyl-amino acids at the Nα -position with a broad specificity regarding the sidechain. Substrates with longer acyl chains, like lauroyl-amino acids, were hydrolyzed to a lesser extent. Purified aminoacylase SgELA specific for the hydrolysis of Nε -acetyl-L-lysine was unstable and lost its enzymatic activity upon storage for a longer period but could initially be characterized. The pH optimum of SgELA was pH 8.0. While synthesis of acyl-amino acids was not observed with SgELA, SgAA catalyzed the synthesis of lauroyl-methionine. KW - Streptomyces lividans KW - recombinant expression KW - Streptomyces griseus KW - ε-lysine acylase KW - α-aminoacylase Y1 - 2023 U6 - https://doi.org/10.1002/2211-5463.13723 SN - 2211-5463 N1 - Corresponding author: Petra Siegert VL - 13 IS - 12 SP - 2224 EP - 2238 PB - Wiley CY - Hoboken, NJ ER - TY - PAT A1 - Degering, Christian A1 - Wieland, Susanne A1 - Islam, Shohana A1 - Lindner, Claudia A1 - Siegert, Petra A1 - Falkenberg, Fabian A1 - Bongaerts, Johannes T1 - Wasch- und Reinigungsmittel enthaltend Protease (aus Fictibacillus arsenicus) N2 - Die Erfindung liegt auf dem Gebiet der Enzymtechnologie. Die Erfindung betrifft Proteasen aus Fictibacillus arsenicus, die insbesondere im Hinblick auf den Einsatz in Wasch- und Reinigungsmitteln verwendet werden können, alle hinreichend ähnlichen Proteasen mit einer entsprechend ähnlichen Sequenz zu SEQ ID NO:1 und für sie codierende Nukleinsäuren. Die Erfindung betrifft ferner deren Herstellung sowie Verfahren zur Verwendung dieser Proteasen, deren Verwendung als solche sowie diese enthaltende Mittel, insbesondere Wasch- und Reinigungsmittel. Y1 - 2024 ER - TY - PAT A1 - Degering, Christian A1 - Wieland, Susanne A1 - Islam, Shohana A1 - Lindner, Claudia A1 - Bongaerts, Johannes A1 - Siegert, Petra A1 - Falkenberg, Fabian T1 - Wasch- und Reinigungsmittel enthaltend Protease (aus Metabacillus indicus) N2 - Die Erfindung liegt auf dem Gebiet der Enzymtechnologie. Die Erfindung betrifft Proteasen aus Metabacillus indicus, die insbesondere im Hinblick auf den Einsatz in Wasch- und Reinigungsmitteln verwendet werden können, alle hinreichend ähnlichen Proteasen mit einer entsprechend ähnlichen Sequenz zu SEQ ID NO:1 und für sie codierende Nukleinsäuren. Die Erfindung betrifft ferner deren Herstellung sowie Verfahren zur Verwendung dieser Proteasen, deren Verwendung als solche sowie diese enthaltende Mittel, insbesondere Wasch- und Reinigungsmittel. Y1 - 2024 ER - TY - JOUR A1 - Ribitsch, Doris A1 - Heumann, Sonja A1 - Karl, Wolfgang A1 - Gerlach, Jochen A1 - Leber, R. A1 - Birner-Gruenberger, R. A1 - Gruber, K. A1 - Eiteljoerg, I. A1 - Remler, Peter A1 - Siegert, Petra A1 - Lange, J. A1 - Maurer, Karl-Heinz A1 - Berg, G. A1 - Guebitz, G. M. A1 - Schwab, H. T1 - Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli JF - Journal of biotechnology N2 - A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 °C. Specific activity of StmPr2 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 17 ± 2 U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2. KW - Alginate beads KW - Stenotrophomonas maltophilia KW - Detergent protease Y1 - 2012 U6 - https://doi.org/10.1016/j.jbiotec.2011.09.025 SN - 1873-4863 (E-Journal); 0168-1656 (Print) VL - 157 IS - 1 SP - 140 EP - 147 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Ribitsch, Doris A1 - Karl, Wolfgang A1 - Birner-Gruenberger, R. A1 - Gruber, K. A1 - Eiteljoerg, I. A1 - Remler, Peter A1 - Wieland, S. A1 - Siegert, Petra A1 - Maurer, Karl-Heinz A1 - Schwab, H. T1 - C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli JF - Journal of biotechnology N2 - Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477. This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40 °C and pH 7–11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40 °C and pH = 9.5 resulted in KM = 0.23 ± 0.01 mM and kcat = 167.5 ± 3.6 s⁻¹. MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease. Y1 - 2010 U6 - https://doi.org/10.1016/j.jbiotec.2010.09.947 SN - 1873-4863 (E-Journal); 0168-1656 (Print) VL - 150 IS - 3 SP - 408 EP - 416 PB - Elsevier CY - Amsterdam ER -