@article{MartinezJakobTuetal.2013, author = {Martinez, Ronny and Jakob, Felix and Tu, Ran and Siegert, Petra and Maurer, Karl-Heinz and Schwaneberg, Ulrich}, title = {Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution}, series = {Biotechnology and bioengineering}, volume = {Vol. 110}, journal = {Biotechnology and bioengineering}, number = {Iss. 3}, publisher = {Wiley}, address = {Weinheim}, issn = {1097-0290 (E-Journal); 0006-3592 (Print); 0368-1467 (Print)}, pages = {711 -- 720}, year = {2013}, language = {en} } @article{RibitschHeumannKarletal.2012, author = {Ribitsch, D. and Heumann, S. and Karl, W. and Gerlach, J. and Leber, R. and Birner-Gruenberger, R. and Gruber, K. and Eiteljoerg, I. and Remler, P. and Siegert, Petra and Lange, J. and Maurer, Karl-Heinz and Berg, G. and Guebitz, G. M. and Schwab, H.}, title = {Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli}, series = {Journal of biotechnology}, volume = {157}, journal = {Journal of biotechnology}, number = {1}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1873-4863 (E-Journal); 0168-1656 (Print)}, doi = {10.1016/j.jbiotec.2011.09.025}, pages = {140 -- 147}, year = {2012}, abstract = {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.}, language = {en} } @article{RibitschKarlBirnerGruenbergeretal.2010, author = {Ribitsch, D. and Karl, W. and Birner-Gruenberger, R. and Gruber, K. and Eiteljoerg, I. and Remler, P. and Wieland, S. and Siegert, Petra and Maurer, Karl-Heinz and Schwab, H.}, title = {C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli}, series = {Journal of biotechnology}, volume = {150}, journal = {Journal of biotechnology}, number = {3}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1873-4863 (E-Journal); 0168-1656 (Print)}, doi = {10.1016/j.jbiotec.2010.09.947}, pages = {408 -- 416}, year = {2010}, abstract = {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.}, language = {en} } @article{SiegertMcLeishBaumannetal.2005, author = {Siegert, Petra and McLeish, Michael J. and Baumann, Martin and Iding, Hans and Kneen, Malea M. and Kenyon, George L. and Pohl, Martina}, title = {Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida}, series = {Protein engineering, design, and selection : peds}, volume = {Vol. 18}, journal = {Protein engineering, design, and selection : peds}, number = {Iss. 7}, issn = {1460-213X (E-Journal); 1741-0134 (E-Journal); 0269-2139 (Print); 1741-0126 (Print)}, pages = {345 -- 357}, year = {2005}, language = {en} } @article{HaegerProbstJaegeretal.2023, author = {Haeger, Gerrit and Probst, Johanna and Jaeger, Karl-Erich and Bongaerts, Johannes and Siegert, Petra}, title = {Novel aminoacylases from Streptomyces griseus DSM 40236 and their recombinant production in Streptomyces lividans}, series = {FEBS Open Bio}, volume = {13}, journal = {FEBS Open Bio}, number = {12}, publisher = {Wiley}, address = {Hoboken, NJ}, issn = {2211-5463}, doi = {10.1002/2211-5463.13723}, pages = {2224 -- 2238}, year = {2023}, abstract = {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.}, language = {en} } @article{HaegerWirgesTanzmannetal.2023, author = {Haeger, Gerrit and Wirges, Jessika and Tanzmann, Nicole and Oyen, Sven and Jolmes, Tristan and Jaeger, Karl-Erich and Sch{\"o}rken, Ulrich and Bongaerts, Johannes and Siegert, Petra}, title = {Chaperone assisted recombinant expression of a mycobacterial aminoacylase in Vibrio natriegens and Escherichia coli capable of N-lauroyl-L-amino acid synthesis}, series = {Microbial Cell Factories}, journal = {Microbial Cell Factories}, number = {22}, publisher = {Springer Nature}, issn = {1475-2859}, doi = {10.1186/s12934-023-02079-1}, pages = {Article number: 77 (2023)}, year = {2023}, abstract = {Background Aminoacylases are highly promising enzymes for the green synthesis of acyl-amino acids, potentially replacing the environmentally harmful Schotten-Baumann reaction. Long-chain acyl-amino acids can serve as strong surfactants and emulsifiers, with application in cosmetic industries. Heterologous expression of these enzymes, however, is often hampered, limiting their use in industrial processes. Results We identified a novel mycobacterial aminoacylase gene from Mycolicibacterium smegmatis MKD 8, cloned and expressed it in Escherichia coli and Vibrio natriegens using the T7 overexpression system. The recombinant enzyme was prone to aggregate as inclusion bodies, and while V. natriegens Vmax™ could produce soluble aminoacylase upon induction with isopropyl β-d-1-thiogalactopyranoside (IPTG), E. coli BL21 (DE3) needed autoinduction with lactose to produce soluble recombinant protein. We successfully conducted a chaperone co-expression study in both organisms to further enhance aminoacylase production and found that overexpression of chaperones GroEL/S enhanced aminoacylase activity in the cell-free extract 1.8-fold in V. natriegens and E. coli. Eventually, E. coli ArcticExpress™ (DE3), which co-expresses cold-adapted chaperonins Cpn60/10 from Oleispira antarctica, cultivated at 12 °C, rendered the most suitable expression system for this aminoacylase and exhibited twice the aminoacylase activity in the cell-free extract compared to E. coli BL21 (DE3) with GroEL/S co-expression at 20 °C. The purified aminoacylase was characterized based on hydrolytic activities, being most stable and active at pH 7.0, with a maximum activity at 70 °C, and stability at 40 °C and pH 7.0 for 5 days. The aminoacylase strongly prefers short-chain acyl-amino acids with smaller, hydrophobic amino acid residues. Several long-chain amino acids were fairly accepted in hydrolysis as well, especially N-lauroyl-L-methionine. To initially evaluate the relevance of this aminoacylase for the synthesis of N-acyl-amino acids, we demonstrated that lauroyl-methionine can be synthesized from lauric acid and methionine in an aqueous system. Conclusion Our results suggest that the recombinant enzyme is well suited for synthesis reactions and will thus be further investigated.}, language = {en} } @article{FalkenbergKohnBottetal.2023, author = {Falkenberg, Fabian and Kohn, Sophie and Bott, Michael and Bongaerts, Johannes and Siegert, Petra}, title = {Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822ᵀ}, series = {FEBS Open Bio}, volume = {13}, journal = {FEBS Open Bio}, number = {11}, publisher = {Wiley}, address = {Hoboken, NJ}, issn = {2211-5463}, doi = {10.1002/2211-5463.13701}, pages = {2035 -- 2046}, year = {2023}, abstract = {Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822ᵀ (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5\% (w/v) SDS and 5\% H₂O₂ (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H₂O₂, suggest it has potential for biotechnological applications.}, language = {en} } @article{FalkenbergVossBottetal.2023, author = {Falkenberg, Fabian and Voß, Leonie and Bott, Michael and Bongaerts, Johannes and Siegert, Petra}, title = {New robust subtilisins from halotolerant and halophilic Bacillaceae}, series = {Applied Microbiology and Biotechnology}, volume = {107}, journal = {Applied Microbiology and Biotechnology}, publisher = {Springer Nature}, address = {Berlin}, issn = {1432-0614}, doi = {10.1007/s00253-023-12553-w}, pages = {3939 -- 3954}, year = {2023}, abstract = {The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465ᵀ and Alkalibacillus haloalkaliphilus DSM 5271ᵀ and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976ᵀ served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0-12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5\% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications.}, language = {en} } @article{HaegerBongaertsSiegert2022, author = {Haeger, Gerrit and Bongaerts, Johannes and Siegert, Petra}, title = {A convenient ninhydrin assay in 96-well format for amino acid-releasing enzymes using an air-stable reagent}, series = {Analytical Biochemistry}, journal = {Analytical Biochemistry}, number = {624}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1096-0309}, doi = {10.1016/j.ab.2022.114819}, pages = {Artikel 114819}, year = {2022}, abstract = {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.}, language = {en} } @article{MuschallikKippReckeretal.2020, author = {Muschallik, Lukas and Kipp, Carina Ronja and Recker, Inga and Bongaerts, Johannes and Pohl, Martina and Gelissen, Melanie and Sch{\"o}ning, Michael Josef and Selmer, Thorsten and Siegert, Petra}, title = {Synthesis of α-hydroxy ketones and vicinal diols with the Bacillus licheniformis DSM 13T butane-2, 3-diol dehydrogenase}, series = {Journal of Biotechnology}, volume = {202}, journal = {Journal of Biotechnology}, number = {Vol. 324}, publisher = {Elsevier}, address = {Amsterdam}, isbn = {2590-1559}, doi = {10.1016/j.jbiotec.2020.09.016}, pages = {61 -- 70}, year = {2020}, abstract = {The enantioselective synthesis of α-hydroxy ketones and vicinal diols is an intriguing field because of the broad applicability of these molecules. Although, butandiol dehydrogenases are known to play a key role in the production of 2,3-butandiol, their potential as biocatalysts is still not well studied. Here, we investigate the biocatalytic properties of the meso-butanediol dehydrogenase from Bacillus licheniformis DSM 13T (BlBDH). The encoding gene was cloned with an N-terminal StrepII-tag and recombinantly overexpressed in E. coli. BlBDH is highly active towards several non-physiological diketones and α-hydroxyketones with varying aliphatic chain lengths or even containing phenyl moieties. By adjusting the reaction parameters in biotransformations the formation of either the α-hydroxyketone intermediate or the diol can be controlled.}, language = {en} }