@article{JablonskiMuenstermannNorketal.2021, author = {Jablonski, Melanie and M{\"u}nstermann, Felix and Nork, Jasmina and Molinnus, Denise and Muschallik, Lukas and Bongaerts, Johannes and Wagner, Torsten and Keusgen, Michael and Siegert, Petra and Sch{\"o}ning, Michael Josef}, title = {Capacitive field-effect biosensor applied for the detection of acetoin in alcoholic beverages and fermentation broths}, series = {physica status solidi (a) applications and materials science}, volume = {218}, journal = {physica status solidi (a) applications and materials science}, number = {13}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {1862-6319}, doi = {10.1002/pssa.202000765}, pages = {7 Seiten}, year = {2021}, abstract = {An acetoin biosensor based on a capacitive electrolyte-insulator-semiconductor (EIS) structure modified with the enzyme acetoin reductase, also known as butane-2,3-diol dehydrogenase (Bacillus clausii DSM 8716ᵀ), is applied for acetoin detection in beer, red wine, and fermentation broth samples for the first time. The EIS sensor consists of an Al/p-Si/SiO₂/Ta₂O₅ layer structure with immobilized acetoin reductase on top of the Ta₂O₅ transducer layer by means of crosslinking via glutaraldehyde. The unmodified and enzyme-modified sensors are electrochemically characterized by means of leakage current, capacitance-voltage, and constant capacitance methods, respectively.}, language = {en} } @article{HandtkeVollandMethlingetal.2014, author = {Handtke, Stefan and Volland, Sonja and Methling, Karen and Albrecht, Dirk and Becher, D{\"o}rte and Nehls, Jenny and Bongaerts, Johannes and Maurer, Karl-Heinz and Lalk, Michael and Liesegang, Heiko and Voigt, Birgit and Daniel, Rolf and Hecker, Michael}, title = {Cell physiology of the biotechnological relevant bacterium Bacillus pumilus - An omics-based approach}, series = {Journal of Biotechnology}, journal = {Journal of Biotechnology}, number = {192(A)}, publisher = {Elsevier}, address = {Amsterdam}, issn = {1873-4863 (E-Journal); 0168-1656 (Print)}, doi = {10.1016/j.jbiotec.2014.08.028}, pages = {204 -- 214}, year = {2014}, abstract = {Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on exoproteome analysis, B. pumilus strain Jo2, possessing a high secretion capability, was chosen for an omics-based investigation. The proteome and metabolome of B. pumilus cells growing either in minimal or complex medium was analyzed. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43\% of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC-MS, IP-LC/MS and H NMR methods numerous metabolites were analyzed and assigned to reconstructed metabolic pathways. In the genome sequence a functional secretion system including the components of the Sec- and Tat-secretion machinery was found. Analysis of the exoproteome revealed secretion of about 70 proteins with predicted secretion signals. In addition, selected production-relevant genome features such as restriction modification systems and NRPS clusters of B. pumilus Jo2 are discussed.}, language = {en} } @article{HandtkeSchroeterJuergenetal.2014, author = {Handtke, Stefan and Schroeter, Rebecca and J{\"u}rgen, Britta and Methling, Karen and Schl{\"u}ter, Rabea and Albrecht, Dirk and Hijum, Sacha A. F. T. van and Bongaerts, Johannes and Maurer, Karl-Heinz and Lalk, Michael and Schweder, Thomas and Hecker, Michael and Voigt, Birgit}, title = {Bacillus pumilus reveals a remarkably high resistance to hydrogen peroxide provoked oxidative stress}, series = {PLOS one}, volume = {9}, journal = {PLOS one}, number = {1}, publisher = {PLOS}, address = {San Francisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0085625}, pages = {e85625}, year = {2014}, abstract = {Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus.}, 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{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{HaegerJolmesOyenetal.2024, author = {Haeger, Gerrit and Jolmes, Tristan and Oyen, Sven and Jaeger, Karl-Erich and Bongaerts, Johannes and Sch{\"o}rken, Ulrich and Siegert, Petra}, title = {Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis}, series = {Applied Microbiology and Biotechnology}, journal = {Applied Microbiology and Biotechnology}, number = {108}, publisher = {Springer}, address = {Berlin}, issn = {1432-0614}, doi = {10.1007/s00253-023-12868-8}, pages = {14 Seiten}, year = {2024}, abstract = {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.}, 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} } @techreport{HaegerBongaertsSiegert2023, author = {Haeger, Gerrit and Bongaerts, Johannes and Siegert, Petra}, title = {Abschlussbericht Teil II: Eingehende Darstellung Neue biobasierte Lipopeptide aus nachhaltiger Produktion (LipoPep)}, pages = {17Seiten}, year = {2023}, language = {de} } @article{GerigkMaassTakorsetal.2000, author = {Gerigk, M. and Maaß, D. and Takors, Ralf and Kreutzer, A. and Wandrey, Christian and Bongaerts, Johannes and Wubbolts, Marcel}, title = {Fermentative Herstellung von L-Phenylalanin im Fed-Batch Verfahren mit E. coli unter Einbindung eines integrierten Aufarbeitungsverfahrens}, series = {Chemie-Ingenieur-Technik (CIT)}, volume = {Vol. 72}, journal = {Chemie-Ingenieur-Technik (CIT)}, number = {Iss. 9}, issn = {1522-2640 (E-Journal); 0009-286X (Print)}, pages = {926 -- 927}, year = {2000}, language = {de} } @article{GerigkMaassKreutzeretal.2002, author = {Gerigk, M. and Maaß, D. and Kreutzer, A. and Sprenger, G. and Bongaerts, Johannes and Wubbolts, Marcel and Takors, Ralf}, title = {Enhanced pilot-scale fed-batch L-phenylalanine production with recombinant Escherichia coli by fully integrated reactive extraction}, series = {Bioprocess and biosystems engineering}, volume = {Vol. 25}, journal = {Bioprocess and biosystems engineering}, number = {Iss. 1}, issn = {1432-0797 (E-Journal); 1615-7605 (E-Journal); 0178-515X (Print); 1615-7591 (Print)}, pages = {43 -- 52}, year = {2002}, language = {en} }