@article{FalkenbergBottBongaertsetal.2022,
  author    = {Falkenberg, Fabian and Bott, Michael and Bongaerts, Johannes and Siegert, Petra},
  title     = {Phylogenetic survey of the subtilase family and a data-mining-based search for new subtilisins from Bacillaceae},
  series = {Frontiers in Microbiology},
  volume    = {2022},
  journal   = {Frontiers in Microbiology},
  number    = {13},
  publisher = {Frontiers},
  address   = {Lausanne},
  issn      = {1664-302X},
  doi       = {10.3389/fmicb.2022.1017978},
  pages     = {Artikel 13:1017978},
  year      = {2022},
  abstract  = {The subtilase family (S8), a member of the clan SB of serine proteases are ubiquitous in all kingdoms of life and fulfil different physiological functions. Subtilases are divided in several groups and especially subtilisins are of interest as they are used in various industrial sectors. Therefore, we searched for new subtilisin sequences of the family Bacillaceae using a data mining approach. The obtained 1,400 sequences were phylogenetically classified in the context of the subtilase family. This required an updated comprehensive overview of the different groups within this family. To fill this gap, we conducted a phylogenetic survey of the S8 family with characterised holotypes derived from the MEROPS database. The analysis revealed the presence of eight previously uncharacterised groups and 13 subgroups within the S8 family. The sequences that emerged from the data mining with the set filter parameters were mainly assigned to the subtilisin subgroups of true subtilisins, high-alkaline subtilisins, and phylogenetically intermediate subtilisins and represent an excellent source for new subtilisin candidates.},
  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{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{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}
}
@misc{DegeringWielandIslametal.2024,
  author    = {Degering, Christian and Wieland, Susanne and Islam, Shohana and Lindner, Claudia and Siegert, Petra and Falkenberg, Fabian and Bongaerts, Johannes},
  title     = {Wasch- und Reinigungsmittel enthaltend Protease (aus Fictibacillus arsenicus)},
  year      = {2024},
  abstract  = {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 k{\"o}nnen, alle hinreichend {\"a}hnlichen Proteasen mit einer entsprechend {\"a}hnlichen Sequenz zu SEQ ID NO:1 und f{\"u}r sie codierende Nukleins{\"a}uren. Die Erfindung betrifft ferner deren Herstellung sowie Verfahren zur Verwendung dieser Proteasen, deren Verwendung als solche sowie diese enthaltende Mittel, insbesondere Wasch- und Reinigungsmittel.},
  language  = {de}
}
@misc{DegeringWielandIslametal.2024,
  author    = {Degering, Christian and Wieland, Susanne and Islam, Shohana and Lindner, Claudia and Bongaerts, Johannes and Siegert, Petra and Falkenberg, Fabian},
  title     = {Wasch- und Reinigungsmittel enthaltend Protease (aus Metabacillus indicus)},
  year      = {2024},
  abstract  = {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 k{\"o}nnen, alle hinreichend {\"a}hnlichen Proteasen mit einer entsprechend {\"a}hnlichen Sequenz zu SEQ ID NO:1 und f{\"u}r sie codierende Nukleins{\"a}uren. Die Erfindung betrifft ferner deren Herstellung sowie Verfahren zur Verwendung dieser Proteasen, deren Verwendung als solche sowie diese enthaltende Mittel, insbesondere Wasch- und Reinigungsmittel.},
  language  = {de}
}
@article{RibitschKarlBirnerGruenbergeretal.2010,
  author    = {Ribitsch, Doris and Karl, Wolfgang and Birner-Gruenberger, Ruth and Gruber, Karl and Eiteljoerg, Inge and Remler, Peter and Wieland, Susanne and Siegert, Petra and Maurer, Karl-Heinz and Schwab, Helmut},
  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}
}