Stephan Kolkenbrock - Academia.edu (original) (raw)

Papers by Stephan Kolkenbrock

Research paper thumbnail of Terminale und Telomer-assoziierte Proteine von pAL1, einem linearen Plasmid von Arthrobacter nitroguajacolicus Rü61a

Research paper thumbnail of Carbohydrate-binding modules interacting with polyethyleneterepthalate

Research paper thumbnail of Corynebacterium glutamicum possesses β-N-acetylglucosaminidase

BMC microbiology, 2016

In Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae,... more In Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae, which includes mycobacteria, cell elongation and peptidoglycan biosynthesis is mainly due to polar growth. C. glutamicum lacks an uptake system for the peptidoglycan constituent N-acetylglucosamine (GlcNAc), but is able to catabolize GlcNAc-6-phosphate. Due to its importance in white biotechnology and in order to ensure more sustainable processes based on non-food renewables and to reduce feedstock costs, C. glutamicum strains have previously been engineered to produce amino acids from GlcNAc. GlcNAc also is a constituent of chitin, but it is unknown if C. glutamicum possesses chitinolytic enzymes. Chitin was shown here not to be growth substrate for C. glutamicum. However, its genome encodes a putative N-acetylglucosaminidase. The nagA 2 gene product was active as β-N-acetylglucosaminidase with 0.27 mM 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside as substrate supporting half-maxima...

Research paper thumbnail of A recombinant fungal chitin deacetylase produces fully defined chitosan oligomers with novel patterns of acetylation

Applied and environmental microbiology, Jan 2, 2016

Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential a... more Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e. their degrees of polymerization and acetylation as well as their pattern of acetylation. However, paCOS generated via chemical N-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDA) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDA. However, compared to bacterial CDA, we know little about the ability of fungal CDA to produce defined paCOS with known patterns of acetylation. Therefore, we optimised the expression of a chitin deacetylase from the fungus Puccinia graminis f.sp. tritici in E. coli The best yield of functional enzyme was obtained as a...

Research paper thumbnail of Identification and in vitro deoxynucleotidylation of the terminal protein of the linear plasmid pAL1 of Arthrobacternitroguajacolicus Rü61a

Fems Microbiology Letters, 2010

The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a is a linear replicon, characterized by i... more The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a is a linear replicon, characterized by inverted terminal repeats and terminal proteins (TPs) covalently bound to its 5 0 -ends. Previous sequence analysis and predictions of possible secondary structures formed by telomeric 3 0 -overhangs indicated significant differences of the 'left' and 'right' telomere of pAL1, raising the question of whether each terminus is recognized by a specific protein. The genes pAL1.102 and pAL1.103, located close to a terminus, code for possible DNA-binding proteins; however, only the pORF102 protein encoded by pAL1.102 shows a weak similarity to known TPs of Streptomyces linear replicons. pORF102, purified from recombinant A. nitroguajacolicus Rü61a as a fusion with maltose-binding protein (MBP), was specifically associated with terminal pAL1 DNA, whereas MBP-pORF103 was devoid of DNA, suggesting that pORF102 represents the protein attached to both ends of the linear plasmid. In electrophoretic mobility shift assays, the MBP-pORF102 protein was not capable of specifically recognizing telomeric DNA sequences. Consistent with its proposed role as a protein primer in DNA synthesis, pORF102 was deoxynucleotidylated in vitro with dCMP, complementary to the 3 0 -ends (. . . GCAGG) of pAL1.

Research paper thumbnail of Stability, Unfolding, and Structural Changes of Cofactor-Free 1 H -3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase

Biochemistry Usa, 2007

Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the... more Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the oxidatively stable C69S variant (HodC) of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) have been determined by classical and pressure modulation scanning calorimetry (DSC and PMDSC, respectively), circular dichroism (CD) spectroscopy, differential scanning densimetry (DSD), and dynamic light scattering measurements. At 25°C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of R-helical structure of ∼60%, based on deconvolution of CD spectra. The percentage of -sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the R/ -hydrolase fold superfamily. His 6 HodC exhibits threestate unfolding (N T I T D) with an intermediate state I that exhibits at the transition temperature a volume larger than that of the native or denatured state. The intermediate state I is also associated with the highest isothermal expansion coefficient, R P , of the three states and exhibits a significantly lower percentage of R-helical structure than the native state. The stability difference between the native and intermediate state is rather small which makes I a potential candidate for reactions with various ligands, particularly those having a preference for the apparently preserved -type motifs.

Research paper thumbnail of Broad host range expression vector for diverse prokaryotes

Research paper thumbnail of N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Ru ¨61a, an /-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant†

J Bacteriol, 2006

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldi... more N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the ␣/␤-hydrolase-fold superfamily of enzymes; inactivation of (His 6 -tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat /K m ‫؍‬ 208 mM ؊1 s ؊1 ), while among the aryl-acetylamides, o-carboxy-or o-nitrosubstituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His 6 Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His 6 AmqC22A/ C63A markedly differed from those of His 6 Amq. The replacements effected some changes in K m s of the enzyme and increased k cat s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in ␣-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

Research paper thumbnail of Copper-saturation of recombinant ToPPO-2

Research paper thumbnail of Towards Biotechnological Production of Chitosans

Research paper thumbnail of Chitin and Chitosan Modifying Enzymes: Versatile Novel Tools for the Analysis of Structure-Function Relationships of Partially Acetylated Chitosans

ABSTRACT The past decade has seen remarkable progress in understanding structure-function relatio... more ABSTRACT The past decade has seen remarkable progress in understanding structure-function relationships of partially acetylated chitosans. In particular, the roles of the degree of polymerisation (DP) and degree of acetylation (DA) of chitosans in defining their physico-chemical properties and their biological activities have been investigated. In contrast, the role of the pattern of acetylation (PA) has not yet been studied, mostly due to a lack of analytical methods to determine PA, and to the non-availability of chitosans with non-random PA. We here describe how chitin and chitosan modifying enzymes, such as chitin deacetylases and chitosan hydrolases, can be used for analysis and synthesis of chitosans. Chitin deacetylases can convert chitin oligomers or highly acetylated chitosan polymers with random PA into chitosan oligomers or less acetylated chitosan polymers with defined, non-random PAs. Sequence specific chitosan hydrolases can be used in enzymatic / mass-spectrometric fingerprinting assays to determine the PA of chitosan polymers, while the PA of chitosan oligomers even in complex mixtures can be determined using quantitative mass spectrometric sequencing. Thus, bio-engineering of designer chitosans with known structures and defined functions becomes feasible, as a prerequisite for the development of reliable chitosan-based products, e.g. for plant disease protection or scar-free wound healing.

Research paper thumbnail of Affinity protein-based FRET tools for cellular tracking of chitosan nanoparticles and determination of the polymer degree of acetylation

Biomacromolecules, Jan 14, 2014

Chitosan (CS) is a family of linear polysaccharides with diverse applications in medicine, agricu... more Chitosan (CS) is a family of linear polysaccharides with diverse applications in medicine, agriculture, and industry. Its bioactive properties are determined by parameters such as the degree of acetylation (DA), but current techniques to measure the DA are laborious and require large amounts of substrate and sophisticated equipment. It is also challenging to monitor the fate of chitosan-based nanoparticles (CS-NPs) in vitro because current tools cannot measure their enzymatic or chemical degradation. We have developed a method based on the Förster resonance energy transfer (FRET) that occurs between two independent fluorescent proteins fused to a CS-binding domain, who interact with CS polymers or CS-NPs. We used this approach to calibrate a simple and rapid analytical method that can determine the DA of CS substrates. We showed unequivocally that FRET occurs on the surface of CS-NPs and that the FRET signal is quenched by enzymatic degradation of the CS substrate. Finally, we provi...

Research paper thumbnail of Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

Scientific Reports, 2015

Chitin and chitosan oligomers have diverse biological activities with potentially valuable applic... more Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture.

Research paper thumbnail of Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample

Journal of Biotechnology, 2014

Chitin and its derivative chitosan are abundant natural polysaccharides with many potential indus... more Chitin and its derivative chitosan are abundant natural polysaccharides with many potential industrial applications. Metagenomic analysis of chitin-enriched soil samples using the Roche Genome Sequencer FLX platform led to the identification of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which may be used to produce novel chitosans. The sequencing approach yielded 2,281,090 reads with an average length of 378bp amounting to a total sequence information of approximately 851Mb. Assembly of the obtained sequences comprised 699,710 reads representing 30.68% of all reads. A total of 6625 contigs larger than 500bp containing 16,289 predicted genes are included in the assembly. Taxonomic profiling of the indigenous microbial community by applying the software CARMA revealed that 96.1% of the reads were of bacterial origin including 17% assigned to the family Xanthomonadaceae. Several putative genes encoding CCMEs were identified by comparison against the GenBank database, inclusive a full-length chitinase gene which was codon optimized for Escherichia coli and heterologously synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector system. Approximately 5mg of the novel active chitinase was purified as demonstrated by dot assay analysis using glycol chitin as a substrate. Next generation metagenomic sequencing, thus, emerges as a new and powerful tool for the identification of potentially novel biocatalysts of biotechnological value.

Research paper thumbnail of Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases

Journal of Experimental Botany, 2014

Polygalacturonases (PGs) are hydrolytic enzymes employed by several phytopathogens to weaken the ... more Polygalacturonases (PGs) are hydrolytic enzymes employed by several phytopathogens to weaken the plant cell wall by degrading homopolygalacturonan, a major constituent of pectin. Plants fight back by employing polygalacturonase-inhibitor proteins (PGIPs). The present study compared the inhibition potential of pearl millet PGIP (Pennisetum glaucum; PglPGIP1) with the known inhibition of Phaseolus vulgaris PGIP (PvPGIP2) against two PGs, the PG-II isoform from Aspergillus niger (AnPGII) and the PG-III isoform from Fusarium moniliforme (FmPGIII). The key rationale was to elucidate the relationship between the extent of sequence similarity of the PGIPs and the corresponding PG inhibition potential. First, a pearl millet pgip gene (Pglpgip1) was isolated and phylogenetically placed among monocot PGIPs alongside foxtail millet (Setaria italica). Upstream sequence analysis of Pglpgip1 identified important cis-elements responsive to light, plant stress hormones, and anoxic stress. PglPGIP1, heterologously produced in Escherichia coli, partially inhibited AnPGII non-competitively with a pH optimum between 4.0 and 4.5, and showed no inhibition against FmPGIII. Docking analysis showed that the concave surface of PglPGIP1 interacted strongly with the N-terminal region of AnPGII away from the active site, whereas it weakly interacted with the C-terminus of FmPGIII. Interestingly, PglPGIP1 and PvPGIP2 employed similar motif regions with few identical amino acids for interaction with AnPGII at non-substrate-binding sites; however, they engaged different regions of AnPGII. Computational mutagenesis predicted D126 (PglPGIP1)-K39 (AnPGII) to be the most significant binding contact in the PglPGIP1-AnPGII complex. Such protein-protein interaction studies are crucial in the future generation of designer host proteins for improved resistance against everevolving pathogen virulence factors.

Research paper thumbnail of Catabolic Linear Plasmids

Microbiology Monographs, 2007

Page 1. Microbiol Monogr (7) F. Meinhardt · R. Klassen: Microbial Linear Plasmids DOI 10.1007/717... more Page 1. Microbiol Monogr (7) F. Meinhardt · R. Klassen: Microbial Linear Plasmids DOI 10.1007/7171_2007_091/Published online: 1 June 2007 © Springer-Verlag Berlin Heidelberg 2007 Catabolic Linear Plasmids Susanne Fetzner (u) · Stephan Kolkenbrock · Katja Parschat ...

Research paper thumbnail of Parameters That Enhance the Bacterial Expression of Active Plant Polyphenol Oxidases

PLoS ONE, 2013

Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper proteins that enzymatically convert dip... more Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper proteins that enzymatically convert diphenolic compounds into their corresponding quinones. Although there is significant interest in these enzymes because of their role in food deterioration, the lack of a suitable expression system for the production of soluble and active plant PPOs has prevented detailed investigations of their structure and activity. Recently we developed a bacterial expression system that was sufficient for the production of PPO isoenzymes from dandelion (Taraxacum officinale). The system comprised the Escherichia coli Rosetta 2 (DE3) [pLysSRARE2] strain combined with the pET-22b(+)-vector cultivated in auto-induction medium at a constant low temperature (26uC). Here we describe important parameters that enhance the production of active PPOs using dandelion PPO-2 for proof of concept. Low-temperature cultivation was essential for optimal yields, and the provision of CuCl 2 in the growth medium was necessary to produce an active enzyme. By increasing the copper concentration in the production medium to 0.2 mM, the yield in terms of PPO activity per mol purified protein was improved 2.7-fold achieving a v max of 0.4860.1 mkat per mg purified PPO-2 for 4-methylcatechol used as a substrate. This is likely to reflect the replacement of an inactive apo-form of the enzyme with a correctly-folded, copper-containing counterpart. We demonstrated the transferability of the method by successfully expressing a PPO from tomato (Solanum lycopersicum) showing that our optimized system is suitable for the analysis of further plant PPOs. Our new system therefore provides greater opportunities for the future of research into this economically-important class of enzymes.

Research paper thumbnail of A Novel Replicative Enzyme Encoded by the Linear Arthrobacter Plasmid pAL1

Journal of Bacteriology, 2010

The soil bacterium Arthrobacter nitroguajacolicus Rü61a contains the linear plasmid pAL1, which c... more The soil bacterium Arthrobacter nitroguajacolicus Rü61a contains the linear plasmid pAL1, which codes for the degradation of 2-methylquinoline. Like other linear replicons of actinomycetes, pAL1 is characterized by short terminal inverted-repeat sequences and terminal proteins (TP pAL1 ) covalently attached to its 5 ends. TP pAL1 , encoded by the pAL1.102 gene, interacts in vivo with the protein encoded by pAL1.101. Bioinformatic analysis of the pAL1.101 protein, which comprises 1,707 amino acids, suggested putative zinc finger and topoisomerase-primase domains and part of a superfamily 2 helicase domain in its N-terminal and central regions, respectively. Sequence motifs characteristic of the polymerization domain of family B DNA polymerases are partially conserved in a C-terminal segment. The purified recombinant protein catalyzed the deoxycytidylation of TP pAL1 in the presence of single-stranded DNA templates comprising the 3-terminal sequence (5-GCAGG-3), which in pAL1 forms the terminal inverted repeat, but also at templates with 5-(G/T)CA(GG/GC/CG)-3 ends. Enzyme assays suggested that the protein exhibits DNA topoisomerase, DNA helicase, and DNA-and protein-primed DNA polymerase activities. The pAL1.101 protein, therefore, may act as a replicase of pAL1.

Research paper thumbnail of N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Ru61a, an  / -Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

Journal of Bacteriology, 2006

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldi... more N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the ␣/␤-hydrolase-fold superfamily of enzymes; inactivation of (His 6 -tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat /K m ‫؍‬ 208 mM ؊1 s ؊1 ), while among the aryl-acetylamides, o-carboxy-or o-nitrosubstituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His 6 Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His 6 AmqC22A/ C63A markedly differed from those of His 6 Amq. The replacements effected some changes in K m s of the enzyme and increased k cat s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in ␣-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

Research paper thumbnail of Identification and in vitro deoxynucleotidylation of the terminal protein of the linear plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a

FEMS Microbiology Letters, 2010

The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a is a linear replicon, characterized by i... more The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a is a linear replicon, characterized by inverted terminal repeats and terminal proteins (TPs) covalently bound to its 5 0 -ends. Previous sequence analysis and predictions of possible secondary structures formed by telomeric 3 0 -overhangs indicated significant differences of the 'left' and 'right' telomere of pAL1, raising the question of whether each terminus is recognized by a specific protein. The genes pAL1.102 and pAL1.103, located close to a terminus, code for possible DNA-binding proteins; however, only the pORF102 protein encoded by pAL1.102 shows a weak similarity to known TPs of Streptomyces linear replicons. pORF102, purified from recombinant A. nitroguajacolicus Rü61a as a fusion with maltose-binding protein (MBP), was specifically associated with terminal pAL1 DNA, whereas MBP-pORF103 was devoid of DNA, suggesting that pORF102 represents the protein attached to both ends of the linear plasmid. In electrophoretic mobility shift assays, the MBP-pORF102 protein was not capable of specifically recognizing telomeric DNA sequences. Consistent with its proposed role as a protein primer in DNA synthesis, pORF102 was deoxynucleotidylated in vitro with dCMP, complementary to the 3 0 -ends (. . . GCAGG) of pAL1.

Research paper thumbnail of Terminale und Telomer-assoziierte Proteine von pAL1, einem linearen Plasmid von Arthrobacter nitroguajacolicus Rü61a

Research paper thumbnail of Carbohydrate-binding modules interacting with polyethyleneterepthalate

Research paper thumbnail of Corynebacterium glutamicum possesses β-N-acetylglucosaminidase

BMC microbiology, 2016

In Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae,... more In Gram-positive Corynebacterium glutamicum and other members of the suborder Corynebacterianeae, which includes mycobacteria, cell elongation and peptidoglycan biosynthesis is mainly due to polar growth. C. glutamicum lacks an uptake system for the peptidoglycan constituent N-acetylglucosamine (GlcNAc), but is able to catabolize GlcNAc-6-phosphate. Due to its importance in white biotechnology and in order to ensure more sustainable processes based on non-food renewables and to reduce feedstock costs, C. glutamicum strains have previously been engineered to produce amino acids from GlcNAc. GlcNAc also is a constituent of chitin, but it is unknown if C. glutamicum possesses chitinolytic enzymes. Chitin was shown here not to be growth substrate for C. glutamicum. However, its genome encodes a putative N-acetylglucosaminidase. The nagA 2 gene product was active as β-N-acetylglucosaminidase with 0.27 mM 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside as substrate supporting half-maxima...

Research paper thumbnail of A recombinant fungal chitin deacetylase produces fully defined chitosan oligomers with novel patterns of acetylation

Applied and environmental microbiology, Jan 2, 2016

Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential a... more Partially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e. their degrees of polymerization and acetylation as well as their pattern of acetylation. However, paCOS generated via chemical N-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDA) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDA. However, compared to bacterial CDA, we know little about the ability of fungal CDA to produce defined paCOS with known patterns of acetylation. Therefore, we optimised the expression of a chitin deacetylase from the fungus Puccinia graminis f.sp. tritici in E. coli The best yield of functional enzyme was obtained as a...

Research paper thumbnail of Identification and in vitro deoxynucleotidylation of the terminal protein of the linear plasmid pAL1 of Arthrobacternitroguajacolicus Rü61a

Fems Microbiology Letters, 2010

The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a is a linear replicon, characterized by i... more The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a is a linear replicon, characterized by inverted terminal repeats and terminal proteins (TPs) covalently bound to its 5 0 -ends. Previous sequence analysis and predictions of possible secondary structures formed by telomeric 3 0 -overhangs indicated significant differences of the 'left' and 'right' telomere of pAL1, raising the question of whether each terminus is recognized by a specific protein. The genes pAL1.102 and pAL1.103, located close to a terminus, code for possible DNA-binding proteins; however, only the pORF102 protein encoded by pAL1.102 shows a weak similarity to known TPs of Streptomyces linear replicons. pORF102, purified from recombinant A. nitroguajacolicus Rü61a as a fusion with maltose-binding protein (MBP), was specifically associated with terminal pAL1 DNA, whereas MBP-pORF103 was devoid of DNA, suggesting that pORF102 represents the protein attached to both ends of the linear plasmid. In electrophoretic mobility shift assays, the MBP-pORF102 protein was not capable of specifically recognizing telomeric DNA sequences. Consistent with its proposed role as a protein primer in DNA synthesis, pORF102 was deoxynucleotidylated in vitro with dCMP, complementary to the 3 0 -ends (. . . GCAGG) of pAL1.

Research paper thumbnail of Stability, Unfolding, and Structural Changes of Cofactor-Free 1 H -3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase

Biochemistry Usa, 2007

Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the... more Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the oxidatively stable C69S variant (HodC) of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) have been determined by classical and pressure modulation scanning calorimetry (DSC and PMDSC, respectively), circular dichroism (CD) spectroscopy, differential scanning densimetry (DSD), and dynamic light scattering measurements. At 25°C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of R-helical structure of ∼60%, based on deconvolution of CD spectra. The percentage of -sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the R/ -hydrolase fold superfamily. His 6 HodC exhibits threestate unfolding (N T I T D) with an intermediate state I that exhibits at the transition temperature a volume larger than that of the native or denatured state. The intermediate state I is also associated with the highest isothermal expansion coefficient, R P , of the three states and exhibits a significantly lower percentage of R-helical structure than the native state. The stability difference between the native and intermediate state is rather small which makes I a potential candidate for reactions with various ligands, particularly those having a preference for the apparently preserved -type motifs.

Research paper thumbnail of Broad host range expression vector for diverse prokaryotes

Research paper thumbnail of N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Ru ¨61a, an /-Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant†

J Bacteriol, 2006

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldi... more N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the ␣/␤-hydrolase-fold superfamily of enzymes; inactivation of (His 6 -tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat /K m ‫؍‬ 208 mM ؊1 s ؊1 ), while among the aryl-acetylamides, o-carboxy-or o-nitrosubstituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His 6 Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His 6 AmqC22A/ C63A markedly differed from those of His 6 Amq. The replacements effected some changes in K m s of the enzyme and increased k cat s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in ␣-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

Research paper thumbnail of Copper-saturation of recombinant ToPPO-2

Research paper thumbnail of Towards Biotechnological Production of Chitosans

Research paper thumbnail of Chitin and Chitosan Modifying Enzymes: Versatile Novel Tools for the Analysis of Structure-Function Relationships of Partially Acetylated Chitosans

ABSTRACT The past decade has seen remarkable progress in understanding structure-function relatio... more ABSTRACT The past decade has seen remarkable progress in understanding structure-function relationships of partially acetylated chitosans. In particular, the roles of the degree of polymerisation (DP) and degree of acetylation (DA) of chitosans in defining their physico-chemical properties and their biological activities have been investigated. In contrast, the role of the pattern of acetylation (PA) has not yet been studied, mostly due to a lack of analytical methods to determine PA, and to the non-availability of chitosans with non-random PA. We here describe how chitin and chitosan modifying enzymes, such as chitin deacetylases and chitosan hydrolases, can be used for analysis and synthesis of chitosans. Chitin deacetylases can convert chitin oligomers or highly acetylated chitosan polymers with random PA into chitosan oligomers or less acetylated chitosan polymers with defined, non-random PAs. Sequence specific chitosan hydrolases can be used in enzymatic / mass-spectrometric fingerprinting assays to determine the PA of chitosan polymers, while the PA of chitosan oligomers even in complex mixtures can be determined using quantitative mass spectrometric sequencing. Thus, bio-engineering of designer chitosans with known structures and defined functions becomes feasible, as a prerequisite for the development of reliable chitosan-based products, e.g. for plant disease protection or scar-free wound healing.

Research paper thumbnail of Affinity protein-based FRET tools for cellular tracking of chitosan nanoparticles and determination of the polymer degree of acetylation

Biomacromolecules, Jan 14, 2014

Chitosan (CS) is a family of linear polysaccharides with diverse applications in medicine, agricu... more Chitosan (CS) is a family of linear polysaccharides with diverse applications in medicine, agriculture, and industry. Its bioactive properties are determined by parameters such as the degree of acetylation (DA), but current techniques to measure the DA are laborious and require large amounts of substrate and sophisticated equipment. It is also challenging to monitor the fate of chitosan-based nanoparticles (CS-NPs) in vitro because current tools cannot measure their enzymatic or chemical degradation. We have developed a method based on the Förster resonance energy transfer (FRET) that occurs between two independent fluorescent proteins fused to a CS-binding domain, who interact with CS polymers or CS-NPs. We used this approach to calibrate a simple and rapid analytical method that can determine the DA of CS substrates. We showed unequivocally that FRET occurs on the surface of CS-NPs and that the FRET signal is quenched by enzymatic degradation of the CS substrate. Finally, we provi...

Research paper thumbnail of Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

Scientific Reports, 2015

Chitin and chitosan oligomers have diverse biological activities with potentially valuable applic... more Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture.

Research paper thumbnail of Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample

Journal of Biotechnology, 2014

Chitin and its derivative chitosan are abundant natural polysaccharides with many potential indus... more Chitin and its derivative chitosan are abundant natural polysaccharides with many potential industrial applications. Metagenomic analysis of chitin-enriched soil samples using the Roche Genome Sequencer FLX platform led to the identification of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which may be used to produce novel chitosans. The sequencing approach yielded 2,281,090 reads with an average length of 378bp amounting to a total sequence information of approximately 851Mb. Assembly of the obtained sequences comprised 699,710 reads representing 30.68% of all reads. A total of 6625 contigs larger than 500bp containing 16,289 predicted genes are included in the assembly. Taxonomic profiling of the indigenous microbial community by applying the software CARMA revealed that 96.1% of the reads were of bacterial origin including 17% assigned to the family Xanthomonadaceae. Several putative genes encoding CCMEs were identified by comparison against the GenBank database, inclusive a full-length chitinase gene which was codon optimized for Escherichia coli and heterologously synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector system. Approximately 5mg of the novel active chitinase was purified as demonstrated by dot assay analysis using glycol chitin as a substrate. Next generation metagenomic sequencing, thus, emerges as a new and powerful tool for the identification of potentially novel biocatalysts of biotechnological value.

Research paper thumbnail of Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases

Journal of Experimental Botany, 2014

Polygalacturonases (PGs) are hydrolytic enzymes employed by several phytopathogens to weaken the ... more Polygalacturonases (PGs) are hydrolytic enzymes employed by several phytopathogens to weaken the plant cell wall by degrading homopolygalacturonan, a major constituent of pectin. Plants fight back by employing polygalacturonase-inhibitor proteins (PGIPs). The present study compared the inhibition potential of pearl millet PGIP (Pennisetum glaucum; PglPGIP1) with the known inhibition of Phaseolus vulgaris PGIP (PvPGIP2) against two PGs, the PG-II isoform from Aspergillus niger (AnPGII) and the PG-III isoform from Fusarium moniliforme (FmPGIII). The key rationale was to elucidate the relationship between the extent of sequence similarity of the PGIPs and the corresponding PG inhibition potential. First, a pearl millet pgip gene (Pglpgip1) was isolated and phylogenetically placed among monocot PGIPs alongside foxtail millet (Setaria italica). Upstream sequence analysis of Pglpgip1 identified important cis-elements responsive to light, plant stress hormones, and anoxic stress. PglPGIP1, heterologously produced in Escherichia coli, partially inhibited AnPGII non-competitively with a pH optimum between 4.0 and 4.5, and showed no inhibition against FmPGIII. Docking analysis showed that the concave surface of PglPGIP1 interacted strongly with the N-terminal region of AnPGII away from the active site, whereas it weakly interacted with the C-terminus of FmPGIII. Interestingly, PglPGIP1 and PvPGIP2 employed similar motif regions with few identical amino acids for interaction with AnPGII at non-substrate-binding sites; however, they engaged different regions of AnPGII. Computational mutagenesis predicted D126 (PglPGIP1)-K39 (AnPGII) to be the most significant binding contact in the PglPGIP1-AnPGII complex. Such protein-protein interaction studies are crucial in the future generation of designer host proteins for improved resistance against everevolving pathogen virulence factors.

Research paper thumbnail of Catabolic Linear Plasmids

Microbiology Monographs, 2007

Page 1. Microbiol Monogr (7) F. Meinhardt · R. Klassen: Microbial Linear Plasmids DOI 10.1007/717... more Page 1. Microbiol Monogr (7) F. Meinhardt · R. Klassen: Microbial Linear Plasmids DOI 10.1007/7171_2007_091/Published online: 1 June 2007 © Springer-Verlag Berlin Heidelberg 2007 Catabolic Linear Plasmids Susanne Fetzner (u) · Stephan Kolkenbrock · Katja Parschat ...

Research paper thumbnail of Parameters That Enhance the Bacterial Expression of Active Plant Polyphenol Oxidases

PLoS ONE, 2013

Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper proteins that enzymatically convert dip... more Polyphenol oxidases (PPOs, EC 1.10.3.1) are type-3 copper proteins that enzymatically convert diphenolic compounds into their corresponding quinones. Although there is significant interest in these enzymes because of their role in food deterioration, the lack of a suitable expression system for the production of soluble and active plant PPOs has prevented detailed investigations of their structure and activity. Recently we developed a bacterial expression system that was sufficient for the production of PPO isoenzymes from dandelion (Taraxacum officinale). The system comprised the Escherichia coli Rosetta 2 (DE3) [pLysSRARE2] strain combined with the pET-22b(+)-vector cultivated in auto-induction medium at a constant low temperature (26uC). Here we describe important parameters that enhance the production of active PPOs using dandelion PPO-2 for proof of concept. Low-temperature cultivation was essential for optimal yields, and the provision of CuCl 2 in the growth medium was necessary to produce an active enzyme. By increasing the copper concentration in the production medium to 0.2 mM, the yield in terms of PPO activity per mol purified protein was improved 2.7-fold achieving a v max of 0.4860.1 mkat per mg purified PPO-2 for 4-methylcatechol used as a substrate. This is likely to reflect the replacement of an inactive apo-form of the enzyme with a correctly-folded, copper-containing counterpart. We demonstrated the transferability of the method by successfully expressing a PPO from tomato (Solanum lycopersicum) showing that our optimized system is suitable for the analysis of further plant PPOs. Our new system therefore provides greater opportunities for the future of research into this economically-important class of enzymes.

Research paper thumbnail of A Novel Replicative Enzyme Encoded by the Linear Arthrobacter Plasmid pAL1

Journal of Bacteriology, 2010

The soil bacterium Arthrobacter nitroguajacolicus Rü61a contains the linear plasmid pAL1, which c... more The soil bacterium Arthrobacter nitroguajacolicus Rü61a contains the linear plasmid pAL1, which codes for the degradation of 2-methylquinoline. Like other linear replicons of actinomycetes, pAL1 is characterized by short terminal inverted-repeat sequences and terminal proteins (TP pAL1 ) covalently attached to its 5 ends. TP pAL1 , encoded by the pAL1.102 gene, interacts in vivo with the protein encoded by pAL1.101. Bioinformatic analysis of the pAL1.101 protein, which comprises 1,707 amino acids, suggested putative zinc finger and topoisomerase-primase domains and part of a superfamily 2 helicase domain in its N-terminal and central regions, respectively. Sequence motifs characteristic of the polymerization domain of family B DNA polymerases are partially conserved in a C-terminal segment. The purified recombinant protein catalyzed the deoxycytidylation of TP pAL1 in the presence of single-stranded DNA templates comprising the 3-terminal sequence (5-GCAGG-3), which in pAL1 forms the terminal inverted repeat, but also at templates with 5-(G/T)CA(GG/GC/CG)-3 ends. Enzyme assays suggested that the protein exhibits DNA topoisomerase, DNA helicase, and DNA-and protein-primed DNA polymerase activities. The pAL1.101 protein, therefore, may act as a replicase of pAL1.

Research paper thumbnail of N-Acetylanthranilate Amidase from Arthrobacter nitroguajacolicus Ru61a, an  / -Hydrolase-Fold Protein Active towards Aryl-Acylamides and -Esters, and Properties of Its Cysteine-Deficient Variant

Journal of Bacteriology, 2006

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldi... more N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the ␣/␤-hydrolase-fold superfamily of enzymes; inactivation of (His 6 -tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k cat /K m ‫؍‬ 208 mM ؊1 s ؊1 ), while among the aryl-acetylamides, o-carboxy-or o-nitrosubstituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His 6 Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His 6 AmqC22A/ C63A markedly differed from those of His 6 Amq. The replacements effected some changes in K m s of the enzyme and increased k cat s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k cat for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in ␣-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.

Research paper thumbnail of Identification and in vitro deoxynucleotidylation of the terminal protein of the linear plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a

FEMS Microbiology Letters, 2010

The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a is a linear replicon, characterized by i... more The plasmid pAL1 of Arthrobacter nitroguajacolicus Rü61a is a linear replicon, characterized by inverted terminal repeats and terminal proteins (TPs) covalently bound to its 5 0 -ends. Previous sequence analysis and predictions of possible secondary structures formed by telomeric 3 0 -overhangs indicated significant differences of the 'left' and 'right' telomere of pAL1, raising the question of whether each terminus is recognized by a specific protein. The genes pAL1.102 and pAL1.103, located close to a terminus, code for possible DNA-binding proteins; however, only the pORF102 protein encoded by pAL1.102 shows a weak similarity to known TPs of Streptomyces linear replicons. pORF102, purified from recombinant A. nitroguajacolicus Rü61a as a fusion with maltose-binding protein (MBP), was specifically associated with terminal pAL1 DNA, whereas MBP-pORF103 was devoid of DNA, suggesting that pORF102 represents the protein attached to both ends of the linear plasmid. In electrophoretic mobility shift assays, the MBP-pORF102 protein was not capable of specifically recognizing telomeric DNA sequences. Consistent with its proposed role as a protein primer in DNA synthesis, pORF102 was deoxynucleotidylated in vitro with dCMP, complementary to the 3 0 -ends (. . . GCAGG) of pAL1.