Publications by authors named "Meike Ballschmiter"

9 Publications

  • Page 1 of 1

Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering.

Protein Eng Des Sel 2011 May 27;24(5):439-46. Epub 2011 Jan 27.

Institute of Biochemistry, University of Leipzig, Deutscher Platz 5b, 04103 Leipzig, Germany.

A focused library comprising nine residues of the active site of P450cam monooxygenase resulting in ∼ 300,000 protein variants was screened for activity on diphenylmethane (DPM). The assay was based on the depletion of NADH by an in vitro reconstituted P450cam system in a 96-well scale. The throughput was increased by the parallel cultivation, purification and analysis of 20 variants per well (cluster screening). Thus ∼ 20,000 protein variants could be screened in summary of which five were found to transform DPM with a specific activity of up to 75% of the wild-type activity on d-camphor and a coupling rate of 7-18%. One variant converting DPM to 4-hydroxydiphenylmethane (4HDPM) was subjected to site-directed mutagenesis and saturation mutagenesis, which revealed the particular importance of positions F87, Y96 and L244 for substrate selectivity and the possibility for further improvements of this variant. Moreover, a reduction in size of the amino acid at position 396 decreased specific activity dramatically but increased coupling and switched the main product formation from 4HDPM towards diphenylmethanol.
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http://dx.doi.org/10.1093/protein/gzq119DOI Listing
May 2011

Hyperthermostable acetyl xylan esterase.

Microb Biotechnol 2010 Jan 18;3(1):84-92. Epub 2009 Sep 18.

Institut f. Mikrobiologie und Genetik, Georg-August-Universität, Grisebachstr. 8, D-37077 Goettingen, Germany.

An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C. Thermotoga maritima AxeA represents the most thermostable acetyl xylan esterase known to date. In a 10 min assay at its optimum pH of 6.5 the enzyme's activity peaked at 90 °C. The inactivation half-life of AxeA at a protein concentration of 0.3 µg µl(-1) in the absence of substrate was about 13 h at 98 °C and about 67 h at 90°C. Differential scanning calorimetry analysis of the thermal stability of AxeA corroborated its extreme heat resistance. A multi-phasic unfolding behaviour was found, with two apparent exothermic peaks at approximately 100-104 °C and 107.5 °C. In accordance with the crystal structure, gel filtration analysis at ambient temperature revealed that the enzyme has as a homohexameric oligomerization state, but a dimeric form was also found.
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http://dx.doi.org/10.1111/j.1751-7915.2009.00150.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815950PMC
January 2010

Genome sequence of the polysaccharide-degrading, thermophilic anaerobe Spirochaeta thermophila DSM 6192.

J Bacteriol 2010 Dec 8;192(24):6492-3. Epub 2010 Oct 8.

Department of Genomic and Applied Microbiology, Göttingen Genomics Laboratory, Institute of Microbiology and Genetics, Georg-August-Universität, Grisebachstr 8, D-37077 Goettingen, Germany.

Spirochaeta thermophila is a thermophilic, free-living anaerobe that is able to degrade various α- and β-linked sugar polymers, including cellulose. We report here the complete genome sequence of S. thermophila DSM 6192, which is the first genome sequence of a thermophilic, free-living member of the Spirochaetes phylum. The genome data reveal a high density of genes encoding enzymes from more than 30 glycoside hydrolase families, a noncellulosomal enzyme system for (hemi)cellulose degradation, and indicate the presence of a novel carbohydrate-binding module.
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http://dx.doi.org/10.1128/JB.01023-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3008529PMC
December 2010

Characterization and engineering of a novel pyrroloquinoline quinone dependent glucose dehydrogenase from Sorangium cellulosum So ce56.

Mol Biotechnol 2011 Mar;47(3):253-61

Institute of Biochemistry, University of Leipzig, Deutscher Platz 5b, 04103 Leipzig, Germany.

A novel pyrroloquinoline quinone dependent glucose dehydrogenase like enzyme (PQQ GDH) was isolated from Sorangium cellulosum So ce56. The putative coding region was cloned, over expressed in E. coli and the resulting enzyme was characterized. The recombinant protein has a relative molecular mass of 63 kDa and shows 43% homology to PQQ GDH-B from Acinetobacter calcoaceticus. In the presence of PQQ and CaCl₂ the enzyme has dehydrogenase activity with the substrate glucose as well as with other mono- and disaccharides. The thermal stability and its pH activity profile mark the enzyme as a potential glucose biosensor enzyme. In order to decrease the activity on maltose, which is unwanted for a potential application in biosensors, the protein was rationally modified at three specified positions. The best variant showed a 59% reduction in activity on maltose compared to the wild type enzyme. The catalytic efficiency (k(cat)/K(M)) was reduced fivefold but the specific activity still amounted to 63% of the wild type activity.
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http://dx.doi.org/10.1007/s12033-010-9339-5DOI Listing
March 2011

A nitrilase from a metagenomic library acts regioselectively on aliphatic dinitriles.

Appl Microbiol Biotechnol 2011 Jan 20;89(1):91-8. Epub 2010 Aug 20.

Junior Research Group White Biotechnology, Institute of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Deutscher Platz 5b, 04103 Leipzig, Germany.

Several novel nitrilases were selected from metagenomic libraries using cinnamonitrile and a mixture of six different nitriles as substrates. The nitrilase gene nit1 was expressed in Escherichia coli and the resulting protein was further examined concerning its biochemical properties. Nit1 turned out to be an aliphatic nitrilase favoring dinitriles over mononitriles. Stereochemical analysis revealed that Nit1 converted the dinitrile 2-methylglutaronitrile regioselectively. Hydrolysis at the ω-nitrile group of a dinitrile, such as catalyzed by Nit1, leads to ω-cyanocarboxylic acids, which are important precursors for chemical and pharmaceutical products. Nit1 metabolized 2-methylglutaronitrile to the corresponding ω-cyanocarboxylic acid 4-cyanopentanoic acid can be used for the production of the fine chemical 1,5-dimethyl-2-piperidone.
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http://dx.doi.org/10.1007/s00253-010-2831-9DOI Listing
January 2011

Indication for a new lipolytic enzyme family: isolation and characterization of two esterases from a metagenomic library.

J Mol Microbiol Biotechnol 2010 8;18(3):181-7. Epub 2010 Jun 8.

Institute of Biochemistry, University of Leipzig, Germany.

We have isolated several novel esterase genes from a sheep rumen metagenomic library using the activity-based cluster screening approach as a highly efficient screening technology. The two most remarkable esterase genes, designated estGK1 and estZ3, were further examined. Sequence analysis of estGK1 and estZ3 revealed that they encoded proteins covering 322 and 317 amino acids, respectively. Both proteins were biochemically characterized. EstGK1 and EstZ3 have only minor overall sequence similarity to known esterases. We propose that, together with other hypothetical enzymes, they constitute a new family of lipolytic enzymes. EstGK1 harbors the catalytic serine in the conserved pentapeptide GHSQG, which is typical for lipases, whereas EstZ3 and several other hypothetical proteins contain the pentapeptide SHSQG, a new variation of the conserved motif in lipolytic enzyme families.
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http://dx.doi.org/10.1159/000315459DOI Listing
September 2010

Identification and characterization of a novel intracellular alkaline alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8.

Appl Environ Microbiol 2006 Mar;72(3):2206-11

Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany.

The gene for a novel alpha-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 alpha-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an alpha-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90 degrees C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.
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http://dx.doi.org/10.1128/AEM.72.3.2206-2211.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1393205PMC
March 2006

Structure of the novel alpha-amylase AmyC from Thermotoga maritima.

Acta Crystallogr D Biol Crystallogr 2006 Mar 22;62(Pt 3):262-70. Epub 2006 Feb 22.

Abteilung Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität, Göttingen, Germany.

alpha-Amylases are essential enzymes in alpha-glucan metabolism and catalyse the hydrolysis of long sugar polymers such as amylose and starch. The crystal structure of a previously unidentified amylase (AmyC) from the hyperthermophilic organism Thermotoga maritima was determined at 2.2 Angstroms resolution by means of MAD. AmyC lacks sequence similarity to canonical alpha-amylases, which belong to glycosyl hydrolase families 13, 70 and 77, but exhibits significant similarity to a group of as yet uncharacterized proteins in COG1543 and is related to glycerol hydrolase family 57 (GH-57). AmyC reveals features that are characteristic of alpha-amylases, such as a distorted TIM-barrel structure formed by seven beta-strands and alpha-helices (domain A), and two additional but less well conserved domains. The latter are domain B, which contains three helices inserted in the TIM-barrel after beta-sheet 2, and domain C, a five-helix region at the C-terminus. Interestingly, despite moderate sequence homology, structure comparison revealed significant similarities to a member of GH-57 with known three-dimensional structure, Thermococcus litoralis 4-glucanotransferase, and an even higher similarity to a structure of an enzyme of unknown function from Thermus thermophilus.
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http://dx.doi.org/10.1107/S0907444905041363DOI Listing
March 2006

AmyA, an alpha-amylase with beta-cyclodextrin-forming activity, and AmyB from the thermoalkaliphilic organism Anaerobranca gottschalkii: two alpha-amylases adapted to their different cellular localizations.

Appl Environ Microbiol 2005 Jul;71(7):3709-15

Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany.

Two alpha-amylase genes from the thermophilic alkaliphile Anaerobranca gottschalkii were cloned, and the corresponding enzymes, AmyA and AmyB, were investigated after purification of the recombinant proteins. Based on their amino acid sequences, AmyA is proposed to be a lipoprotein with extracellular localization and thus is exposed to the alkaline milieu, while AmyB apparently represents a cytoplasmic enzyme. The amino acid sequences of both enzymes bear high similarity to those of GHF13 proteins. The different cellular localizations of AmyA and AmyB are reflected in their physicochemical properties. The alkaline pH optimum (pH 8), as well as the broad pH range, of AmyA activity (more than 50% activity between pH 6 and pH 9.5) mirrors the conditions that are encountered by an extracellular enzyme exposed to the medium of A. gottschalkii, which grows between pH 6 and pH 10.5. AmyB, on the other hand, has a narrow pH range with a slightly acidic pH optimum at 6 to 6.5, which is presumably close to the pH in the cytoplasm. Also, the intracellular AmyB is less tolerant of high temperatures than the extracellular AmyA. While AmyA has a half-life of 48 h at 70 degrees C, AmyB has a half-life of only about 10 min at that temperature, perhaps due to the lack of stabilizing constituents of the cytoplasm. AmyA and AmyB were very similar with respect to their substrate specificity profiles, clearly preferring amylose over amylopectin, pullulan, and glycogen. Both enzymes also hydrolyzed alpha-, beta-, and gamma-cyclodextrin. Very interestingly, AmyA, but not AmyB, displayed high transglycosylation activity on maltooligosaccharides and also had significant beta-cyclodextrin glycosyltransferase (CGTase) activity. CGTase activity has not been reported for typical alpha-amylases before. The mechanism of cyclodextrin formation by AmyA is unknown.
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http://dx.doi.org/10.1128/AEM.71.7.3709-3715.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1169011PMC
July 2005