Christoph Winterhalter
Technische Universität München
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Molecular Microbiology | 1995
Christoph Winterhalter; Peter Heinrich; Anton Candussio; Günther Wich; Wolfgang Liebl
A segment of Thermotoga maritima strain MSB8 chromosomal DNA was isolated which encodes an endo‐1,4‐β‐D‐xylanase, and the nucleotide sequence of the xylanase gene, designated xynA, was determined. With a half‐life of about 40 min at 90°C at the optimal pH of 6.2, purified recombinant XynA is one of the most thermostable xylanases known. XynA is a 1059‐amino‐acid (˜120 kDa) modular enzyme composed of an N‐terminal signal peptide and five domains, in the order A1‐A2‐B‐C1‐C2. By comparison with other xylanases of family 10 of glycosyl hydrolases, the central ˜340‐amino‐acid part (domain B) of XynA represents the catalytic domain. The N terminal ˜150‐amino‐acid repeated domains (A1‐A2) have no significant similarity to the C‐terminal ˜170‐amino‐acid repeated domains (C1‐C2). Cellulose‐binding studies with truncated XynA derivatives and hybrid proteins indicated that the C‐terminal repeated domains mediate the binding of XynA to microcrystalline cellulose and that C2 alone can also promote cellulose binding. C1 and C2 did not share amino acid sequence similarity with any other known cellulose‐binding domain (CBD) and thus are CBDS of a novel type. Structurally related protein segments which are probably also CBDs were found in other multi‐domain xylanolytic enzymes. Deletion of the N‐terminal repeated domains or of all the non‐catalytic domains resulted In substantially reduced tbermostability while a truncated xylanase derivative lacking the C‐terminal tandem repeat was as thermostable as the full‐length enzyme. It is argued that the multidomain organization of some enzymes may be one of the strategies adopted by thermophiles to protect their proteins against thermal denaturation.
Molecular Microbiology | 2000
Tobias Dassler; Thomas Maier; Christoph Winterhalter; August Böck
A chromosomal fragment has been identified in a gene bank from Escherichia coli, which augmented the yield of cysteine in an industrial production strain. Subcloning and genetic analysis showed that an open reading frame coding for a product of 299 amino acids (Orf299) was responsible. Orf299 was synthesized in the T7 polymerase/promoter system and exhibited the properties of an integral membrane protein. Mutational interruption of orf299 did not cause a distinct phenotype; however, transformants overexpressing orf299 had lost the ability to grow in minimal medium unless it was supplemented with a source of reduced sulphur compounds, and they excreted considerable amounts of cysteine and O‐acetyl‐l‐serine, especially in the presence of thiosulphate. Most of the cysteine was found to be masked in 2‐methyl‐2,4‐thiazolidinedicarboxylic acid. N‐acetyl‐l‐serine was also present in the medium, but it is open to question whether it represents a primary excretion product. Measurement of the induction status of the cysteine regulon by means of a cysK′–′lacZ gene fusion demonstrated that the regulon is not induced upon growth in the presence of a poor sulphur source and that the introduction of a constitutive cysB allele alleviates this deficiency. The results indicate that orf299 codes for an export pump for different metabolites of the cysteine pathway. Its relation to other efflux systems and the physiological role are discussed.
Molecular Microbiology | 1997
Peter Ruile; Christoph Winterhalter; Wolfgang Liebl
This is the first report describing the analysis of a gene encoding an α‐glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for α‐glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The α‐glucuronidase gene (aguA) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli, and the start of the coding region was defined by amino‐terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674‐amino‐acid, largely hydrophilic polypeptide with a calculated molecular mass of 78 593 Da. The α‐glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS–PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (<630kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2‐O‐(4‐O‐methyl‐α‐d‐glucopyranosyluronic acid)‐d‐xylobiose (MeGlcAX2) to xylobiose and 4‐O‐methylglucuronic acid. The Km for MeGlcAX2 was 0.95mM. The pH optimum was 6.3. Maximum activity was measured at 85°C, about 25°C or more above the values reported for all other α‐glucuronidases known to date. When incubated at 55–75°C, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.
Journal of Bacteriology | 2008
Wolfgang Liebl; Christoph Winterhalter; Wolfgang Baumeister; Martin Armbrecht; Michael Valdez
Archive | 1999
Thomas Maier; Christoph Winterhalter
Archive | 2004
Thomas Dr. Maier; Christoph Winterhalter; Kerstin Pfeiffer
Archive | 2004
Thomas Maier; Christoph Winterhalter; Kerstin Pfeiffer
Archive | 2004
Thomas Dr. Maier; Kerstin Pfeiffer; Christoph Winterhalter
Archive | 2004
Thomas Dr. Maier; Christoph Winterhalter; Kerstin Pfeiffer
Archive | 2000
Thomas Maier; Christoph Winterhalter