From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p-nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET-hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2-hydroxyethyl benzoate (HEB), and mono-(2-hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET-hydrolase was isolated from non-denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC-MS/MS analysis. BsEstB was expressed in Escherichia coli with C-terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p-nitrophenyl acetate and 108 U/mg on p-nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half-life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2-hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The kcat values decreased with increasing complexity of the substrate from 6 and 8 (s-1) for p-nitrophenyl-acetate (4NPA) and p-nitrophenyl-butyrate (4NPB), respectively, to 0.14 (s-1) for bis(2-hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water-contact angle (WCA) from 68.2°±1.7° to 62.6°±1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2-(bromomethyl)naphthalene.

A novel biocatalytic protocol for CC bond formation is described and is an equivalent to Friedel–Crafts alkylation. S‐Adenosyl‐L‐methionine (SAM), the major methyl donor for biological methylation catalyzed by methyltransferases (Mtases), can perform alkylations (see scheme). These enzymes can accept non‐natural cofactors and transfer functionalities other than methyl onto aromatic substrates.

Eine neuartige biokatalytische C‐C‐Verknüpfung, die äquivalent zur Friedel‐Crafts‐Alkylierung ist, wird vorgestellt. S‐Adenosyl‐L‐methionin (SAM), der Haupt‐Methyldonor bei Methyltransferase(Mtase)‐katalysierten biologischen Methylierungen, kann Alkylierungen bewirken (siehe Schema). Diese Enzyme akzeptieren nichtnatürliche Cofaktoren und können andere Funktionalitäten als Me auf aromatische Substrate übertragen.

No abstract

We have cloned and sequenced a cluster of six open reading frames containing gene kdsA from Escherichia coli K-12. The gene encodes 3-deoxy-D-manno-octulosonate 8-phosphate synthetase (KDO-8-phosphate synthetase), which catalyzes formation of 3-deoxy-D-manno-octulosonic acid (KDO), an essential component of enterobacterial lipopolysaccharide. We have also identified two other genes, hemA and prfA, at the beginning of the cluster. Deletion analysis shows that kdsA, the terminal gene of this putative operon, is transcribed from its own promoter located within the cluster rather than from two promoters preceding this group of six open reading frames. Northern (RNA) blot analysis as well as lacZ operon fusion experiments reveal that the expression of gene kdsA occurs maximally in the early log phase and falls to a low level in the late log and stationary phases. Hence, this gene is subjected to growth phase-dependent regulation at the transcriptional level. Similarly, we show that expression of gene kdsB, which codes for the CTP:CMP-3-deoxy-D-mannooctulosonate cytidyltransferase (CMP-KDO-synthetase), is also growth regulated. This enzyme catalyzes the activation of KDO via formation of CMP-KDO, which is necessary for the incorporation of KDO into lipid A. We have identified the promoter of gene kdsB, whose expression is growth regulated in the same way as that of kdsA. Despite the fact that transcription of genes kdsA and kdsB is shut off as cells enter stationary phase, KDO-8-phosphate synthetase as well as CMP-KDO-synthetase activities are still present at various levels during stationary-phase growth of an E. coli K-12 culture.A major component of the outer membrane of enterobacteria is lipopolysaccharide (LPS), the so-called endotoxin, which is responsible for the pathogenic properties of gramnegative bacteria (38). This macromolecule consists of two structural segments, the hydrophobic lipid A part and the hydrophilic core oligosaccharide chain to which the O-antigenic polysaccharide chains may be attached. Lipid A and the core oligosaccharides are linked through two or three molecules of the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid (KDO) (31,38). KDO is an essential component of LPS. This is reflected by the fact that only conditionally lethal KDO biosynthesis mutants can be isolated (25,36,37). KDO is enzymatically synthesized in the cytoplasm in two steps, i.e., formation of KDO-8-phosphate by the condensation of Darabinose-5-phosphate and phosphoenol pyruvate and subsequent dephosphorylation by a specific phosphatase to give KDO. The enzymes involved in this biosynthetic pathway are 3-deoxy-D-manno-octulosonate 8-phosphate synthetase (KDO-8-phosphate synthetase) (32) and KDO-8-phosphate phosphatase (33). Subsequent incorporation into lipid A requires activation of KDO by CTP in a reaction catalyzed by the cytosolic enzyme CTP:CMP-3-deoxy-D-manno-octulosonate cytidyltransferase (CMP-KDO-synthetase) (17,34,35). KDO is finally incorporated into lipid A by a KDO-transferase(s) (7). KDO-8-phosphate sy...