To broaden the applicability of chemically modified DNAs in nano- and biotechnology, material science, sensor development, and molecular recognition, strategies are required for introducing a large variety of different modifications into the same nucleic acid sequence at once. Here, we investigate the scope and limits for obtaining functionalized dsDNA by primer extension and PCR, using a broad variety of chemically modified deoxynucleotide triphosphates (dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand were substituted with up to four different base-modified analogues. We studied the sequence dependence of enzymatic amplification to yield high-density functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found that GC-rich sequences are amplified with decreased efficiency as compared to AT-rich ones. There is also a strong dependence on the polymerase used. While family A polymerases generally performed poorly on "demanding" templates containing consecutive stretches of a particular base, family B polymerases were better suited for this purpose, in particular Pwo and Vent (exo-) DNA polymerase. A systematic analysis of fDNAs modified at increasing densities by CD spectroscopy revealed that single modified bases do not alter the overall B-type DNA structure, regardless of their chemical nature. A density of three modified bases induces conformational changes in the double helix, reflected by an inversion of the CD spectra. Our study provides a basis for establishing a generally applicable toolbox of enzymes, templates, and monomers for generating high-density functionalized DNAs for a broad range of applications.
Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) utilize phosphatidylinositols containing D-3-position phosphates as substrates to form phosphatidylinositol 3,4-bisphosphate. In addition, type I PIP5Ks phosphorylate phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, while type II kinases have less activity toward this substrate. Remarkably, these kinases can convert phosphatidylinositol 3-phosphate to phosphatidylinositol 3,4,5-trisphosphate in a concerted reaction. Kinase activities toward the 3-position phosphoinositides are comparable with those seen with phosphatidylinositol 4-phosphate as the substrate. Therefore, the PIP5Ks can synthesize phosphatidylinositol 4,5-bisphosphate and two 3-phosphate-containing polyphosphoinositides. These unexpected activities position the PIP5Ks as potential participants in the generation of all polyphosphoinositide signaling molecules.Two distinct pathways have been characterized for agoniststimulated signal transduction involving phosphatidylinositol (PtdIns).1 One pathway entails activation of phosphatidylinositol-specific phospholipase C by extracellular agonists resulting in the hydrolysis of phosphoinositides to generate soluble inositol phosphates including inositol 1,4,5-trisphosphate and diacylglycerol (reviewed in Refs. 1 and 2). The other pathway involves receptor-mediated activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) to produce the second messengers, phosphatidylinositol 3,4-bisphosphate (PtdIns 3,4-P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P 3 ) (reviewed in Refs. 3 and 4).A pathway for the formation of D-3-phosphatidylinositols, proposed based on kinetic studies of intact human neutrophils, is through phosphorylation of the D-3 position of the myoinositol ring of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P 2 ) by a PtdIns 4,5-P 2 3-kinase and subsequent dephosphorylation of PtdIns 3,4,5-P 3 to produce PtdIns 3,4-P 2 (5). This pathway has been supported by the existence of the extensively characterized PtdIns 3-kinase enzyme family, which can catalyze in vitro phosphorylation of phosphatidylinositol 4-phosphate (PtdIns 4-P) and PtdIns 4,5-P 2 . Evidence for a different pathway for the formation of D-3 phosphatidylinositols has been found in human platelets, NIH 3T3 cells, and plants in which phosphorylation of the D-3-position of PtdIns to form PtdIns 3-P is followed by phosphorylation of the D-4-position to give PtdIns 3,4-P 2 and then of the D-5-position to form PtdIns 3,4,5-P 3 (6 -
Commercial products for personal care, generally perceived as cosmetics, have an important impact on everyday life worldwide. Accordingly, the market for both consumer products and specialty chemicals comprising their ingredients is considerable. Lipases have started to play a minor role as active ingredients in so-called 'functional cosmetics' as well as a major role as catalysts for the industrial production of various specialty esters, aroma compounds and active agents. Interestingly, both applications almost always require preparation by appropriate immobilisation techniques. In addition, for catalytic use special reactor concepts often have to be employed due to the mostly limited stability of these preparations. Nevertheless, these processes show distinct advantages based on process simplification, product quality and environmental footprint and are therefore apt to more and more replace traditional chemical processes. Here, for the first time a review on the various aspects of using immobilised lipases in the cosmetics industry is given.
To overcome limitations due to the high viscosity in the solvent free esterification of polyglycerol-3 and related polyols, such as poly(ethylene glycol)s, an alternative reactor concept was developed. The new reactor comprises a bubble column that prevents mechanical erosion of Novozym 435 (lipase B from Candida antarctica) found by mechanical stirring of the mixture. That way polyglycerol-3 laurate was synthesized at a space time yield (sty) of 3042 g/L/d and PEG-55-propylene glycol dioleate at a sty of 738 g/L/d. To proof the broad application range of this system, low-viscosity myristyl myristate was synthesized at a sty of 6731 g/L/d, thus outperforming conventional methods such as stirred tank or fixed bed. The newly developed reactor concept is universally applicable to esterification reactions and can be advantageously applied in the synthesis of a broad range of high quality surfactants.
Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.
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