Enterobacter sp. BK2K, screened from soil samples, can enantioselectively reduce 2-oxo-4-phenylbutanoic acid into (S)-2-hydroxy-4-phenylbutanoic acid. alpha-Hydroxy acid dehydrogenase (HADH) (specific activity 62.
View Article and Find Full Text PDFStreptomyces coelicolor has a quorum sensing (QS) system triggered by small diffusible signaling molecules, i.e. butanolides (or gamma-butyrolactones) and their cognate DNA-binding receptors.
View Article and Find Full Text PDFA novel beta-transaminase gene was cloned from Mesorhizobium sp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.
View Article and Find Full Text PDFThe effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.
View Article and Find Full Text PDFJ Chromatogr B Analyt Technol Biomed Life Sci
May 2007
A method for the rapid identification of proteins and their N-glycans was developed through the use of two parallel columns directly connected to a mass spectrometer. Both porous graphitic carbon (PGC) and C18 capillary columns were connected in parallel with two switching valves for the simultaneous analysis of glycans and peptides, respectively. To verify the efficiency of the analytical system, profiling of N-glycans and proteins from human serum was demonstrated.
View Article and Find Full Text PDFAn important prerequisite for successful construction of a metagenome library is an efficient procedure for extracting DNA from environmental samples. We compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity, and time requirement. A special focus was on methods that are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.
View Article and Find Full Text PDFWe have carried out a simultaneous thermal denaturation and trypsin digestion of proteins using a temperature-controllable microreactor. This is a simple and rapid sample preparation technique for use before matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In contrast to a conventional sample preparation method, which involves several chemical treatments, our sample preparation was performed using only trypsin digestion with the thermal denaturation of the target protein.
View Article and Find Full Text PDFA new growth-based screening method for the identification of enantioselective hydrolases, such as lipases and esterases, using pro-antibiotic substrates was devised. An enantioselective hydrolase could be identified by measuring growth rates of cells in liquid media containing (R)- or (S)-2-phenylbutyric chloramphenicol esters. This method can be applied to the screening of novel enantioselective microbes and to the high-throughput screening for the directed evolution of enantioselective hydrolytic enzymes.
View Article and Find Full Text PDFThe amyloid beta (Abeta) peptide is known to increase free radical production in nerve cells, leading to cell death. To investigate the effect of Rosa laevigata against Abeta-induced oxidative damage, in vitro assays and in vivo behavioral tests were performed. R.
View Article and Find Full Text PDFA combination of microfluidic protein patterning and quantitative microfluidic handling has been used to analyze the binding kinetics of protein-ligand interactions on the nanoliter scale. The microfluidic handling method employing hydrophobic valving and pneumatic control allowed us to control nanoliter volumes of ligand or protein on a microfluidic chip. A hydrophobic and inert fluorocarbon thin film was patterned on a silicon nitride substrate to prevent non-specific binding on the background.
View Article and Find Full Text PDFAn enzymatic asymmetric synthesis was carried out for the preparation of enantiomerically pure L-diphenylalanine using the rationally engineered aromatic L-amino acid transaminase (eAroATEs) obtained from Enterobacter sp. BK2K-1. To rationally redesign the enzyme, structural model was constructed by the homology modeling.
View Article and Find Full Text PDFTylosin polyketide synthase (Tyl PKS) was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin PKS gene cluster using two compatible low-copy plasmids, each under the control of a pikAI promoter. The mutant strain produced 0.5 mg/l of the 16-membered ring macrolactone, tylactone, after a 4-day culture, which is a considerably reduced culture period to reach the maximum production level compared to other Streptomyces hosts.
View Article and Find Full Text PDFThe immunogenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney were identified and characterized using MALDI-TOF MS and ESI-QTOF-MS. The MALDI profile, investigated by incubation with exoglycosidases, showed a series of about 40 carbohydrates that were identified as high mannose glycans (Man(3-9)GlcNAc2) and complex bi-, tri-, and tetra-antennary glycans with and without core fucose. The antennae of many of the complex glycans were terminated with alpha-galactose residues, with the numbers of these residues ranging from one up to the number of antennae.
View Article and Find Full Text PDFLipopolysaccharide (LPS)-binding peptides were enriched by using epoxy beads as a novel support to immobilize LPS for a phage displayed peptide library screening. The sequence of Phe-Ala-Pro-Trp (FAPW) was the most significant consensus motif of 10 selected clones, and Pro-Phe (PF) was the key dipeptide for binding at the apex of the loop to form a characteristic structure of CXXPFXXXC. Moreover, AWLPWAK, one of the highly conserved heptamer peptides, could detect specifically Gram-negative bacteria via a whole cell binding test at 10(6) cells ml(-1).
View Article and Find Full Text PDFThe fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor.
View Article and Find Full Text PDFSyntheses of trans-(1R,2R) and cis-(1S,2R)-1-amino-2-indanol (AI) were accomplished by a series of enantioselective enzymatic reactions using lipase and transaminase (TA). Lipase catalysed enantioselective hydrolysis of 2-acetoxyindanone was employed to prepare (R)-2-hydroxy indanone (HI). trans-AI (5 mM) (de > 98%) was produced from 20 mM (R)-2- HI using omega-TA and 50 mM (S)-1-aminoindan as an amino donor in water-saturated ethyl acetate.
View Article and Find Full Text PDFdTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy-D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4-keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6-dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials.
View Article and Find Full Text PDFA cluster of genes for ribostamycin (Rbm) biosynthesis was isolated from Streptomyces ribosidificus ATCC 21294. Sequencing of 31.892 kb of the genomic DNA of S.
View Article and Find Full Text PDFA beacon aptamer-based biosensor for the detection of thrombin was developed using electrochemical transduction method. Gold surface was modified with a beacon aptamer covalently linked at 5'-terminus with a linker containing a primary aliphatic amine. Methylene blue (MB) was intercalated into the beacon sequence, and used as an electrochemical marker.
View Article and Find Full Text PDFGamma-butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry.
View Article and Find Full Text PDFA novel high-throughput screening method that overcame product inhibition was used to isolate a mutant omega-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, omega-TAmla, showed significantly reduced product inhibition by aliphatic ketone.
View Article and Find Full Text PDFBiochem Biophys Res Commun
September 2005
We report a new membrane surface display system based on molecular chaperon, prsA, of Bacillus subtilis. Clostridium thermocellum cellulase, celA, was fused to C-terminal end of PrsA. Cellulase activity of B.
View Article and Find Full Text PDFMost of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures.
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