Molecular cloning and expression of an endo-β-1,4-d-glucanase I (avicelase I) gene from Bacillus cellulyticus K-12 and characterization of the recombinant enzyme

Bacillus cellulyticus K-12 Avicelase (Avicelase I; EC 3.2.1.4) gene (ace A) has been cloned in Escherichia coli by using the vector pT7T3U19 and HindIII-HindIII libraries of the chromosomal inserts. The libraries were screened for the expression of avicelase by monitoring the immunoreaction of the antiavicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5-kb HindIII fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constitutively using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide that showed a carboxymethylcellulase (CMCase) activity with an M _r , of 64,000 was detected. The recombinant endo 1,4-β- _d -glucanase I was purified to homogeneity from an intracellular fraction of E. coli by DEAE-Toyopearl M650, Phenyl Toyoperal M650, and TSK gel HW50S chromatography. The enzyme had a monomeric structure, its relative molecular mass being 65 kDa by gel filtration and 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.3 and the optimal pH was 4.6, and the enzyme was stable at pH 4.0–10.5. The enzyme had a temperature optimum of 50°C and was stable at 55°C for 48 h, and retained approx 20% of its activity after 30 min at 70°C. It showed high activity toward carboxymethylcellulose (CMC) as well as p-nitrophenyl-β-d-cellobioside, 4-methylumbelliferyl cellobioside, Avicel, filter paper, and some cellooligosaccharides. K _m values for CMC and Avicel were 7.6 and 85.2 mg/mL, respectively, whereas V _max values were 201 and 9.2 μmol · min⁻¹ · mg⁻¹, respectively. Cellotetraose (G4) was preferentially cleaved into cellobiose (G2) and cellopentaose (G5) was cleaved into G2 + cellotriose (G3), whereas cellohexaose (G6) was cleaved into G4 + G2 and, to a lesser extent, into G3 + G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. G2 inhibited whereas Mg⁺⁺ stimulated the activity of CMCase and Avicelase. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars. Based on these results, it appeared that the cellulase should be regarded as endo type, although it hydrolyzed Avicel.     

The influences of solid-phase organic constituents on the partition of aliphatic and aromatic organic contaminants

The influence of natural organic matter (NOM) constituents on contaminant distribution coefficients was evaluated by determining the Koc values of aromatic and aliphatic organic compounds (solutes) with clays modified with both aromatic- and aliphatic-rich organic constituents. The studied compounds consisted of naphthalene, phenanthrene, n-pentane, and 2,3,4-trimethylmethane; the solid samples comprised two clays with little organic content, kaolinite and Ca-montmorillonite. Two aliphatic surfactants and three aromatic dyes, sorbed to the clays, served as reference NOM constituents. For solutes of comparable water solubilities, the organic-carbon normalized distribution coefficients (Koc) of the aliphatic solutes between sorbed aliphatic organic matter and aqueous solution slightly exceed those of the aromatic solutes. By contrast, the aromatic solutes exhibited higher Koc values than did the aliphatic compounds with sorbed aromatic-rich organic matter. The difference in Koc values could be attributed to either comparable solubility parameters or the difference in the chemical structure between nonionic organic solutes and specific components of the simulated NOM. The much higher Koc values observed for the aromatic solutes indicate that the NOM composition is a major factor determining the NOC environmental distribution.     

Amperometric Glucose Biosensor Based on Glucose Oxidase Encapsulated in Carbon Nanotube–Titania–Nafion Composite Film on Platinized Glassy Carbon Electrode

A highly sensitive and selective glucose biosensor has been developed based on immobilization of glucose oxidase within mesoporous carbon nanotube–titania–Nafion composite film coated on a platinized glassy carbon electrode. Synergistic electrocatalytic activity of carbon nanotubes and electrodeposited platinum nanoparticles on electrode surface resulted in an efficient reduction of hydrogen peroxide, allowing the sensitive and selective quantitation of glucose by the direct reduction of enzymatically‐liberated hydrogen peroxide at ?0.1 V versus Ag/AgCl (3 M NaCl) without a mediator. The present biosensor responded linearly to glucose in the wide concentration range from 5.0×10^?5 to 5.0×10^?3 M with a good sensitivity of 154 mA M^?1cm^?2. Due to the mesoporous nature of CNT–titania–Nafion composite film, the present biosensor exhibited very fast response time within 2 s. In addition, the present biosensor did not show any interference from large excess of ascorbic acid and uric acid.     

Chiral resolution of diphenylalanine by high-performance liquid chromatography on a crown-ether-based chiral stationary phase and by NMR spectroscopy

Summary The enantiomers of diphenylalanine (DPA) were well separated by chiral HPLC and NMR spectroscopy on the chiral stationary phase (CSP) derived from (18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TA). The chromatographic parameters such as separation factors and retention times were greatly influenced by the mobile phase conditions. The (+)-18-C-6-TA used in the CSP was also employed as a chiral solvating agent for the enantiodiscrimination of the DPA enantiomers by NMR spectroscopy. The proton of the DPA analyte showing the chemical shift nonequivalences was used in determining the enantiomeric composition of the analyte.     

Syntheses of methylene-bridged ansa-zirconocene complexes and copolymerization studies of ethylene and norbornene

Methylene-bridged ansa-metallocene complexes bearing substituents on the cyclopentadienyl (Cp) and fluorenyl (Flu) moieties, namely methylene[9-(2,7-di-tert-butyl)fluorenyl(2-(1,3-dimethylcyclopentadienyl))]zirconium dichloride (1a) and its analogue, methylene[(9-(2,7-di-tert-butyl)fluorenyl(2-(1-methyl-3-phenyl)cyclopentadienyl))]zirconium dichloride (2a), have been prepared from (2,7-di-tert-butyl)-9-prop-2-ynyl-9H-fluorene (2). This procedure includes the use of 3-bromo-1-propyne which affords the methylene bridging unit by way of an intermolecular Pauson-Khand reaction in which norbornadiene and a pendant alkyne cyclize to form a ring that later becomes a substituted cyclopentadienyl group. Ethylene-norbornene (E-N) copolymerization was then carried out using these new complexes (1a and 1b) in the presence of methylaluminoxane (MAO) as a cocatalyst; these activities can be compared to that of isopropylene[9-fluorenyl-cyclopentadienyl]zirconium dichloride (3a). The activity of catalyst 1a was comparable to that of 3a but much higher than that of 2a. In addition, 1a shows higher norbornene insertion performance, and gives an E-N copolymer with a higher glass transition temperature (T_g) than 2a under identical conditions; both 1a and 2a give a lower T_g polymer than 3a does.     

Total synthesis of erythromycin B

We report the details of the first total synthesis of erythromycin B using two different strategies for the end game. The first of these follows a classical approach in which the desosamine and cladinose residues are sequentially appended to a macrocyclic lactone, which was formed by cyclization of a seco acid derivative, to give a bis-glycosylated macrolide intermediate that is converted into erythromycin B. The second strategy features an abiotic approach in which a seco acid bearing a desosamine residue is cyclized to give a monoglycosylated macrocyclic lactone that is then transformed into erythromycin B via a sequence of steps involving refunctionalizations and a glycosylation to introduce the cladinose moiety. Attempts to prepare a bis-glycosylated seco acid by de novo synthesis were unsuccessful. The syntheses of the key seco acid intermediates feature the oxidative transformation of a furan containing C(3)-C(10) to provide a dioxabicyclo[3.3.1]nonenone that served as a template on which to create the stereocenters at C(6) and C(8). A stereoselective aldol reaction was used to establish the C(11)-C(15) segment, and a stereoselective crotylation was implemented to introduce the propionate subunit comprising C(1)-C(2).     

Essential of Proline and Valine Residues in the Peptide Derived from Lactoferrin for Angiotensin Converting Enzyme Inhibition

We synthesized Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu, the peptide contained in lactoferrin (Lf), to identify the angiotensin converting enzyme (ACE) inhibition. In an attempt to know the structure‐activity relationship of this peptide, we replaced Pro (the third amino acid residues from N‐terminal) or Val (the fourth amino acid residues from N‐terminal) with Ala (neutral amino acid), Glu (acidic amino acid) or Lys (basic amino acid) to produce six peptides. From the in vitro ACE inhibition (IC₅₀) of these synthesized peptides, the original peptide (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu) showed higher ACE inhibition than the replaced six peptides. Thus, replacement of Pro at the third amino acid residues or Val at the fourth position with Ala, Glu or Lys revealed the ACE inhibition to be lower than the original form of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu. Otherwise, we added one peptide at the C‐terminal of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu and found both products with an addition of Val (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Val) or Ile (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Ile) showing a lower ACE inhibition than the original one. The ACE inhibitions produced by both replaced peptides were without significance. Also, deletion of the last peptide at the C‐terminal (Leu‐Arg‐Pro‐Val‐Ala‐Ala) failed to produce a marked change of ACE inhibition as compared to the original one. These results suggest that Pro and Val are essential in the peptide for inhibition of ACE activity.     

Construction of diverse peptide structural architectures via chemoselective peptide ligation

Herein, we report the development of a facile synthetic strategy for constructing diverse peptide structural architectures via chemoselective peptide ligation. The key advancement involved is to utilize the benzofuran moiety as the peptide salicylaldehyde ester surrogate, and Dap–Ser/Lys–Ser dipeptide as the hydroxyl amino functionality, which could be successfully introduced at the side chain of peptides enabling peptide ligation. With this method, the side chain-to-side chain cyclic peptide, branched/bridged peptides, tailed cyclic peptides and multi-cyclic peptides have been designed and successfully synthesized with native peptidic linkages at the ligation sites. This strategy has provided an alternative strategic opportunity for synthetic peptide development. It also serves as an inspiration for the structural design of PPI inhibitors with new modalities.

Methods of introducing peptide salicylaldehyde esters and hydroxyl amine functionality into the peptide side chain have been developed. Diverse peptide structural motifs were constructed via ligation with native amide linkages at the ligation sites.