The evolution of cyclodextrin glucanotransferase product specificity

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, β- and γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity

Catalytic activity and stability of laccase entrapped in sol-gel silica with additives

This study investigated the effects of different additives and precursors on the catalytic activity of laccase entrapped in sol-gel silica. It was found that the laccase catalytic activity and stability of sol-gel laccase could be enhanced if the entrapment was performed in the presence of additives such as PVA, PEG and APTS. The use of TEOS as a precursor showed slightly higher laccase catalytic activity compared to TMOS. The PVA as an additive showed a better catalytic activity enhancement compared to the PEG and APTMS with the optimum PVA concentration of 0.03 mg/mL. The optimal temperatures of sol-gel laccase without and with additives were found to be at 40 and 27°C, respectively. After 70 days of storage at 27°C, the catalytic activity of the immobilized sol-gel laccase with additives maintained its catalytic activity compared to only 30% of its original catalytic activity for the sol-gel laccase without additives

Fluorobenzoyl dipeptidyl derivatives as inhibitors of the Fasciola hepatica cysteine protease cathepsin L1

Cathepsins are known to have many important physiological roles and provide a viable target for inhibition. Fluorobenzoyl dipeptide derivatives were synthesized and tested for biological activity in an effort to find an efficient inhibitor of the cysteine protease cathepsin L. Thirty-six novel inhibitors (1-36) were synthesized from protected amino acids via the standard DCC/HOBt coupling protocol, containing a benzyl ester or a nitrile as an electrophilic warhead. The activity of the inhibitors was evaluated against cathepsin L and IC50 values calculated. Modification of both amino acids and terminal groups afforded compounds with single digit micromolar inhibition. Results utilizing the benzoyl-L-leucine-glycine nitrile backbone are comparable to that for the commercially available inhibitor 39. © 2010 Informa UK Ltd

Design and characterization of redox enzyme electrodes: new perspectives on established techniques with application to an extremeophilic hydrogenase

Biofuel cells promise power generation with low cost and almost unlimited biocatalyst supply. This paper reports new perspectives on the characterization of immobilized thermostable (Pyrococcus,furiosus) hydrogenase electrodes in working configuration, using a combination of well defined electrochemical and spectrophotometric techniques. Electrodes based on porous pyrolytic carbon paper (PCP) and packed graphite columns (PGC) were fabricated for hydrogenase immobilization, which used a direct (hydrophilic adsorption) technique. Potentiostatic de polarization, dynamic potentiometry, and electrochemical impedance spectroscopy (EIS) were combined with spectrophotometric detection of enzyme activity in order to characterize the electrodes and differentiate the relative contributions from enzyme loading, charge transfer, and mass transport to the limiting current density, which is a critical consideration for electrodes intended for fuel cell and bioelectrocatalytic application. Dynamic potentiometry proved useful as a rapid screening and characterization procedure for both blank electrodes and the presence of bound hydrogenase post-immobilization. Combining current density data determined via dc polarization (hydrogen oxidation currents of 30 muA cm(-2) for PCP electrodes and 90-120 muA cm(-2) PGC electrodes at 75 degreesC) with hydrogen mass transport limits determined via modeling and platinum electrode polarization (480 muA cm(-2) at 75 degreesC), allowed determination that insufficient active enzyme was present to reach system mass transport limits to hydrogen supply for all electrodes. Additionally, spectrophotometric enzyme determination on the PCP electrode (0.034 units cm(-2)) was used to derive a theoretical maximum in hydrogen oxidation current (110 muA cm(-2), assuming 100% enzyme/support charge transfer efficiency), which when combined with current density data (30 muA cm(-2)), allowed determination that actual charge transfer efficiency per unit total bound enzyme was significant (28%). The EIS scans on immobilized PGC type electrodes suggest that mass transport might attribute the most to cell impedance at the low frequency regime. These combined results indicated that low bulk enzyme loading was the limiting factor to current density for thermostable hydrogenase electrodes fabricated for the study. (C) 2004 Elsevier Inc. All rights reserved

Evaluation of Protein Modification during Anti-Viral Heat Bioprocessing by Electrospray Ionization Mass Spectrometry

During the preparation of therapeutic plasma and recombinant protein biopharmaceuticals heat-treatment is routinely applied as a means of viral inactivation. However, as most proteins denature and aggregate under heat stress, it is necessary to add thermostabilizing excipients to protein formulations destined for anti-viral heat-treatment in order to prevent protein damage. Anti-viral heat-treatment bioprocessing therefore requires that a balance be found between the bioprocessing conditions, virus kill and protein integrity. In this study we have utilized a simple model protein, beta -lactoglobulin, to investigate the relationship between virucidal heat-treatment conditions (protein formulation and temperature) and the type and extent of protein modification in the liquid state. A variety of industrially relevant heat-treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) we show here that protein mo difications do occur with increasingly harsh heat-treatment. The predominant modification under these conditions was protein glycation by either glucose or fructose derived from hydrolyzed sucrose. Advanced glycation end products and additional unidentified products were also present in beta -lactoglobulin protein samples subjected to extended heat-treatment. These findings have implications for the improvement of anti-viral heat-treatment bioprocesses to ensure the safety and efficacy of protein biopharmaceuticals