Improved operational stability of peroxidases by coimmobilization with glucose oxidase

The operational stability of peroxidases was considerably enhanced by generating hydrogen peroxide in situ from glucose and oxygen. For example, the total turnover number of microperoxidase-11 in the oxidation of thioanisole was increased sevenfold compared with that obtained with continuous addition of H(2)O(2). Coimmobilization of peroxidases with glucose oxidase into polyurethane foams afforded heterogeneous biocatalysts in which the hydrogen peroxide is formed inside the polymeric matrix from glucose and oxygen. The total turnover number of chloroperoxidase in the oxidation of thioanisole and cis-2-heptene was increased to new maxima of 250. 10(3) and 10. 10(3), respectively, upon coimmobilization with glucose oxidase. Soybean peroxidase, which normally shows only classical peroxidase activity, was transformed into an oxygen-transfer catalyst when coimmobilized with glucose oxidase. The combination catalyst mediated the enantioselective oxidation of thioanisole [50% ee (S)] with 210 catalyst turnovers.     

Engineering chloroperoxidase for activity and stability

Random mutagenesis of Caldariomyces fumago was carried out to produce a chloroperoxidase (CPO) with enhanced activity in mixtures of aqueous buffers and organic cosolvents. A mutant CPO with a 3.4-fold increased activity in 40% aqueous tert-butyl alcohol was obtained.

A CPO-surfactant conjugate, prepared by colyophilisation, mediated the oxidation of thioanisole to its (R)-sulfoxide by TBHP in water-saturated isooctane. Copolymerisation of CPO and a toluenediisocyanate prepolymer resulted in a stable preparation that mediated the oxidation of indole to 2-oxindole in a range of organic solvents. The highest yield was obtained in 1-octanol.     

Chemical modification of chloroperoxidase for enhanced stability and activity

Chloroperoxidase from Caldariomyces fumago (CPO, EC 1.11.1.10) is one of the most interesting enzymes from the group of heme peroxidases and has been extensively applied in synthetic processes. Nevertheless, the practical application of CPO is limited due to its very low operational stability, especially in the presence of peroxidative compounds. For this reason, effect of chemical modifications of CPO in the stability of the enzyme was studied. Side-chain selective modifications of amino groups of Lys residues, and carboxyl groups of Asp and Glu residues, as well as crosslinking and periodate oxidation of sugar moiety were carried out. The stability of modified CPOs was evaluated at elevated pH and temperature, and in the presence of tert-butyl hydroperoxide. Effect of modification of CPO on the performance of the reaction of Cbz-ethanolamine oxidation was studied as well. Those modifications that involved carboxyl groups via carbodiimide coupled method and the periodate oxidation of the sugar moiety produced better catalysts than native CPO in terms of stability and activity at elevated pH values and temperatures.     

Improved insulin stability through amino acid substitution.

Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.     

Improvement of chloroperoxidase stability by covalent immobilization on chitosan membranes

Chloroperoxidase (CPO) from Caldariomyces fumago was optimally covalently immobilized on chitosan membranes pretreated with 0.8 M glutaraldehyde at pH 3.5 to give 3.18 mg CPO g⁻¹ support. Using monochlorodimedone (MCD) as assay substrate, the immobilized-CPO retained 40% activity at 50°C after 40 min whereas free CPO retained only 0.02%. The residual activity for immobilized-CPO was 99 and 58% compared with 68 and 43% for free CPO in the presence of 1.5 M urea and 300 μM H₂O₂, respectively, after 20 h.     

Improvement of activity and stability of chloroperoxidase by chemical modification

Background  

Enzymes show relative instability in solvents or at elevated temperature and lower activity in organic solvent than in water. These limit the industrial applications of enzymes.     

Improved protein identification through the use of unstained gels

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.     

Kinetic and stability studies on the chloroperoxidase complexes in presence of tert-butyl hydroperoxide

The inactivation of native chloroperoxidase (CPO) from Caldariomyces fumago in the presence of tert-butyl hydroperoxide (tert-BuOOH) was investigated. A kinetic analysis was made and the inactivation constants (V(3) and K(3)) were evaluated. In prolonged times, uni-exponential equation describes the enzyme time course inactivation. A method based on the rate of inactivation of the enzyme in the presence of the inactivating molecule tert-BuOOH was also performed. A second group of inactivation constants (j(3) and K) was obtained, which is sufficiently close to the first two, thus verifying that the decreasing of enzyme absorbance corresponds to the decay of activity.     

Improved stability of a protein vaccine through elimination of a partially unfolded state

Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide-linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N-acetylgalactosamine-binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267-amino-acid A chain consisting of residues 1-33/44-198 or 1-198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier-transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule-like state at 45 degrees C, rRTA 1-33/44-198 and 1-198 are significantly more thermostable, remaining completely folded at temperatures up to 53 degrees C and 51 degrees C, respectively. Deleting both an exposed loop region (amino acids 34-43) and the C-terminal domain (199-267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three-state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C-terminal domain. Removing the C-terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens.     

Cross-linked crystals of chloroperoxidase

Chloroperoxidase from Caldariomyces fumago was crystallized. The crystals were modified with several cross-linkers, but only glurataldehyde was able to produce catalytically active and insoluble crystals. Unlike other immobilized chloroperoxidase preparations, these catalytic crystals are more thermostable than the unmodified soluble enzyme. The enhanced stability is probably due to the structure conservation in the crystalline matrix. In addition, non-cross-linked chloroperoxidase crystals retained more activity than the soluble enzyme after incubation in an organic solvent with low water content. Although the cross-linked crystals were catalytically active, they showed lower specific activity than the soluble enzyme. This low activity may be due to non-specific reactions between the cross-linker and essential residues for catalysis. Alternative cross-linking strategies are discussed.     

Enhancement of operational stability of immobilized whole cell D-hydantoinase

Summary A bacteriumPseudomonas sp. KBEL 101 isolated from soil was immobilized within polyacrylamide gel and used for the synthesis of D-p-hydroxyphenylglycine from DL-5-substituted hydantoin. The half-life of immobilized whole cell D-hydantoinase was found to be about 50 hrs. In order to increase the operational stability of immobilized whole cell D-hydantoinase, a carbon or nitrogen source was supplied with the reaction mixture in the continuous stirred tank reactor. As a sole source of carbon, glycerol was found to be most effective, and the activity of immobilized whole cell enzyme was maintained stably during 7 days when 0.1%(W/V) glycerol solution was provided. In the case of nitrogen source, supplying of 0.1%(W/V) yeast extract prolonged the half-life of immobilized whole cell D-hydantoinase to about 25 days.     

Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway

The metabolic pathways of the central carbon metabolism in Saccharomyces cerevisiae are well studied and consequently S. cerevisiae has been widely evaluated as a cell factory for many industrial biological products. In this study, we investigated the effect of engineering the supply of precursor, acetyl‐CoA, and cofactor, NADPH, on the biosynthesis of the bacterial biopolymer polyhydroxybutyrate (PHB), in S. cerevisiae. Supply of acetyl‐CoA was engineered by over‐expression of genes from the ethanol degradation pathway or by heterologous expression of the phophoketolase pathway from Aspergillus nidulans. Both strategies improved the production of PHB. Integration of gapN encoding NADP⁺‐dependent glyceraldehyde‐3‐phosphate dehydrogenase from Streptococcus mutans into the genome enabled an increased supply of NADPH resulting in a decrease in glycerol production and increased production of PHB. The strategy that resulted in the highest PHB production after 100 h was with a strain harboring the phosphoketolase pathway to supply acetyl‐CoA without the need of increased NADPH production by gapN integration. The results from this study imply that during the exponential growth on glucose, the biosynthesis of PHB in S. cerevisiae is likely to be limited by the supply of NADPH whereas supply of acetyl‐CoA as precursor plays a more important role in the improvement of PHB production during growth on ethanol. Biotechnol. Bioeng. 2013; 110: 2216–2224. © 2013 Wiley Periodicals, Inc.     

Inactivation of chloroperoxidase by arginine

Inactivation of chloroperoxidase (CPO) from Caldariomyces fumago by arginine was investigated. It was found that the red native CPO solution was turned into a stable green species with a concomitant shift of the Soret band from 398 to 425 nm in the presence of arginine. The green CPO lost almost all of its catalytic activity, and this inactivation was irreversible.Differential UV–vis spectrophotometry was used to examine the binding properties of arginine to CPO. The formation of CPO-arginine (1:1) complex was highly pH-dependent. Fluorescence investigation revealed the exposure degree of prosthetic group increased. Kinetic analysis indicated that CPO has both a high affinity and specificity to arginine.This inactivation may be caused mainly by the binding of guanidinium group in arginine to the acid–base catalytic group Glu183 in CPO. The change of surrounding environment around heme induced by the interaction of heme propionates with arginine and the occupying of the sixth axial ligand position of heme iron by hydroxyl are also reasons bringing on this inactivation.     

The ferric-hydroperoxo complex of chloroperoxidase

The hydroperoxo-ferric complex, or Compound 0 (Cpd 0), is an unstable transient intermediate common for oxygen activating heme enzymes such as the cytochromes P450, nitric oxide synthases, and heme oxygenases, as well as the peroxidases and catalases which utilize hydrogen peroxide as a source of oxygen and reducing equivalents. Detailed understanding of the mechanism of oxygen activation and formation of the higher valent catalytically active intermediates in heme enzyme catalysis requires the structural and spectroscopic characterization of this immediate precursor, Cpd 0. Using the method of cryoradiolytic reduction of the oxy-ferrous heme complex, we have prepared and characterized hydroperoxo-ferric complex in chloroperoxidase (CPO) and compared this to the same intermediate generated in cytochrome P450 CYP101. Optical absorption spectrum of Cpd 0 in CPO has a Soret band at 449 nm and poorly resolved α, β bands at 576 and 546 nm.     

Improved sensitivity of profile searches through the use of sequence weights and gap excision

Position-specific substitution matrices, known as profiles,derived from multiple sequence alignments are currently usedto search sequence databases for distantly related members ofprotein families. The performance of the database searches isenhanced by using (i) a sequence weighting scheme which assignshigher weights to more distantly related sequences based onbranch lengths derived from phylogenetic trees, (ii) exclusionof positions with mainly padding characters at sites of insertionsor deletions and (iii) the BLOSUM62 residue comparison matrix.A natural consequence of these modifications is an improvementin the alignment of new sequences to the profiles. However,the accuracy of the alignments can be further increased by employinga similarity residue comparison matrix. These developments areimplemented in a program called PROFILEWEIGHT which runs onUnix and Vax computers. The only input required by the programis the multiple sequence alignment. The output from PROFILEWEIGHTis a profile designed to be used by existing searching and alignmentprograms. Test results from database searches with four differentfamilies of proteins show the improved sensitivity of the weightedprofiles.     

Spectrum of chloroperoxidase compound I

When compound I of chloroperoxidase is formed from the native enzyme the absorption peak in the Soret region diminishes in intensity, and shifts to a maximum absorbance at 367 nm. This unusual Soret spectrum decreases in intensity in a linear fashion as the wavelength increases. The first visible spectrum of chloroperoxidase compound I is reported which has a peak at 689 nm as its most prominent feature.     

Macrokinetics and operational stability of immobilized glucose oxidase and catalase

Glucose oxidation by immobilized glucose oxidase (GlO) and catalase (Cat) has been investigated in batch and continuous reactions for operational studies. The macrokinetics of the process depend on coupled reaction steps and diffusion rates. The problem may be approximated by a simple pseudohomogeneous model taking into account both substrates of glucose oxidase and the intermediate reaction product H₂O₂. The effectiveness of both enzymes is enhanced in the coupled reaction path, the overall effectiveness nevertheless is very low. H₂O₂ causes the inactivation of both GlO and Cat. The rates of deactivation depend on the oxidation rates of glucose that give different quasistationary levels of H₂O₂ concentration. As a first approximation, the deactivation rates may be described by first-order reactions with respect to H₂O₂.