Palle Schneider

Directed evolution of a fungal peroxidase

The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50°C), and high peroxide concentration (5–10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.

Redox Chemistry in Laccase-Catalyzed Oxidation of N-Hydroxy Compounds

ABSTRACT 1-Hydroxybenzotriazole, violuric acid, andN-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an “outer-sphere” type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation.

The identification and characterization of four laccases from the plant pathogenic fungusRhizoctonia solani

Four distinct laccase genes,lcc1, lcc2, lcc3 andlcc4, have been identified in the fungusRhizoctonia solani. Both cDNA and genomic copies of these genes were isolated and characterized. Hybridization analyses indicate that each of the four laccase genes is present in a single copy in the genome. TheR. solani laccases can be divided into two groups based on their protein size, intron/exon organization, and transcriptional regulation. Three of these enzymes have been expressed in the fungusAspergillus oryzae. Two of the recombinant laccases, r-lccl and r-lcc4, as well as the native lcc4 enzyme were purified and characterized. The purified proteins are homodimeric, comprised of two subunits of approximately 66 kDa for lcc4 and 50–100 kDa for the recombinant lccl protein. These laccases have spectral properties that are consistent with other blue copper proteins. With syringaldazine as a substrate, lcc4 has optimal activity at pH 7, whereas lcc1 has optimal activity at pH 6.

Characterization of a Coprinus cinereus laccase

A wild-type Coprinus cinereus laccase and its recombinant form expressed in an Aspergillus oryzae host have been purified and characterized. The mature laccase had a molecular mass of 58 kDa by mass spectrometry, an isoelectric point near 4, and two absorption maxima at 278 and 614 nm. Photometric titration with 2,2′-biquinoline showed a Cu/protein(subunit) stoichiometry of ≈4. The electron paramagnetic resonance spectrum showed typical type 1 and type 2 Cu signals, and the circular dichroism showed a typical coordination geometry of the type 1 Cu(II). At pH 5.5, the enzyme had a redox potential of 0.55 V vs. normal hydrogen electrode at its type 1 site. The laccase could oxidize 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) and syringaldazine with optimum pH of 4 and 6.5, respectively. Halides inhibited the laccase. At pH 8.5, the laccase had an optimum temperature between 60°C and 70°C. At the same pH, the laccase had a half-life of >200 or 21.8 min in the presence of 0 or 2 mM H2O2, respectively, at 40°C. Mediated by several phenols and phenothiazines, the laccase was able to oxidatively bleach Direct Blue 1 dye at alkaline pH, making it a promising industrial enzyme candidate.

Comparison of structure and activities of peroxidases from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus

Initial structural and kinetic data suggested that peroxidases from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus were similar. Therefore they were characterized more fully. The three peroxidases were purified to RZ 2.5 and showed immunochemical identity as well as an identical M(r) of 38,000, pI about 3.5 and similar amino acid compositions. The N-termini were blocked for amino acid sequencing. The peroxidases had similar retention volumes by anion-exchange and gel-filtration chromatography. All peroxidases showed multiple peaks by Concanavalin A-Sepharose chromatography. The Concanavalin A-Sepharose profiles were different and depended furthermore on a fermentation batch. Tryptic peptide maps were very similar except for one peptide. This peptide contained an N-linked glycan composed of varying ratios of glucosamine and mannose for the three peroxidases. Rate constants and their pH dependence were the same for the three peroxidases using guaiacol or iodide as reducing substrates. We conclude that peroxidases from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus are most likely identical in their amino acid sequences, but deviate in glycosylation which, apparently, has no influence on the reaction rates of the enzyme. We suggest, that the Coprinus fungi express one peroxidase only in contrast to the lignin-degrading white-rot Basidiomycetes, which produce multiple peroxidase isozymes.

Isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes, exemplified by purification of glucoamylase.

A highly purified preparation of glucoamylase G1 from Aspergillus niger was found, by isoelectric focusing in immobilized pH gradients, to contain a major form with a pI of 3.50 and a number of minor, more acidic and more basic contaminants. The major isoform was purified to homogeneity by recycling isoelectric focusing in a multicompartment electrolyzer, by confining this form in between two zwitterionic membranes, with pI 3.49 at the anodic side and pI 3.52 at the cathodic side. Recoveries were high (90%) and, notwithstanding the rather low operational pH, the electrosmotic flow was minimal and no protein precipitation occurred up to concentrations of 2.5 mg/ml (at the isoelectric point). The forms resolved in an analytical focusing gel were subjected to two types of in situ enzyme detections, by the glucose oxidase peroxidase (GOP) test and by the starch-iodine test. By both criteria all resolved zones exhibited enzyme activity, the GOP assay, however, following more closely the Coomassie blue stained protein profile. By computer modelling, it is shown that it is impossible to obtain linear pH gradients at such low pH values (pH 2.5-4.5 intervals) when the mixture has a low buffering power (beta = 2.0 mequiv.l-1 pH-1). When the beta power was gradually raised (beta = 4, beta = 6, beta = 8) the pH gradient became progressively linear until, in a recipe with beta = 10 mequiv.l-1 pH-1 full linearity of the pH gradient could be obtained. This is shown to be due to the substantial buffering power of bulk water in the pH 2.5-3.5 region.

Crystallization and preliminary X-ray analysis of the laccase from Coprinus cinereus.

The laccase from the fungus Coprinus cinereus has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small plate-like crystals of an enzymatically deglycosylated form of the enzyme have been grown by the hanging-drop method using polyethylene glycol as precipitant. These crystals diffract to at least 2.2 A. They belong to the space group P2(1)2(1)2(1) with cell dimensions a = 45.4, b = 85.7, c = 143.1 A with a single molecule of laccase in the asymmetric unit.

Specificity and kinetic parameters of recombinant Microdochium nivale carbohydrate oxidase

Abstract A new carbohydrate oxidase from Microdochium nivale heterologously expressed in Aspergillus oryzae (rMnO) has been characterized. The carbohydrate oxidase is a flavoenzyme which oxidizes glucose and other mono- or oligosaccharides. It shows a broad substrate specificity towards carbohydrates reacting with aldoses in the 1-position. The rMnO oxidizes the β-form of d -glucose, and the product of d -glucose oxidation is d -gluconic acid. The mechanism of carbohydrate oxidation by oxygen and artificial electron acceptors has been described by a ping-pong scheme. Compared to Aspergillus niger glucose oxidase (GOx) the reactivity of rMnO at pH 7.0 is significantly lower; k cat is 20, k ox 11 and k red 22 times less, using oxygen as electron acceptor. Also with other two electron acceptors, like DPIP, the activity is low. However, compared to oxygen the rMnO shows 2–10 times higher activity towards some artificial single electron acceptors (AAs). The enzyme activity increases at higher ionic strength of the solution, if positively-charged AAs are used. The high activity towards AAs and low rate for oxygen as well as broad specificity to carbohydrates indicates that rMnO may have some advantages compared to the most used GOx in connection with enzyme use for analytical devices and for biotechnological purposes.

Expression and characterization of a recombinant Fusarium spp. galactose oxidase.

The Fusarium spp. (Dactylium dendroides) galactose oxidase was expressed in Aspergillus oryzae and Fusarium venenatum hosts. Under the control of an A. niger α-amylase or a Fusarium trypsin promoter, high level galactose oxidase expression was achieved. The recombinant oxidase expressed in the A. oryzae host was purified and characterized. The purified enzyme had a molecular weight of 66 k Da on sodium dodecyl sulfate-polymerase gel electrophoresis (SDS-PAGE) and 0.4 mol copper atom per mole protein. The stoichiometry increased to 1.2 after a Cu saturation. Based on a peroxidase-coupled assay, the enzyme preparation showed an activity of 440 turnover per second toward d-galactose (0.1 M) at pH7 and 20°C. The enzyme had an optimal temperature of 60°C at pH 6.0 and an activation free Gibbs energy of 33 kJ/mol. A series of d-galactose derivatives was tested as the reducing substrate for the oxidase. The difference in activity was interpreted by the stereospecificity of the oxidase toward the substituents in the pyranose substrate, particularly on the C5 and the cyclic hemiacetal O sites. The recombinan toxidase could act on some galactose-containing polysaccharides, such as guar gum, but was not able to oxidize several common redox compounds that lacked a primary alcohol functional group.

The development of an improved glucose biosensor using recombinant carbohydrate oxidase from Microdochium nivale

Biosensors containing recombinant carbohydrate oxidase from Microdochium nivale (rMnO) were developed using either a chemically modified carbon paste or a graphite electrode. 1-(N,N-dimethylamine)-4-(4-morpholine)benzene (AMB) and 1,1′-dimethylferrocene (DMFc) were used as the mediators. The biosensors showed a linear calibration graph up to 0.018 mol dm−3 of glucose when operated at 0.04–0.36 V vs. SCE. Almost no change was detected in the sensitivity of the biosensors at pH 7.2–8.1. The biosensors responded to a range of D-aldoses, but maximal sensitivity of the biosensor was with D-glucose. The biosensors gave no response to polyhydroxylic compounds such as D-mannitol, D-sorbitol and inositol. The advantage of the biosensor in comparison to the biosensor based on Aspergillus niger glucose oxidase is a wide linear range, low sensitivity to oxygen and (in some cases) broad specificity.