Peanut protein structure, polyphenol content and immune response to peanut proteins in vivo are modulated by laccase.

Food texture can be improved by enzyme-mediated covalent cross-linking of different food components, such as proteins and carbohydrates. Cross-linking changes the biological and immunological properties of proteins and may change the sensitizing potential of food allergens. In this study we applied a microbial polyphenol oxidase, laccase, to cross-link peanut proteins. The size and morphology of the obtained cross-linked proteins were analyzed by electrophoresis and electron microscopy. Structural changes in proteins were analyzed by CD spectroscopy and by using specific antibodies to major peanut allergens. The bioavailability of peanut proteins was analyzed using a Caco-2 epithelial cell model. The in vivo sensitizing potential of laccase-treated peanut proteins was analyzed using a mouse model of food allergy. Finally, peanut polyphenols were analyzed by UHPLC-MS/MS, before and after the enzymatic reaction with laccase. Laccase treatment of peanut proteins yielded a covalently cross-linked material, with the modified tertiary structure of peanut proteins, improved bioavailability of Ara h 2 (by 70 fold, p < 0.05) and modulated allergic immune response in vivo. The modulation of the immune response was related to the increased production of IgG2a antibodies 11 fold (p < 0.05) and reduced IL-13 secretion in in vitro cultured splenocytes 7 fold (p < 0.05). Analysis of the peanut polyphenol content and profile by HPLC-MS/MS revealed that laccase treatment depleted the peanut extract of polyphenol compounds leaving mostly isorhamnetin derivatives and procyanidin dimer B-type in detectable amounts. Treatment of complex food extracts rich in polyphenols with laccase results in both protein cross-linking and modification of polyphenol compounds. These extensively cross-linked proteins have unchanged potency to induce allergic sensitization in vivo, but certain immunomodulatory changes were observed.

Tyrosinase-aided protein cross-linking: effects on gel formation of chicken breast myofibrils and texture and water-holding of chicken breast meat homogenate gels.

UNLABELLED: The effects of Trichoderma reesei tyrosinase-catalyzed cross-linking of isolated chicken breast myofibril proteins as a simplified model system were studied with special emphasis on the thermal stability and gel formation of myofibrillar proteins. In addition, tyrosinase-catalyzed cross-linking was utilized to modify the firmness, water-holding capacity (WHC), and microstructure of cooked chicken breast meat homogenate gels. According to SDS-PAGE, the myosin heavy chain (MHC) and troponin T were the most sensitive proteins to the action of tyrosinase, whereas actin was not affected to the same extent. Calorimetric enthalpy (DeltaH) of the major thermal transition associated with myosin denaturation was reduced and with actin denaturation increased in the presence of tyrosinase. Low-amplitude viscoelastic measurements at constant temperatures of 25 degrees C and 40 degrees C showed that tyrosinase substantially increased the storage modulus (G') of the 4% myofibrillar protein suspension in the 0.35 M NaCl concentration. The effect was the most pronounced with high-enzyme dosages and at 40 degrees C. Without tyrosinase, the G' increase was low. Tyrosinase increased the firmness of the cooked phosphate-free and low-meat chicken breast meat homogenate gels compared to the corresponding controls. Tyrosinase maintained gel firmness at the control level of the low-salt homogenate gel and weakened it when both salt and phosphate levels were low. Tyrosinase improved the WHC of the low-meat and low-salt homogenate gels and maintained it at the level of the corresponding controls of phosphate-free and low-salt/low-phosphate homogenate gels. Microstructural characterization showed that a collagen network was formed in the presence of tyrosinase. KEYWORDS: Chicken myofibrillar proteins; protein modification; cross-linking; tyrosinase; gelation; thermal stability; texture; water-holding capacity; microstructure.

Colanic acid is an exopolysaccharide common to many enterobacteria isolated from paper-machine slimes.

In this study, polysaccharide-producing bacteria were isolated from slimes collected from two Finnish and one Spanish paper mill and the exopolysaccharides (EPSs) produced by 18 isolates were characterised. Most of the isolates, selected on the bases of slimy colony morphology, were members of the family Enterobacteriaceae most frequently belonging to the genera Enterobacter and Klebsiella including Raoultella. All of the EPSs analysed showed the presence of charged groups in the form of uronic acid or pyruvate revealing the polyanionic nature of these polysaccharides. Further results of the carbohydrate analysis showed that the EPS produced by nine of the enterobacteria was colanic acid.

Enzymatic determination of biogenic amines with transglutaminase.

Tyramine, histamine, putrescine and cadaverine, the most common biogenic amines indicating the food quality, were studied in the transglutaminase-catalyzed reaction. Transglutaminase (protein-glutamine gamma-glutamyltransferase EC 2.3.2.13) catalyzes an acyl transfer reaction between a donor substrate and an acceptor substrate (e.g. biogenic amine) and forms a cross-linkage between substrates with a release of ammonia. The reaction can be monitored by measuring the ammonia produced in the reaction. The concentration of produced ammonia was found to be proportional to the concentration of biogenic amine and could hence be used to determination of biogenic amines in food matrixes.

Enzyme-aided modification of chicken-breast myofibril proteins: effect of laccase and transglutaminase on gelation and thermal stability.

The effect of laccase and transglutaminase (TG) on cross-linking, gelation, and thermal stability of salt-soluble chicken-breast myofibril proteins was investigated at pH 6. Both enzymes modified the protein pattern detected by SDS-PAGE. Identification of proteins by peptide mass mapping showed that myosin heavy chain (MHC) and troponin T were the most affected proteins. These proteins faded or disappeared as a function of the incubation time with both enzymes on SDS-PAGE. The molecular weight of actin was not, however, affected by either enzyme. The effects that the enzymes had on the gel formation of chicken-breast myofibrils were studied in 0.35 and 0.60 M NaCl solutions at 3% protein content and a constant temperature of 40 degrees C by using a small deformation viscoelastic measurement. TG substantially increased the storage modulus (G') of 3% protein in 0.35 M NaCl. Without the enzymes, gelation was insignificant in 0.35 M NaCl. The increased solubility of the proteins at 0.60 M NaCl intensified gelation with TG. G' increased 32 and 64% at dosages of 10 and 100 nkat of TG, respectively. Also, laccase increased G' of the gel in 0.60 M salt concentration. However, a high laccase dosage decreased the magnitude of G' below the control level. Differential scanning calorimetric (DSC) measurements indicated slightly reduced myosin heat stability after TG pretreatment and increased actin heat stability with both enzymes. Maximum transition temperatures did not alter with either enzyme.

Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase.

In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O(2) during laccase and tyrosinase treatment and of H(2)O(2) during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20-120 min after the addition of enzyme).

Action of purified Trichoderma reesei cellulases on cotton fibers and yarn.

In this work the possibility and potential of treating cotton fibers and yarns instead of fabrics with monocomponent cellulases was investigated. Different pretreatments on fibers were performed and tested in order to improve the accessibility of cotton to enzymatic modification. The enzymatic treatments were evaluated microscopically and by analysing the effects of treated fibers on spinnability, yarn evenness, tenacity and pilling. The accessibility of the cotton fibers for cellulases could be increased by different pretreatments. Steaming of fibers prior to enzymatic treatment was found to be an efficient way to increase hydrolysis levels. Cellulase treatments of carded yarns resulted in modification of yarn properties. Decrease in yarn hairiness was observed and the knitted fabric made of the treated yarn showed a lowered tendency towards pilling. In all cases endoglucanase activity rather than cellobiohydrolase activity was responsible for these modifications.

Reactivity of Trametes laccases with fatty and resin acids.

Lipophilic extractives commonly referred to as wood pitch or wood resin can have a negative impact on paper machine runnability and product quality. The lipophilic extractives are composed mainly of fatty acids, resin acids, sterols, steryl esters and triglycerides. In this work, the suitability of laccases for the modification of fatty and resin acids was studied, using two model fractions. In the treatments, resin and fatty acid dispersions were treated with two different laccases, i.e. laccases from Trametes hirsuta and T. villosa. Different chromatographic methods were used to elucidate the effects of laccase treatments on the chemistry of the fatty and resin acids. Both laccases were able to modify the fatty and resin acids to some extent. In the case of fatty acids, a decrease in the amount of linoleic, oleic and pinolenic acids was observed, whereas the modification of resin acids resulted in a reduced amount of conjugated resin acids.

Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and cellulases.

Effects of recycling ECF-bleached softwood kraft pulp on pulp properties were evaluated in the laboratory. The tensile strength, fiber flexibility and WRV lost during drying of the pulp were recovered by refining between the cycles which, however, resulted in deteriorated drainage properties. The recycled pulps were treated with purified Trichoderma reesei cellulases and hemicellulases and the changes in fiber properties due to enzymatic treatments were characterized. The endoglucanases (EG I and EG II) significantly improved pulp drainage already at low dosage levels, and EG II was found to be more effective at a given level of carbohydrate solubilization. Combining hemicellulases with the endoglucanase treatments increased the positive effects of the endoglucanases on pulp drainage. However, as a result of the endoglucanase treatments a slight loss in strength was observed. Combining mannanase with endoglucanase treatments appeared to increase this negative effect, whereas the impact of xylanase was not significant. Although the drainage properties of the pulps could be improved by selected enzymes, the water retention capacity of the dried hornified fibers could not be recovered by any of the enzymes tested.

Structure of dicarboxyl malto-oligomers isolated from hypochlorite-oxidised potato starch studied by 1H and 13C NMR spectroscopy.

The main oxidised component in hypochlorite-oxidised potato starch was isolated by anion-exchange chromatography after enzymatic hydrolysis. The primary structure of the isolated oligosaccharides was determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The isolated pentamer and hexamer contained one glucose unit oxidised to a dicarboxyl residue. As the hypochlorite oxidation has occurred at positions C-2 and C-3 of a glucose unit, the introduced carboxyl groups caused ring cleavage between the carbons C-2 and C-3. The ring-cleaved dicarboxyl residue had glycosidic linkages on both sides, implying that this oxidation pathway does not result in depolymerisation. The vicinal coupling constant between H-4 and H-5 in the ring-cleaved dicarboxyl residue was 3.2 Hz, showing that the gauche orientations are preferred. As a result, a different bending of the starch chain is observed and is probably, therefore, one of the reasons why hypochlorite oxidation reduces the tendency to retrogradation. The pKa values (3.0) were determined from the pH-dependent chemical shifts of H-1, H-4 and H-5 of the dicarboxylic residue.

Hemicellulases in the bleaching of chemical pulps.

Hemicellulase-aided bleaching is the first full-scale biotechnical application in the pulp and paper industry which truly exploits the unique specificity and safety of biocatalysts. Hemicellulases are used to modify the structure of xylan and glucomannan in pulp fibers in order to enhance the chemical delignification. This technology can be combined with various types of kraft pulping processes and bleaching sequences. The aims of the enzymatic treatment depend on the actual mill conditions, and may be related to environmental demands, reduction of chemical costs, or maintenance or even improvement of product quality. The technology is applied on the mill scale in several countries. This review describes the principles of the enzyme-aided bleaching, the composition of the fiber substrates, the basic enzymology involved, and the present knowledge of the mechanisms of the action of enzymes, as well as the practical results and advantages obtained on the laboratory and industrial scale.

4-O-methyl-beta-L-idopyranosyluronic acid linked to xylan from kraft pulp: isolation procedure and characterisation by NMR spectroscopy.

The tetrasaccharide 2"-O-(4-O-methyl-beta-L-idopyranosyluronic acid)xylotriose was isolated from enzymatically hydrolysed, unbleached, birch kraft pulp by anion-exchange chromatography in two steps. The primary structure of the tetrasaccharide was determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. NOE data and 3JH,H coupling constants show that the 4-O-methyl-beta-L-idopyranosyluronic acid in the tetrasaccharide is predominantly in the 1C4 chair conformation. The pKa value (3.17) for 4-O-methyliduronic acid attached beta-(1-->2) to xylose was determined from the pH-dependent chemical shift of H-5. The amount of 4-O-methyliduronic acid (0.1-0.5 mol%) in surface xylan of unbleached birch and pine kraft pulps was determined by extensive xylanase treatment and further analysis by NMR spectroscopy and high-performance anion-exchange chromatography.

Characterisation of 4-deoxy-beta-L-threo-hex-4-enopyranosyluronic acid attached to xylan in pine kraft pulp and pulping liquor by 1H and 13C NMR spectroscopy.

A new acidic sidegroup in xylans, from both kraft pulp and pulping liquor, was identified by NMR spectroscopy. Unmodified oligosaccharides from kraft pulp xylan were obtained by enzymatic hydrolysis with xylanase (Trichoderma reesei). The acidic oligosaccharides were separated from the natural forms on an anion exchange resin. The new acidic sidegroup was identified as 4-deoxy-beta-L-threo-hex-4-enopyranosyluronic acid (hexenuronic acid) by 1H and 13C NMR spectroscopy. Hexenuronic acid is a beta-elimination product of 4-O-methylglucuronic acid and is formed during kraft pulping. HMBC and NOESY experiments showed that hexenuronic acid is attached beta-(1 --> 2) to xylose. The NOESY data further indicated that hexenuronic acid protrudes from the main xylan chain. The pKa values for hexenuronic acid (3.03) and 4-O-methylglucuronic acid (3.14) attached (1 --> 2) to xylose were determined from pH-dependent chemical shifts.

Large-scale applicable purification and characterization of a membrane-bound PQQ-dependent aldose dehydrogenase.

A membrane-bound xylose oxidizing PQQ-dependent dehydrogenase from Gluconobacter oxydans was purified with a simple large-scale applicable purification procedure. The activity recovery from membrane extract was 33% with 130-fold purification. Important characteristic with respect to the application of the dehydrogenase in biosensor technology were studied. The purified enzyme was most stable in the pH range 3.5-6.5. The pH optimum for xylose oxidation was in the range 7.5-8 for the solubilized enzyme. Optimal pH for the electrochemical detection of xylose oxidation was 6.5. Dimethyl and carboxylic acid derivatives of ferrocene were able to mediate electrons transferred in xylose oxidation from the enzyme immobilized on graphite electrode to the electrode. Hence the purified enzyme appeared to be suitable for biosensor applications.

Ultrahigh repetition rate picosecond pulses from a passively mode-locked neodymium glass laser by solid-state saturable dye films.

Ultrashort pulses with ultrahigh tunable repetition rates up to 50 GHz are produced by the use of a solid-state saturable absorber film of polymethine dyes in a polymer matrix to mode lock a neodymium glass laser. The energy and the pulse duration of the ultrashort pulses and the stability of the train are measured. The shortest duration pulses produced are ~4 ps with no noticeable change in the optical properties of solid-state dye films after several hundred shots of operation.

Picosecond kinetic absorption and fluorescence studies of bovine rhodopsin with a fixed 11-ene.

A synthetic retinal having a fixed 11-cis geometry has been used to prepare a nonbleachable analogue of bovine rhodopsin. Marked differences in the picosecond absorption and fluorescence behavior of this analogue at room temperature, compared with that of natural rhodopsin, were observed. This not only indicates that the 11-cis to trans isomerization of the retinal moiety is the crucial primary event in the photolysis of rhodopsin, but also it establishes that this isomerization must occur on the picosecond time scale or faster.

A fast method for arranging DNA sequence fragments.

A method is described which allows efficient arrangement of DNA sequence fragments and, based on this arrangement, reconstruction of a complete DNA sequence. The concepts and algorithms used are based on the mathematical theory of graphs. The amount of human interaction required is considerably reduced compared to existing methods. An experiment with a set of 168 fragments yields a DNA sequence of about 5800 bases almost automatically.