Carlos Regalado

Biotechnological applications of peroxidases

Peroxidases are widely distributed in nature. Reduction of peroxides at the expense of electron donating substrates, make peroxidases useful in a number of biotechnological applications. Enzymes such as lignin peroxidase and manganese peroxidase, both associated with lignin degradation, may be successfully used for biopulping and biobleaching in the paper industry, and can produce oxidative breakdown of synthetic azo dyes. Oxidative polymerization of phenols and aromatic amines conducted by horseradish peroxidase (HRP) in water and water-miscible organic solvents, may lead to new types of aromatic polymers. Site directed mutagenesis of HRP has been used to improve the enantioselectivity of arylmethylsulfide oxidations. Peroxidase has a potential for soil detoxification, while HRP as well as soybean and turnip peroxidases have been applied for the bioremediation of wastewater contaminated with phenols, cresols, and chlorinated phenols. Peroxidase based biosensors have found use in analytical systems for determination of hydrogen peroxide and organic hydroperoxides, while co-immobilized with a hydrogen peroxide producing enzyme, they can be used for determination of glucose, alcohols, glutamate and choline. Peroxidase has also been used for practical analytical applications in diagnostic kits, such as quantitation of uric acid, glucose, cholesterol, lactose, and so on. Enzyme linked immunorbent assay (ELISA) tests on which peroxidase is probably the most common enzyme used for labeling an antibody, are a simple and reliable way of detecting toxins, pathogens, cancer risk in bladder and prostate, and many other analytes. Directed evolution methods, appear to be a valuable alternative to engineer new catalyst forms of plant peroxidases from different sources to overcome problems of stability and to increase thermal resistance.

Antimicrobial Edible Films and Coatings for Meat and Meat Products Preservation

Animal origin foods are widely distributed and consumed around the world due to their high nutrients availability but may also provide a suitable environment for growth of pathogenic and spoilage microorganisms. Nowadays consumers demand high quality food with an extended shelf life without chemical additives. Edible films and coatings (EFC) added with natural antimicrobials are a promising preservation technology for raw and processed meats because they provide good barrier against spoilage and pathogenic microorganisms. This review gathers updated research reported over the last ten years related to antimicrobial EFC applied to meat and meat products. In addition, the films gas barrier properties contribute to extended shelf life because physicochemical changes, such as color, texture, and moisture, may be significantly minimized. The effectiveness showed by different types of antimicrobial EFC depends on meat source, polymer used, film barrier properties, target microorganism, antimicrobial substance properties, and storage conditions. The perspective of this technology includes tailoring of coating procedures to meet industry requirements and shelf life increase of meat and meat products to ensure quality and safety without changes in sensory characteristics.

Polyethylene glycol improves phenol removal by immobilized turnip peroxidase.

Purified peroxidase from turnip (Brassica napus L. var. esculenta D.C.) was immobilized by entrapment in spheres of calcium alginate and by covalent binding to Affi-Gel 10. Both immobilized Turnip peroxidase (TP) preparations were assayed for the detoxification of a synthetic phenolic solution and a real wastewater effluent from a local paints factory. The effectiveness of phenolic compounds (PCs) removal by oxidative polymerization was evaluated using batch and recycling processes, and in the presence and in the absence of polyethylene glycol (PEG). The presence of PEG enhances the operative TP stability. In addition, reaction times were reduced from 3h to 10 min, and more effective phenol removals were achieved when PEG was added. TP was able to perform 15 reaction cycles with a real industrial effluent showing PCs removals >90% PCs during the first 10 reaction cycles. High PCs removal efficiencies (>95%) were obtained using both immobilized preparations at PCs concentrations <1.2mM. Higher PCs concentrations decreased the removal efficiency to 90% with both preparations after the first reaction cycle, probably due to substrate inhibition. On the other hand, immobilized TP showed increased thermal stability when compared with free TP. A large-scale enzymatic process for industrial effluent treatment is expected to be developed with immobilized TP that could be stable enough to make the process economically feasible.

Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme

A neutral peroxidase isozyme (TP) purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated, using chemical and enzymatic treatment. A 32% carbohydrate removal was achieved by exposing TP to a mixture of PNGase F, O-glycosidase, NANase, GALase III and HEXase I, while m-periodate treatment removed about 88% of TP carbohydrate moiety. The glycoprotein fraction of the TP contained a relatively high mannose and fucose content (37 and 31%, w/w, respectively), 16% (w/w) galactose, and 15% (w/w) GlcNAc. Thus, the carbohydrate moiety was classified as a hybrid type. Partially deglycosylated TP had reduced activity (by 50-85%), was more susceptible to proteolysis, and showed a slight decrease in thermostability compared to the native enzyme. Circular dichroism studies strongly suggested that although the carbohydrate moiety of TP did not influence the conformation of the polypeptide backbone, its presence considerably enhanced protein conformational stability toward heat. Removal of oligosaccharide chains from TP caused a decrease in K(m) and V(max) for hydrogen peroxide. Native and chemically deglycosylated TP were similarly immunodetected by rabbit polyclonal antibodies raised against TP. The results suggest that the carbohydrate moiety of TP is important for peroxidase activity and stability.

Chemical Modification of Turnip Peroxidase with Methoxypolyethylene Glycol Enhances Activity and Stability for Phenol Removal Using the Immobilized Enzyme

Peroxidase from turnip roots (TP) was isolated followed by modification with methoxypolyethylene glycol (MPEG). The catalytic activity of the modified TP (MTP) on ABTS increased 2.5 times after 80 min of reaction. MTP showed a KM similar value to that of TP, but a significantly greater kcat for ABTS oxidation, in aqueous buffer. Chemical modification produced an enhanced stability in organic solvents and increased thermal stability of about 4 times that of TP, in aqueous buffer at 70 degrees C. Circular dichroism showed that MPEG modification decreased TP alpha-helical structure from 26 to 16% and increased beta-turns from 26 to 34%, resulting in an enhanced conformational stability. The temperature at the midpoint of thermal denaturation (melting temperature) increased from 57 to 63 degrees C after modification. MTP was immobilized in alginate beads (IMTP) and tested for oxidative polymerization of concentrated phenolic synthetic solutions, achieving 17 effective contact cycles removing >65% phenols. IMTP may be useful for the development of an enzymatic process for wastewater effluent treatment.

Cloning, Heterologous Expression and Properties of a Recombinant Active Turnip Peroxidase

Turnip (Brassica napus) roots peroxidase isoforms have been used in diagnostic kits and can also efficiently polymerize phenolic compounds from wastewaters. Heterologous expression of a turnip acidic peroxidase (BnPA) was investigated to increase availability of this widely used enzyme. The mature BnPA was ligated into the pET28a(+) vector and used to transform Escherichia coli Rosetta 2. Recombinant BnPA peroxidase was overexpressed and accumulated in inclusion bodies from which it was purified to homogeneity by immobilized metal affinity chromatography under denaturing conditions. Peroxidase activity was observed after a refolding process under oxidative conditions. The yield of pure recombinant BnPA was 29 mg L(-1) of culture with a specific activity of 981 ± 20 ABTS units mg(-1) at optimal conditions (pH 6, 45 °C). Recombinant BnPA showed similar kinetic properties compared to native turnip peroxidase, and its secondary structure evaluated by circular dichroism comprised 20% α-helix, 32% β-sheet and 48% random structure. Recombinant BnPA showed high yield and good kinetic properties which are key steps for future structure-function studies and biotechnological applications.

On-site hydrolytic enzymes production from fungal co-cultivation of Bermuda grass and corn cob.

Solid state fermentation (SSF) is used to produce industrial enzymes. The objective of this study was to use a co-culture of Aspergillus niger GS1 and Trichoderma reesei, grown on a mixture of Bermuda grass and corn cob to obtain fermented forage (FF) rich in hydrolytic enzymes, as a value added ingredient for animal feed. FPase, amylase and xylanase productivities (dry matter, DM) were 8.8, 181.4, and 42.1Ug(-1)h(-1), respectively (1U=reducing sugars released min(-1)), after 12-16h of SSF with C/N=60. Cellulose, hemicellulose and lignin decreased 1.6-, 2.7- and 1.9-fold (DM), respectively. In vitro ruminal and true digestibility of DM was improved 2.4- and 1.4-fold. Ruminal digestion of FF reduced 1.32-fold the acetate:propionate ratio, which may reduce the environmental impact of ruminants feeding. On-site hydrolytic enzymes productivity using SSF without enzymes extraction could be of economic potential for digestibility improvement in animal feed.

Use of urea-polyacrylamide electrophoresis for discrimination of A1 and A2 beta casein variants in raw cow’s milk

Abstract Beta-casein (BC) in cow’s milk occurs in several genetic variants, where BC A1 (BCA1) and BC A2 (BCA2) are the most frequent. This work deals with a method based on modified polyacrylamide gel electrophoresis using urea PAGE to discriminate BCA1 and BCA2 variants from Holstein Friesian (HF) and genetically selected Jersey A2/A2 (JA2) cow’s milk. Two well defined bands were obtained from BC fraction of HF milk, while that of JA2 showed a single band. Proteins from these bands were sequenced by HPLC-quadrupole linear ion trap/mass spectrometry, resulting in BCA1 and BCA2 separation from the BC fraction of HF milk, whereas BCA2 was the only constituent of JA2 fraction. This method represents a feasible and useful tool to on site phenotyping of BC fraction of cow’s milk for pharmaceutical and food industries applications.

Characterization of Magnetic Nanoparticles Coated with Chitosan: A Potential Approach for Enzyme Immobilization

Cross-linking of magnetic nanoparticles with proteins plays a significant role in the preparation of new materials for biotechnological applications. The aim was the maximization of the magnetic mass attracted and protein loading of magnetic iron oxide nanoparticles coated with chitosan, synthesized in a single step by alkaline precipitation. Chitosan-coated magnetite particles (Fe3O4@Chitosan) were cross-linked to a xylanase and a cellulase (Fe3O4@Chitosan@Proteins), showing a 93% of the magnetic saturation of the magnetite. X-ray diffraction pattern in composites corresponds to magnetite. Thermogravimetry and differential scanning calorimetry showed that 162 mg of chitosan was coating one gram of composite and 12 mg of protein was cross-linked to each gram of magnetic support. Cross-linking between enzymes and Fe3O4@Chitosan was confirmed by infrared spectroscopy with Fourier transform, X-ray energy, and X-ray photoelectron spectroscopy dispersion analysis. From dynamic light scattering, transmission and electron microscopy the average particle size distribution was 230 nm and 430 nm for Fe3O4@Chitosan and Fe3O4@Chitosan@Proteins, showing agglomerates of individual spherical particles, with an average diameter of 8.5 nm and 10.8 nm, respectively. The preparation method plays a key role in determining the particle size and shape, size distribution, surface chemistry, and, therefore, the applications of the superparamagnetic nanoparticles.