Chitin Nanocrystal Hydrophobicity Adjustment by Fatty Acid Esterification for Improved Polylactic Acid Nanocomposites.

Bioplastics may solve environmental issues related to the current linear plastic economy, but they need improvement to be viable alternatives. To achieve this, we aimed to add chitin nanocrystals (ChNC) to polylactic acid (PLA), which is known to alter material properties while maintaining a fully bio-based character. However, ChNC are not particularly compatible with PLA, and surface modification with fatty acids was used to improve this. We used fatty acids that are different in carbon chain length (C4-C18) and degree of saturation (C18:2). We successfully used Steglich esterification and confirmed covalent attachment of fatty acids to the ChNC with FTIR and solid-state 13C NMR. The morphology of the ChNC remained intact after surface modification, as observed by TEM. ChNC modified with C4 and C8 showed higher degrees of substitution compared to fatty acids with a longer aliphatic tail, while particles modified with the longest fatty acid showed the highest hydrophobicity. The addition of ChNC to the PLA matrix resulted in brown color formation that was reduced when using modified particles, leading to higher transparency, most probably as a result of better dispersibility of modified ChNC, as observed by SEM. In general, addition of ChNC provided high UV-protection to the base polymer material, which is an additional feature that can be created through the addition of ChNC, which is not at the expense of the barrier properties, or the mechanical strength.

ß-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin.

Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable ß-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50 °C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55 °C (144 h, 75% activity), 60 °C (48 h, 85% activity; half-life 82 h), and 70 °C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)2-5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)1-3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)2. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MthNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.

Biocatalytic Route for the Synthesis of Oligoesters of Hydroxy-Fatty acids and ϵ-Caprolactone.

Developments of past years placed the bio-based polyesters as competitive substitutes for fossil-based polymers. Moreover, enzymatic polymerization using lipase catalysts has become an important green alternative to chemical polymerization for the synthesis of polyesters with biomedical applications, as several drawbacks related to the presence of traces of metal catalysts, toxicity and higher temperatures could be avoided. Copolymerization of ϵ-caprolactone (CL) with four hydroxy-fatty acids (HFA) from renewable sources, 10-hydroxystearic acid, 12-hydroxystearic acid, ricinoleic acid, and 16-hydroxyhexadecanoic acid, was carried out using commercially available immobilized lipases from Candida antarctica B, Thermomyces lanuginosus, and Pseudomonas stutzeri, as well as a native lipase. MALDI-TOF-MS and 2D-NMR analysis confirmed the formation of linear/branched and cyclic oligomers with average molecular weight around 1200 and polymerization degree up to 15. The appropriate selection of the biocatalyst and reaction temperature allowed the tailoring of the non-cyclic/cyclic copolymer ratio and increase of the total copolymer content in the reaction product above 80%. The catalytic efficiency of the best performing biocatalyst (Lipozyme TL) is evaluated during four reaction cycles, showing excellent operational stability. The thermal stability of the reaction products is assessed based on TG and DSC analysis. This new synthetic route for biobased oligomers with novel functionalities and properties could have promising biomedical applications.

Chitinase Chi1 from Myceliophthora thermophila C1, a Thermostable Enzyme for Chitin and Chitosan Depolymerization.

A thermostable Chitinase Chi1 from Myceliophthora thermophila C1 was homologously produced and characterized. Chitinase Chi1 shows high thermostability at 40 °C (>140 h 90% activity), 50 °C (>168 h 90% activity), and 55 °C (half-life 48 h). Chitinase Chi1 has broad substrate specificity and converts chitin, chitosan, modified chitosan, and chitin oligosaccharides. The activity of Chitinase Chi1 is strongly affected by the degree of deacetylation (DDA), molecular weight (Mw), and side chain modification of chitosan. Chitinase Chi1 releases mainly (GlcNAc)2 from insoluble chitin and chito-oligosaccharides with a polymerization degree (DP) ranging from 2 to 12 from chitosan, in a processive way. Chitinase Chi1 shows higher activity toward chitin oligosaccharides (GlcNAc)4-6 than toward (GlcNAc)3 and is inactive for (GlcNAc)2. During hydrolysis, oligosaccharides bind at subsites -2 to +2 in the enzyme's active site. Chitinase Chi1 can be used for chitin valorisation and for production of chitin- and chito-oligosaccharides at industrial scale.

Chitosan films and blends for packaging material.

An increased interest for hygiene in everyday life as well as in food, feed and medical issues lead to a strong interest in films and blends to prevent the growth and accumulation of harmful bacteria. A growing trend is to use synthetic and natural antimicrobial polymers, to provide non-migratory and non-depleting protection agents for application in films, coatings and packaging. In food packaging, antimicrobial effects add up to the barrier properties of the materials, to increase the shelf life and product quality. Chitosan is a natural bioactive polysaccharide with intrinsic antimicrobial activity and, due to its exceptional physicochemical properties imparted by the polysaccharide backbone, has been recognized as a natural alternative to chemically synthesized antimicrobial polymers. This, associated with the increasing preference for biofunctional materials from renewable resources, resulted in a significant interest on the potential for application of chitosan in packaging materials. In this review we describe the latest developments of chitosan films and blends as packaging material.

Biocatalytic synthesis of δ-gluconolactone and ε-caprolactone copolymers.

The biodegradability and biocompatibility properties of ε-caprolactone homopolymers place it as a valuable raw material, particularly for controlled drug delivery and tissue engineering applications. However, the usefulness of such materials is limited by their low hydrophilicity and slow biodegradation rate. In order to improve polycaprolactone properties and functionalities, copolymerization of ε-caprolactone with δ-gluconolactone was investigated. Since enzymatic reactions involving sugars are usually hindered by the low solubility of these compounds in common organic solvents, finding the best reaction medium was a major objective of this research. The optimal copolymerization conditions were set up by using different organic media (solvent and solvents mixtures), as well as solvent free systems that are able to dissolve (completely or partially) sugars, and are nontoxic for enzymes. Native and immobilized lipases by different immobilization techniques from Candida antarctica B and Thermomyces lanuginosus have been used as biocatalyst at 80°C. Although the main copolymer amount was synthesized in DMSO:t-BuOH (20:80) medium, the highest polymerization degrees, up to 16 for the copolymer product, were achieved in solventless conditions. The products, cyclic and linear polyesters, have been characterized by FT-IR and MALDI-TOF MS analysis. The reaction product analysis revealed the formation of cyclic products that could be the major impediment of further increase of the chain length.

Enzymatic synthesis of oligo- and polysaccharide fatty acid esters.

Amphiphilic oligo- and polysaccharides (e.g. polysaccharide alkyl or alkyl-aryl esters) form a new class of polymers with exceptional properties. They function as polymeric surfactants, whilst maintaining most of the properties of the starting polymeric material such as emulsifying, gelling, and film forming properties combined with partial water solubility or permeability. At present carbohydrate fatty acid esters are generally obtained by chemical methods using toxic solvents and organic and inorganic catalysts that leave residual traces in the final products. Enzymatic reactions offer an attractive alternative route for the synthesis of polysaccharide esters. In this review the state of the art of enzymatic synthesis of oligo- and polysaccharides fatty esters has been described.

Novel surface-active oligofructose fatty acid mono-esters by enzymatic esterification.

This article describes the synthesis of a series of oligofructose monoesters with fatty acids of different chain length (C8, C12, C16 and C18) to obtain food-grade surfactants with a range of amphiphilicity. Reactions were performed in a mixture of DMSO/Bu(t)OH (10/90 v/v) at 60°C and catalysed by immobilised Candida antarctica lipase B. MALDI-TOF-MS analysis showed that the crude reaction products were mixtures of unmodified oligofructose and mostly mono-esters. The conversion into mono-esters increased with the length of the fatty acid chain, reflecting the specificity of the lipase towards more lipophilic substrates. Reverse phase solid phase extraction was used to fractionate the products, which lead to sufficient purity (>93%) of the fatty acid esters for functionality testing. It was shown that derivatives of longer (C16 and C18) fatty acids were more efficient in lowering surface tension and gave a much higher dilatational modulus than derivatives of the shorter (C8 and C12) fatty acids.

Crystallization and preliminary X-ray crystallographic analysis of tyrosinase from the mushroom Agaricus bisporus.

Tyrosinase catalyzes the conversion of tyrosine to dihydroxyphenylalanine quinone, which is the main precursor for the biosynthesis of melanin. The enzyme from Agaricus bisporus, the common button mushroom, was purified and crystallized in two different space groups. Crystals belonging to space group P2(1) (unit-cell parameters a = 104.2, b = 105.0, c = 119.1 Å, ß = 110.6°, four molecules per asymmetric unit) diffracted to 3.0 Å resolution. Crystals belonging to space group P2(1)2(1)2 (unit-cell parameters a = 104.0, b = 104.5, c = 108.4 Å, two molecules per asymmetric unit) diffracted to 2.6 Å resolution. It was essential to include 5 mM HoCl(3) in all crystallization conditions in order to obtain well diffracting crystals.

Proof of principle for the synthesis of hydroxy-aryl esters of glycosidic polyols and non-reducing oligosaccharides with subsequent enzymatic coupling to a tyrosine-containing tripeptide.

To enable enzymatic coupling of saccharides to proteins, several di- and trisaccharides were hydroxy-arylated using anhydrous transesterification with methyl 3-(4-hydroxyphenyl)propionate, catalyzed by potassium carbonate. This transesterification resulted in the attachment of up to 3 hydroxy-aryl units per oligosaccharide molecule, with the monosubstituted product being by far the most abundant. The alkaline reaction conditions, however, resulted in a partial breakdown of reducing sugars. This breakdown could easily be bypassed by a preceding sugar reduction step converting them to polyols. Hydroxy-arylated products were purified by using solid phase extraction, based on the number of hydroxy-aryl moieties attached. Monohydroxy-arylated saccharose was subsequently linked to a tyrosine-containing tripeptide using horseradish peroxidase, as monitored by LC-MS(n). This proof of principle for peptide and protein glycation with a range of possible saccharides and glycosidic polyols can lead to products with unique new properties.

Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis.

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.

Highly stable foams from block oligomers synthesized by enzymatic reactions.

We have synthesized a new amphiphilic block oligomer by the enzymatic linking of a fatty acid (lauric acid) to a fructan oligomer (inulin) and tested the functionality of this carbohydrate derivative in foam stabilization. The structure of the modified oligosaccharide was found to be (Fruc)n(Glc)1CO-C11H23, which implies that on average one lauric acid molecule was linked to one inulin molecule. The new component produces foams with exceptional stability. Our results show that enzymatic acylation can produce an entirely new class of amphiphilic materials, with functionality comparable to that of synthetic block copolymers.

Improvement of lipoxygenase inhibition by octapeptides.

The beta-casein-derived octapeptide RINKKIEK is a noncompetitive inhibitor of soybean lipoxygenase (LOX). To investigate the molecular determinants for the enzyme-peptide interaction, a peptide library containing substitutional analogs of RINKKIEK was prepared by SPOT synthesis and analyzed for interaction with fluorescent-labeled LOX. The positively charged amino acid residues in RINKKIEK appear to be essential for the LOX-peptide interaction. Replacement of the negatively charged glutamic acid by any other amino acid residue improves LOX binding. For both RINKKIPK and RINKKISK this increase in LOX binding is accompanied by a threefold increase in LOX inhibition.

Novel peptides with tyrosinase inhibitory activity.

Tyrosinase inhibition by peptides may find its application in food, cosmetics or medicine. In order to identify novel tyrosinase inhibitory peptides, protein-based peptide libraries made by SPOT synthesis were used to screen for peptides that show direct interaction with tyrosinase. One of the peptide libraries studied consists of overlapping, octameric peptides derived from industrial proteins as beta-casein, alpha-lactalbumin, beta-lactoglobulin, ovalbumin, gliadin, glycinin, and beta-conglycinin. On-membrane activity staining resulted in a set of peptides that are not only able to bind to tyrosinase, but are able to inhibit tyrosinase as well. Peptides containing aspartic or glutamic acid residues usually do not bind very well to tyrosinase. Strong tyrosinase-binding peptides always contain one or more arginine residues, often in combination with phenylalanine, while lysine residues can be found equally among nonbinding peptides as well as moderate tyrosinase-binding peptides. The presence of the hydrophobic, aliphatic residues valine, alanine or leucine appears to be important for tyrosinase inhibition. Therefore, good tyrosinase inhibitory peptides preferably contain arginine and/or phenylalanine in combination with valine, alanine and/or leucine.

Horseradish peroxidase-catalyzed cross-linking of feruloylated arabinoxylans with beta-casein.

Heterologous conjugates of wheat arabinoxylan and beta-casein were prepared via enzymatic cross-linking, using sequential addition of the arabinoxylan to a mixture of beta-casein, peroxidase, and hydrogen peroxide. The maximal formation of adducts between the beta-casein and the feruloylated arabinoxylan was reached at a protein-to-arabinoxylan ratio of 10:1, in combination with a molar ratio hydrogen peroxide to substrate of 2:1 and a molar protein-to-enzyme ratio between 10(2) and 10(4). The protein-arabinoxylan adducts were separated from the arabinoxylan homopolymers by size exclusion and anion exchange chromatography. The molar ratio protein:arabinoxylan in the purified conjugates varied between 0.1 and 5.6. This is the first report on the large-scale enzymatic preparation of heterologous protein-arabinoxylan conjugates.

Horseradish peroxidase-catalyzed oligomerization of ferulic acid on a template of a tyrosine-containing tripeptide.

Ferulic acid (FA) is an abundantly present phenolic constituent of plant cell walls. Kinetically controlled incubation of FA and the tripeptide Gly-Tyr-Gly (GYG) with horseradish peroxidase and H2O2 yielded a range of new cross-linked products. Two predominant series of hetero-oligomers of FA linked by dehydrogenation to the peptidyl tyrosine were characterized by electrospray ionization (tandem) mass spectrometry. One series comprises GYG coupled with 4-7 FA moieties linked by dehydrogenation, of which one is decarboxylated. In the second series 4-9 FA moieties linked by dehydrogenation, of which two are decarboxylated, are coupled to the tripeptide. A third series comprises three hetero-oligomers in which the peptidyl tyrosine is linked to 1-3 FA moieties of which none is decarboxylated. Two mechanisms for the formation of the FA-Tyr oligomers that result from the dualistic, concentration-dependent chemistry of FA and their possible role in the regulation of plant cell wall tissue growth are presented.