Heterogeneous components of chitosans.

The main objectives of the research were to compare the components of partially N-deacetylated chitins prepared identically from native chitin and a chitin regenerated from a heavily deacetylated chitosan. Additionally, to determine if any of the water-soluble components would serve as substrates in a study of a Chitinase isolated from soy bean hull. The brief heating of suspended chitins in 20% (w/w) NaOH resulted in similar degrees of N-deacetylation, the native chitin giving DAc 0.84 and the regenerated chitin DAc 0.79-0.72, with DAc indicating the proportion of glucosamine residues that are acetylated. Evidence for the nature of the hydrolysis of acetamido groups was provided by analyses of the water-soluble and -insoluble Smith degradation products. The water-soluble fraction derived from the native chitin comprised very small amounts of erythrityl N-acetyl glucosaminoside (GlcNAc1E), erythrityl N,N'-diacetyl chitobioside (GlcNAc2E), and erythrityl N,N',N''-triacetyl chitotrioside (GlcNAc3E), each identified by MALDI-TOF mass spectrometry of the butanoyl derivative. The water-insoluble products, as analyzed by light scattering detection method of their butanoyl esters and corrected for their composition, had a molecular weight (Mw) of 25 kDa, corresponding to about 120 N-acetyl glucosaminyl repeating residues (DPw), contrasting to that of 140 kDa with DPw of 680 for the parent chitin. Much of the decrease in the molecular weight of the polymer occurs by the loss of sugar residues by alkaline peeling at reducing terminals. For the regenerated chitin (DAc 1.0), prepared by N-reacetylation of a commercial chitosan (DAc 0.15), the resulting Smith products comprised erythritol and a series of N-acetyl glucosaminyl erythritol homologues of up to at least 39 N-acetyl glucosaminyl repeating residues, reflecting greater heterogeneity in the hydrolysis of acetamido groups along the polymer chain than what was seen for the native chitin. Of the water-soluble Smith products, GlcNAc5-7E were good substrates for chitinase isolated from soybean hull.

Preparation and physical properties of chitin fatty acids esters.

Trifluoroacetic anhydride is an effective promoter for the preparation of chitin single- and mixed-acid esters. Complete dissolution is achieved within 30 min when powdered chitin is heated at 70 degrees C in a mixed solution of carboxylic acid(s) and trifluoroacetic anhydride. Chitin esters prepared are chitin acetate, chitin butyrate, chitin hexanoate and chitin octanoate, chitin co-acetate/butyrate, chitin co-acetate/hexanoate, chitin co-acetate/octanoate, chitin co-acetate/palmitate, each from a solution of the respective reactants. The products have degrees of O-acyl substitution in a range of DS 1-2 depending on the nature of acyl group, as analyzed by gas-liquid and high-pressure liquid chromatography. Acetic acid as a mutual component for the mixed-acid esters increases the total degree of substitution, and the acetyl substitution is close to the relative distribution in the reaction mixture for chitin co-acetate/butyrate. It is favored over hexanoate, octanoate, and palmitate. The parent molecules, as calculated by the composition of the chitin esters and their molecular weights by light-scattering spectroscopy, are 30 kDa for the smallest and 150-151 kDa for the largest. Films of these chitin derivatives when cast from solution are strong and flexible with limited extensibility. By dynamic mechanical analysis of the ester film, it was found that both the glass transition temperature (T(g)) and the tensile modulus (E' at 25 degrees C) are highest for chitin acetate (218 degrees C and 5.8 GPa), and lowest for chitin octanoate (182 degrees C and 1.5 GPa). For the other esters, these values lie between the above-cited values, where the T(g) and the E' decrease with an increase in the chain length of the acyl constituent.

Alkaline degradation of invert sugar from molasses.

Sugar beet and sugar cane molasses have been shown to be suitable starting materials for producing de-icer preparations. The sucrose in the molasses is hydrolyzed to glucose and fructose by invertase. The reducing sugars are then degraded by NaOH, the alkali being neutralized by the sugar acids produced, resulting in an increase of the ionic strength and consequently depression of the freezing point of the resulting solution. For the preparation of de-icers, the desired freezing point depression to a temperature of less than about -20 degrees C can be achieved by adjusting the amount and concentration of the alkali metal hydroxide used. The resulting products are biodegradable and eliminate the corrosive effects associated with the use of conventional chloride salts. Degradation of invert sugar by NaOH has been achieved without an external heat source. The reaction products showed the same freezing point depression as seen in the degradation products from pure glucose.

Asymmetric glycosylation of soybean seed coat peroxidase.

Reanalysis of the tryptic digests of soybean seed coat peroxidase (SBP) and its carboxyamidated peptide derivatives in the light of more complete sequence data has thrown light on the diglycosylated tryptic peptides, TP13 (Leu[183-205]Arg) and TP15 (Cys[208-231]Arg). Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses indicate that although all potential sites carry some glycan substituents, not all sites are fully occupied. Tryptic glycopeptide TP13, carrying two N-glycosylation consensus sequons (Asn185 and Asn197), occurs mainly (85-90%) as the diglycosylated species, the remainder (10-15%) being monoglycosylated. In contrast, tryptic peptide TP15, also with two N-glycosylation sites (Asn211 and Asn216), is primarily monoglycosylated (approximately 90%), with the remainder (10%) being diglycosylated. No non-glycosylated TP13 or TP15 was observed. Some artifacts are noted in the reactions of N-terminal cysteine residues and aspartate/asparagines residues in glycopeptide TP15. Mapping the glycans onto the crystal structure of SBP shows that these are asymmetrically distributed on the molecule, occurring primarily on the substrate-channel face of the enzyme. In contrast, the glycans of HRP, isozyme c, are more uniformly distributed over the enzyme surface.

Oxidation of lactose with bromine.

Oxidation of lactose by bromine in an aqueous buffered solution was conducted as a model experiment to examine the glycosidic linkage cleavage occurring during the oxidation of oligosaccharides and polysaccharides. The resulting oxidation products, after reduction with sodium borodeuteride, were characterized by GLC-MS analyses of the per-O-methyl or per-O-Me3Si derivatives. Most of the products were carboxylic acids, of which lactobionic acid was major. Minor products, identified after partial fractionation on a BioGel P-2 column, comprised oxalic acid; glyceric acid; threonic and erythronic acids; tartaric acid; lyxonic, arabinonic, and xylonic acids; galactonic and gluconic acids; galactosylerythronic acid; galactosylarabinonic acid; galactosylarabinaric acid; galacturonosylarabinonic acid; and galactosylglucaric acid. No keto acids were identified. Galactose was detected as 1-deuteriogalactitol, the presence of which, together with the C6 aldonic acids, supported a galactosidic bond cleavage. Galactosylarabinonic acid was the major constituent (7.5%) among minors, and others constituted 0.2-3.7% of the principal lactobionic acid. These products together comprised 29% of the lactobionic acid, more than half (17%) of which were accounted for by the galactosidic linkage cleavage, supporting the significant decrease in molecular weight seen earlier in the bromine-oxidized polysaccharides by glycosidic cleavage.

Structure and hydrodynamic properties of the extracellular polysaccharide from a mutant strain (RA3W) of Erwinia chrysanthemi RA3.

The structure of the extracellular polysaccharide (EPS) produced by Erwinia chrysanthemi strain RA3W, a mutant strain of E. chrysanthemi RA3, has been determined using low pressure size-exclusion and anion-exchange chromatographies, high pH anion-exchange chromatography, glycosyl linkage analysis, and 1D 1H NMR spectroscopy. The polysaccharide is structurally similar, if not identical, to the family of EPS produced by such as E. chrysanthemi strains Ech9, Ech9Sm6, and SR260. The molecular weight of EPS RA3W by ultracentrifugation (sedimentation equilibrium) and light scattering is compared with those of other E. chrysanthami EPSs, as are the viscometric properties.

Development of biobased products.

Research conducted over the past seven years by the biotechnology byproducts consortium (BBC) addresses its mission to investigate the opportunities to add value to agricultural products, byproducts and coproducts and to manage the wastewater arising from agribusinesses in an environmentally favorable way. Since a wide variety of research approaches have been taken, the results are collected in five topic groups: (1) bioremediation that includes anaerobic fermentations of wastes to produce methane and hydrogen, the genetics of methanogenesis and in situ remediation of contaminated aquifer systems, landfill leachates and industrial effluents; (2) land application of fermentation byproducts and their use in animal feeds; (3) biocatalytic studies of transformations of components of corn and soybean oils, peroxidases present in plant products, such as soybean hulls; (4) biochemical reactions for the production of de-icers from industrial water streams, biodiesel production from fats and greases, biodegradable plastics from polymerizable sugar derivatives, single cell foods derived from fungal growth on waste streams, and bacterial polysaccharides from Erwinia species; (5) separation and recovery of components by membrane technologies.

Hydrodynamic properties of oxidized extracellular polysaccharides from Erwinia chrysanthemi spp.

The molecular weights of the native polysaccharides of Erwinia chrysanthemi strains range from 1.8 to 7.1 x 10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions. The effect on the hydrodynamic properties of the polysaccharides by adding carboxyl groups to increase the charge density is studied, with particular reference to their molecular weight (MW), viscosity and conformation. In general, it is found that periodate oxidation of the extracellular polysaccharides of E. chrysanthemi strains, Ech9Sm6 and Ech6S+, introduces little change in the hydrodynamic properties of the resulting polyaldehydes. However, bromine oxidation at neutral pH of the polyaldehydes results in polycarboxylate biopolymers that show significant reduction in MW and viscosity, but they are still characteristic polyanions.

De-icers derived from corn steep water.

Corn steep water (CSW) and other byproducts derived from fermentations and sugar productions are presently forming the base of compositions for de-icing and anti-icing materials. Since the de-icing and anti-icing values are in part a colligative property, increase in the molar concentration of ionic species has been frequently necessary to decrease further the freezing point of this byproducts stream. In the present study this has been achieved by the generation of biodegradable organic acid salts in situ, without the use of chloride or other inorganic salts, by the alkaline degradation of reducing sugars added to corn steep water, which alone is not an efficient de-icer. Reducing sugars, such as glucose, react with alkali metal hydroxides to produce principally hydroxy carboxylic acids that react with the alkali metal hydroxide to form a mixture of organic acid salts. The ionic strength of the resulting solution is increased since each sugar molecule produces nearly two acid molecules upon degradation. The ionic strength necessary to achieve the desired freezing point depression is determined by the amount and concentration of the alkali metal hydroxide used, with the necessary counter anions being derived from the degradation of the reducing sugar. The amount of the sugar used is that required to result in a near to neutral final solution. The well-known anti-corrosive property of CSW is used in the de-icer preparations, either by conducting the alkaline degradation of the sugar in this medium, or by using water for the degradation of the sugar followed by dilution of the resulting solution with CSW to adjust the viscosity of the final solution to meet the requirements for spraying. The monovalent metal hydroxides are more efficient in producing de-icer solutions than the divalent metal hydroxides.

Purification and characterization of a peroxidase from corn steep water.

Three cationic peroxidases have been detected in early, middle, and late corn steep water, with pI values of approximately 8.9, approximately 9.5, and >10.0. The major cationic corn steep water peroxidase (CSWP), with a pI >10, was purified 36400-fold with a 12% recovery from late steep water by a combination of acetone and ammonium sulfate precipitation and sequential chromatography on CM-cellulose, phenyl-Sepharose, and Sephadex G-75. The UV-vis spectrum of purified CSWP is typical of other plant class III peroxidases. The RZ (A(403)/A(280)) of CSWP was between 2.6 and 2.9. It is not glycosylated and exhibited an M(r) of 30662 +/- 7 by MALDI-TOF MS. The pH optimum of CSWP depends on the substrate, and it is active on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), guaiacol, ferulic acid, o-dianisidine, o-phenylenediamine, and pyrogallol but is not active on either syringaldazine or ascorbate. At 75 degrees C and pH 4.5, the enzyme has half-lives of 22.7 min (0 mM Ca(2+)) and 248 min (1 mM Ca(2+)). The enzyme is stable at room temperature (22-25 degrees C), losing <3% of the activity at pH 4.5 and <10% at pH 6.2 over 400 h in the presence of 1 mM Ca(2+).

Extracellular polysaccharides of Erwinia futululu, a bacterium associated with a fungal canker disease of Eucalyptus spp.

Extracellular polysaccharides (EPSs) produced by an Erwinia spp. associated with a fungal canker disease of Eucalyptus were fractionated into two polysaccharides, one that was identified with that produced by Erwinia stewartii. The other has a similar structure, but with one terminal Glc residue replaced by pyruvic acid to give 4,6-O-[(R)-1-carboxyethylidene)-Galp. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, periodate oxidation, low-pressure gel filtration and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharides, identified as EPS Futululu FF-1 and FF-2, have the following structures:The molecular weights of the polysaccharides range from 1.3-2.1x10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.

Extracellular polysaccharides of a bacterium associated with a fungal canker disease of Eucalyptus sp.

Extracellular polysaccharides (EPSs) produced by an Erwinia sp associated with a fungal canker disease of Eucalyptus were fractionated into one polysaccharide that was identified with that produced by Erwinia chrysanthemi strains SR260, Ech1, and Ech9, and the other distinctively different from any other EPS produced by E. chrysanthemi strains so far studied. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, low pressure gel-filtration, and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharide, identified as EPS Teranera, has the following structure: [structure: see text] The molecular weights of the polysaccharides range from 3.2-6.2 x 10(5) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.