Richard V. Greene

Wood Adhesive from Crosslinked Poly(Vinyl Alcohol) and Partially Gelatinized Starch: Preparation and Properties

An environmentally friendly wood adhesive was developed by crosslinking cornstarch and poly(vinyl alcohol) (PVOH) with hexamethoxy-methylmelamine (Cymel 323). Citric acid was used as a catalyst and latex (UCar 443) was added to improve moisture resistance. The adhesive was evaluated for its utility in plywood manufacture. It exhibited excellent mechanical properties comparable to many of the commercially available urea-formaldehyde plywood adhesives used for interior applications. The viscosity of the adhesive at 27 % (w/v) was 7000 mPas, allowing easy application to wood surfaces by brush. The minimum concentration of crosslinking agent needed to achieve good mechanical properties in plywood was 15 % (w/w proportion of total solids). Optimum curing temperature and curing time were 175 °C and 15 min, respectively. Addition of latex to the adhesive formulation improved both moisture resistance and physical properties of plywood test samples. Samples prepared with an optimal adhesive formulation, when completely immersed in water for 2 h or exposed at 93 % or 50 % relative humidity (RH) for 30 days, exhibited > 90 % failure in the veneer as opposed to < 10 % failure in the adhesive joints.

Substrate specificity of acetylxylan esterase from Schizophyllum commune: mode of action on acetylated carbohydrates

Substrate specificity of a purified acetylxylan esterase from Schizophyllum commune was investigated on a variety of methyl per-O-acetyl glycopyranosides, methyl di-O-acetyl-beta-D-xylopyranosides and acetylated polysaccharides. The enzyme preferentially deacetylated the 3-position of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside. Removal of the 3-acetyl group from the xylopyranoside was accompanied by a slower deacetylation at positions 2 and 4. A similarly slower, accompanying deacetylation occurred primarily at position 2 with the glucopyranoside. Such specificity corresponds well to the expected function of the esterase in acetylxylan degradation. Of the three possible diacetates of methyl beta-D-xylopyranoside, the 3,4-diacetate was found to be the most rapidly deacetylated. Unexpectedly, products of its deacetylation were a mixture of 2- and 4-monoacetate. The formation of the methyl 2-O-acetyl-beta-D-xylopyranoside involved an enzyme-mediated acetyl group transfer because the rate of the enzyme-catalyzed reaction exceeded the rate of spontaneous migration of acetyl groups. This is the likely mechanism for acetyl removal from position 2 in the native substrate. The enzyme exhibited the highest regioselectivity with methyl 2,3,4,6-tetra-O-acetyl-beta-D-mannopyranoside. An 80% conversion of this substrate to methyl 4,6-di-O-acetyl-beta-D-mannopyranoside, a new mannose derivative, was achieved. In contrast to the majority of lipases and esterases exploited for regioselective deacetylation, the S. commune acetylxylan esterase did not attack the C-6 acetyl linkages in methyl hexopyranosides when other acetyl groups were available.

Biodegradation of starch-poly(β-hydroxybutyrate-co-valerate) composites in municipal activated sludge

Injection-molded composites were prepared by blending PHBV5 with native cornstarch (30% and 50%) and with cornstarch precoated with PEO as a binding agent. These composites were evaluated for their biodegradability in municipal activated sludge by measuring changes in their physical and chemical properties over a period of 35 days. All composites lost weight, ranging from 45 to 78% within 35 days. Interestingly, the extent and rate of weight loss were quite similar in PHBV composites with no starch, with 30% starch, and with 50% starch. Weight loss was slowest in PHBV blends prepared with PEO-coated starch. For all samples, the weight loss was accompanied by a rapid deterioration in tensile strength and percentage elongation. The deterioration of these mechanical properties exhibited a relative rate of PHBV>starch-PHBV>PEO-coated starch-PHBV. Changes in starch/PHBV composition after biodegradation were quantified by FTIR spectroscopy. Increasing the starch content resulted in more extensive starch degradation, while the PHBV content in the blends became less susceptible to hydrolytic enzymes.

Biodegradation of Injection Molded Starch-Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) Blends in a Natural Compost Environment

Injection molded specimens were prepared by blending poly (hydroxybutyrate-co-valerate) (PHBV) with cornstarch. Blended formulations incorporated 30% or 50% starch in the presence or absence of poly-(ethylene oxide) (PEO), which enhances the adherence of starch granules to PHBV. These formulations were evaluated for their biodegradability in natural compost by measuring changes in physical and chemical properties over a period of 125 days. The degradation of plastic material, as evidenced by weight loss and deterioration in tensile properties, correlated with the amount of starch present in the blends (neat PHBV < 30% starch < 50% starch). Incorporation of PEO into starch-PHBV blends had little or no effect on the rate of weight loss. Starch in blends degraded faster than PHBV and it accelerated PHBV degradation. Also, PHBV did not retard starch degradation. After 125 days of exposure to compost, neat PHBV lost 7% of its weight (0.056% weight loss/day), while the PHBV component of a 50% starch blend lost 41% of its weight (0.328% weight loss/day). PHB and PHV moieties within the copolymer degraded at similar rates, regardless of the presence of starch, as determined by 1H-NMR spectroscopy. GPC analyses revealed that, while the number average molecular weight (Mn) of PHBV in all exposed samples decreased, there was no significant difference in this decrease between neat PHBV as opposed to PHBV blended with starch. SEM showed homogeneously distributed starch granules embedded in a PHBV matrix, typical of a filler material. Starch granules were rapidly depleted during exposure to compost, increasing the surface area of the PHBV matrix.

Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4‐tri‐O‐acetyl‐β‐d‐xylopyranoside and methyl 2,3,4,6‐tetra‐O‐acetyl‐β‐d‐glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4‐tri‐O‐acetyl‐β‐d‐xylopyranoside and methyl 2,3‐di‐O‐acetyl‐β‐d‐xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl β‐d‐xylo‐ and ‐glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate‐specific AcXE.

Detection of ethanol in a two-component glucose/ethanol mixture using a nonselective microbial sensor and a glucose enzyme electrode.

Chemometric theory was applied to a microbial sensor for determinations of ethanol in the presence of glucose. Microbial sensors, consisting of Gluconobacter oxydans cells immobilized on Clark-type amperometric oxygen electrodes, exhibited good sensitivity but low selectivity toward ethanol and glucose. An Eksan-G commercial glucose analyzer was used as a second sensor for multivariate calibration and analyses. Microbial sensors exhibited nearly complete additivity for total glucose plus ethanol concentrations from 0.0 to 0.6 mM. Within this linear range, chemometric analyses provided estimates of ethanol concentration with measurement errors of less than 8%. Multivariate calibration thus is a promising approach to enhance the usefulness of microbial sensors.

Evaluation of a Gluconobacter oxydans whole cell biosensor for amperometric detection of xylose

Abstract Whole cells of Gluconobacter oxydans were employed in a microbial sensor for xylose determinations using Clark-type electrodes. Bacterial cells were immobilized on chromatographic paper by simple physical adsorption and attached to the surface of the electrodes. The lower limit of xylose detection was approximately 0·5 mM and measurements were useful up to at least 20 mM xylose. Physiological buffers showed little effect on biosensor function. Responses were highly reproducible, showing a standard deviation of 6·7% over 10 consecutive measurements. Whole cell biosensors were relatively stable, retaining 60% of initial activity after 35 days of dry storage at 4°C. Xylose detection was not significantly affected by the presence of xylitol, suggesting that biosensors will be useful in monitoring conversions of these compounds. However, glucose or ethanol elicited a 10-fold higher response than xylose at equal concentrations (1 mM). Such interfering materials will need to be controlled or concurrently monitored in specific sensor applications.

FET-microbial sensor for xylose detection based on Gluconobacter oxydans cells

A potentiometric biosensor for xylose was devised utilizing Gluconobacter oxydans whole cells. Immobilization methods based on physical adsorption were used for G. oxydans cells and extracellular pH changes resulting from xylose dehydrogenation were monitored by a field effect transistor (FET). The G. oxydans, FET-based sensor detected xylose at a lower limit of 0.5 mM. From 5.0 to 30 mM xylose, the response of the sensor was linear. Expectedly, output signals were significantly suppressed by buffer (Tris-HCl). Responses were essentially stable for at least four weeks of storage and showed only a slight loss of initial xylose sensitivity. Xylitol exerted an insignificant influence on the sensors response to xylose. However, the response to glucose was 5 times higher in relation to that of xylose at the same concentration (1 mM). For xylose determinations in the presence of glucose, a two-step assay is discussed.

Isolation and characterization of an alkaline protease from the marine shipworm bacterium

Bacterial isolates from the gland of Deshayes of the marine shipworm (Psiloteredo healdi) produced extracellular protease activity when cultured with 1% cellulose. A protease with a relative molecular mass of 36,000 daltons as determined by SDS-PAGE and a pI of 8.6 was isolated from the medium and purified to electrophoretic homogeneity. No carbohydrate appeared to be associated with the protein. The enzyme was activated and stabilized by relatively high salt concentrations (>0.2M). Below 0.1M salt, significant protein aggregation occurred, as well as autohydrolysis of the protease, both of which resulted in the loss of activity. The specific activity of the enzyme was 65,840 proteolytic units/mg with azocasein substrate of optimal temperature (42°C), pH (9.0), and salt concentration (0.20M NaCl). The activity was stable up to 40°C, from pH 3.0 to pH 11.9, and from 0.1M to 3.5M NaCl. These stabilities, as well as the proteases stability in the presence of chelators, oxidizing agents, and heavy metals, suggest the enzyme has potential for use in relatively low temperature (40°C) industrial applications.

Action of acetylxylan esterase from Trichoderma reesei on acetylated methyl glycosides

Substrate specificity of purified acetylxylan esterase (AcXE) from Trichoderma reesei was investigated on partially and fully acetylated methyl glycopyranosides. Methyl 2,3,4‐tri‐O‐acetyl‐β‐d‐xylopyranoside was deacetylated at positions 2 and 3, yielding methyl 4‐O‐acetyl‐β‐d‐xylopyranoside in almost 90% yield. Methyl 2,3‐di‐O‐acetyl β‐d‐xylopyranoside was deacetylated at a rate similar to the fully acetylated derivative. The other two diacetates (2,4‐ and 3,4‐), which have a free hydroxyl group at either position 3 or 2, were deacetylated one order of magnitude more rapidly. Thus the second acetyl group is rapidly released from position 3 or 2 after the first acetyl group is removed from position 2 or 3. The results strongly imply that in degradation of partially acetylated β‐1,4‐linked xylans, the enzyme deacetylates monoacetylated xylopyranosyl residues more readily than di‐O‐acetylated residues. The T. reesei AcXE attacked acetylated methyl β‐d‐glucopyranosides and β‐d‐mannopyranosides in a manner similar to the xylopyranosides.