Production of specific copolymers of polyhydroxyalkanoates from industrial waste

Polyhydroxyalkanoates, biodegradable plastics with the desired physical and chemical properties of conventional synthetic plastics, are extensively investigated. In this study, specific bacterial strains produced specific copolymers from food waste. Copolymers of HB and HV (poly[3-hydroxybutyrate-co-3-hydroxyvalerate]) were obtained using various ratios of butyric acid (C₄) and valeric acid (C₅) as carbon sources. The C₄ to C₅ ratio affected the melting points of the copolymers. Melting and glass transition temperatures and many other thermal properties are important parameters relative to in-service polymer applications. Higher ratios of butyrate to valerate gave higher melting points. When a mixed culture of activated sludge was employed to produce copolymers using food wastes as nutrients, the obtained copolymers showed various monomer compositions. Copolymers with a higher portion of HV were obtained using soy waste; copolymers with less HV were obtained using malt wastes. Pure strains, (i.e., Alcaligenes latus DSM 1122, and DSM 1124, Staphylococcus spp., Klebsiella spp.) produced specific copolymers from food waste. Only Klebsiella spp. produced different copolymers; the ratios of HB:HV were 93:7 and 79:21 from malt waste and soy waste, respectively. The other strains produced polymers of 100% HB. Selecting industrial food wastes as carbon sources can further reduce the cost of producing copolymers. Open Laboratory of Chirotechnology of the Institute of Molecular Technology for Drug Discovery and Synthesis The University Grants Committee Area of Excellence Scheme, Hong Kong     

Effect of structural and physico-chemical features of cellulosic substrates on the efficiency of enzymatic hydrolysis

Effects of major physicochemical and structural parameters of cellulose on the rate and degree of its enzymatic hydrolysis were tested with cellulosic materials from various sources. Some different pretreatments were: mechanical (milling), physical (X-ray irradiation), and chemical (cadoxen, H₃PO₄, H₂SO₄, NaOH, Fe²+/H₂O₂). The average size of cellulose particles and its degree of polymerization had little effect on the efficiency of enzymatic hydrolysis. For samples of pure cellulose (cotton linter, microcrystalline cellulose, α-cellulose), increase in the specific surface area accessible to protein molecules and decrease in the crystallinity index accelerated the enzymatic hydrolysis (the correlation coefficients were 0.89 and 0.92, respectively). In the case of lignocellulose (bagasse), a quantitative linear relationship only between specific surface area and reactivity was observed.     

Reactivity of Chiral Sesquiterpene Synthons Obtained by the Degradation of Maslinic Acid from Olive‐Pressing Residues

Maslinic acid, a naturally occurring compound isolated from the solid wastes of olive‐oil pressing, was fragmented through the C‐ring via oxidative procedures to obtain two structural fragments. The chemical behaviors of cis‐decalin, from the D and E rings, and of trans‐decalin fragments, from the A and B rings, were investigated in depth using several chemical and enzymatic reactions. These decalin chiral synthons are interesting intermediates to semisynthesize phenanthrene‐ and drimane‐type compounds and natural tricyclic triterpenes.     

Variations in Physicochemical Properties and Bioconversion Efficiency of <Emphasis Type="Italic">Ulva lactuca</Emphasis> Polysaccharides After Different Biomass Pretreatment Techniques

Green macroalgae are an abundant and undervalued biomass with a specific cell wall structure. In this context, different pretreatments, namely ethanol organosolv (Org), alkaline, liquid hot water (LHW), and ionic liquid (IL) pretreatments, were applied to the green macroalgae Ulva lactuca biomass and then evaluated. Their effects on chemical composition, biomass crystallinity, enzymatic digestibility, and theoretical ethanol potential were studied. The chemical composition analysis showed that the Org and LHW pretreatments allowed the highest glucan recovery (80.8 ± 3.6 and 62.9 ± 4.4 g/100 g DM, respectively) with ulvan (80.0 and 99.1%) and hemicellulose (55.0 and 42.3%) removal. These findings were in agreement with both thermogravimetric analysis and scanning electron microscopy results that confirm significant structural changes of the pretreated biomasses. It was found that the employed pretreatments did not significantly affect the cellulose crystallinity; however, they both increased the whole crystallinity and the enzymatic digestibility. This later reached 97.5% in the case of LHW pretreatment. Our results showed high efficiency saccharification of Ulva lactuca biomass that will constitute the key step of the implementation of a biorefinery process.     

Laccase from Trametes versicolor

The enzyme laccase was produced by the white-rot fungus Trametes versicolor in repeated batches cultures with immobilized mycelium. Two different culture conditions were used. Enzymes produced were evaluated regarding their stability a thigh temperatures (55°C and 65°C) and at alkaline conditions (pH 7.0 and pH 8.0) having in view the application of these enzymes in biobleaching of hardwood Kraft pulp. Biobleaching experiments were divided in two parts, enzymatic prebleaching followed by chemical bleaching. In the enzymatic prebleaching the enzyme laccase was used at two conditions of pH and temperature, whereas the reaction time was fixed at 1h in all pretreatments. In the chemical bleaching the DEDED and DEpDED sequences were used. The enzyme action was evaluated by Kappa number, viscosity, and brightness at the end of bleaching sequences. There were obtained values of Kappa numbers lower than control assays, viscosities compatible with industrial pulps, and brightness higher than controls, when pulps were pretreated for 1 h with laccase at pH 8.0 and 55°C.     

Comparison of changes in cellulose ultrastructure during different pretreatments of poplar

One commonly cited factor that contributes to the recalcitrance of biomass is cellulose crystallinity. The present study aims to establish the effect of several pretreatment technologies on cellulose crystallinity, crystalline allomorph distribution, and cellulose ultrastructure. The observed changes in the cellulose ultrastructure of poplar were also related to changes in enzymatic hydrolysis, a measure of biomass recalcitrance. Hot-water, organo-solv, lime, lime-oxidant, dilute acid, and dilute acid-oxidant pretreatments were compared in terms of changes in enzymatic sugar release and then changes in cellulose ultrastructure measured by ¹³C cross polarization magic angle spinning nuclear magnetic resonance and wide-angle X-ray diffraction. Pretreatment severity and relative chemical depolymerization/degradation were assessed through compositional analysis and high-performance anion-exchange chromatography with pulsed amperometric detection. Results showed minimal cellulose ultrastructural changes occurred due to lime and lime-oxidant pretreatments, which at short residence time displayed relatively high enzymatic glucose yield. Hot water pretreatment moderately changed cellulose crystallinity and crystalline allomorph distribution, yet produced the lowest enzymatic glucose yield. Dilute acid and dilute acid-oxidant pretreatments resulted in the largest increase in cellulose crystallinity, para-crystalline, and cellulose-I_β allomorph content as well as the largest increase in cellulose microfibril or crystallite size. Perhaps related, compositional analysis and Klason lignin contents for samples that underwent dilute acid and dilute acid-oxidant pretreatments indicated the most significant polysaccharide depolymerization/degradation also ensued. Organo-solv pretreatment generated the highest glucose yield, which was accompanied by the most significant increase in cellulose microfibril or crystallite size and decrease in relatively lignin contents. Hot-water, dilute acid, dilute acid-oxidant, and organo-solv pretreatments all showed evidence of cellulose microfibril coalescence.     

Comparison of yellow poplar pretreatment between NREL digester and sunds hydrolyzer

Single-stage cocurrent dilute acid pretreatments were carried out on yellow poplar (Liriodendron tulipifera) sawdust using an as-installed and short residence time modified pilot-scale Sunds hydrolyzer and a 4-L bench-scale NREL digester (steam explosion reactor). Pretreatment conditions for the Sunds hydrolyzer, installed in the NREL process development unit (PDU), which operates at 1 t/d (bone-dry t) feed rate, spanned the temperature range of 160 – 210°C, 0.1 – 1.0% (w/w) sulfuric acid, and 4-10-min residence times. The batch pretreatments of yellow poplar sawdust in the bench-scale digester were carried out at 210 and 230°C, 0.26% (w/w) sulfuric acid, and 1-, 3-, and 4-min residence times. The dilute acid prehydrolysis solubilized more than 90% of the hemicellulose, and increased the enzymatic digestibility of the cellulose that remained in the solids. Compositional analysis of the pretreated solids and liquors and mass balance data show that the two pretreatment devices had similar pretreatment performance.     

The Effect of Varying Organosolv Pretreatment Chemicals on the Physicochemical Properties and Cellulolytic Hydrolysis of Mountain Pine Beetle-Killed Lodgepole Pine

Mountain pine beetle-killed lodgepole pine (Pinus contorta) chips were pretreated using the organosolv process, and their ease of subsequent enzymatic hydrolysis was assessed. The effect of varying pretreatment chemicals and solvents on the substrate’s physicochemical characteristics was also investigated. The chemicals employed were MgCl₂, H₂SO₄, SO₂, and NaOH, and the solvents were ethanol and butanol. It was apparent that the different pretreatments resulted in variations in both the chemical composition of the solid and liquid fractions as well in the extent of cellulolytic hydrolysis (ranging from 21% to 82% hydrolysis after 12 h). Pretreatment under acidic conditions resulted in substrates that were readily hydrolyzed despite the apparent contradiction that pretreatment under alkaline conditions resulted in increased delignification (approximately 7% and 10% residual lignin for alkaline conditions versus 17% to 19% for acidic conditions). Acidic pretreatments also resulted in lower cellulose degree of polymerization, shorter fiber lengths, and increased substrate porosity. The substrates generated when butanol/water mixtures were used as the pretreatment solvent were also hydrolyzed more readily than those generated with ethanol/water. This was likely due to the limited miscibility of the solvents resulting in an increased concentration of pretreatment chemicals in the aqueous layer and thus a higher pretreatment severity.     

Organic Solvent‐Free Enzymatic Transformation of Poly(ϵ‐caprolactone) into Repolymerizable Oligomers in Supercritical Carbon Dioxide

The enzymatic transformation of poly(ϵ‐caprolactone) (PCL) into repolymerizable oligomers in supercritical carbon dioxide (scCO₂) using an enzyme was carried out in order to establish a sustainable chemical recycling system for PCL, which is a typical biodegradable synthetic plastic. The enzymatic conversion of PCL beads having an M̄_n of 110 000 using Candida antarctica lipase (lipase CA) in scCO₂ containing small amounts of water quantitatively afforded CL oligomers at 40°C. The CL oligomers were readily repolymerized using the same enzyme to produce high‐molecular weight PCL.     

Furostane-Type Steroidal Saponin from <Emphasis Type="Italic">Digitalis ciliata</Emphasis>

A new furostane-type steroidal glycoside and derivative of tigogenin (1) was isolated from aqueous wastes from production of the cardiac drug acetyldigitoxin from leaves of Digitalis ciliata Trautv. (Scrophulariaceae) and characterized. The structure of the glycoside was established using physical constants, chemical transformations, and spectral data as 3-O-β-D-glucopyranosyl-(1→3) [β-D-fucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranosyl-(25R)-5α-furostan-3β,22α,26-triol-26-O-β-D-glucopyranoside.     

Steroidal glycosides from Digitalis ciliata leaves

Two new steroidal glycosides of the spirostane and furostane classes, derivatives of gitogenin, were found in wastes from producing acetyldigitoxin preparation from Digitalis ciliata Trautv. (Scrophulariaceae). The structures of the glucosides were established using physical constants, chemical transformations, and IR, mass, and NMR spectra. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 452–455, September–October, 2006.     

An Oxidant- and Solvent-Stable Protease Produced by <Emphasis Type="Italic">Bacillus cereus</Emphasis> SV1: Application in the Deproteinization of Shrimp Wastes and as a Laundry Detergent Additive

The current increase in amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, an extracellular organic solvent- and oxidant-stable metalloprotease was produced by Bacillus cereus SV1. Maximum protease activity (5,900 U/mL) was obtained when the strain was grown in medium containing 40 g/L shrimp wastes powder as a sole carbon source. The optimum pH, optimum temperature, pH stability, and thermal stability of the crude enzyme preparation were pH 8.0, 60 °C, pH 6–9.5, and <55 °C, respectively. The crude protease was extremely stable toward several organic solvents. No loss of activity was observed even after 60 days of incubation at 30 °C in the presence of 50% (v/v) dimethyl sulfoxide and ethyl ether; the enzyme retained more than 70% of its original activity in the presence of ethanol and N,N-dimethylformamide. The protease showed high stability toward anionic (SDS) and non-ionic (Tween 80, Tween 20, and Triton X-100) surfactants. Interestingly, the activity of the enzyme was significantly enhanced by oxidizing agents. In addition, the enzyme showed excellent compatibility with some commercial liquid detergents. The protease of B. cereus SV1, produced under the optimal culture conditions, was tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio E/S of 20 was about 88%. The novelties of the SV1 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition, the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.     

Synthesis and CD Spectra of Fluoro‐ and Hydroxy‐Substituted β‐Peptides

β‐Amino acids 1 – 3 with OH and F substituents in the α‐position have been prepared (Scheme) from the natural (S)‐α‐amino acids alanine, valine, and leucine, and incorporated into β‐hexa‐ and β‐heptapeptides 4 – 12 . The peptide syntheses were performed according to a conventional solution strategy (Boc/Bn protection) with fragment coupling. The new β‐peptides with (series a ) and without (series b ) terminal protection were isolated in HPLC‐pure form and characterized by NMR spectroscopy and MALDI mass spectrometry. The chemical properties as well as the patterns of the CD spectra (Figs. 3–5) depend upon constitution (OH, F, F₂ substitution) and configuration (l or u) of the amino acid residues, upon the total number of OH and F substituents in the peptide chain, and upon the solvent used (H₂O, MeOH, CF₃CH₂OH, (CF₃)₂CHOH). No reliable clues regarding the structures can be obtained from these CD spectra. Only a full NMR analysis will be able to answer the questions: a) with which known secondary structures (Figs. 1 and 2) of β‐peptides are the OH and F derivatives compatible? b) Are new secondary structures enforced by the polar and/or H‐bonding backbone substituents? Furthermore, the β‐peptides described here will enable us to study changes in chemical, enzymatic, and metabolic stability, and in physiological properties caused by the heteroatoms.     

Degradation of triterpenic compounds from olive-pressing residues. Synthesis of trans-decalin type chiral synthons

Three seco-C-ring triterpenic compounds were obtained from oleanolic or maslinic acids from olive-mill solid wastes by photochemical and chemical reactions. From these oleantriene compounds, different remarkable sesquiterpene and nor-sesquiterpene fragments such as 3β-hydroxydrimenol (13) and epoxydecalone (16) were achieved through oxidative cleavages of the double bonds in the opened C-ring.     

Quantitative Relationships of Structure-Activity and Volume Fraction For Selected Nonpolar and Polar Narcotic Chemicals

Abstract

The relative toxicity of selected industrial organic chemicals was secured from the literature for the static 48-h Tetrahymena pyriformis 50% population growth impairment and the flow-through 96-h Pimephales promelas 50% mortality endpoints. Chemicals were selected to represent the nonpolar narcosis (aliphatic alcohols and aliphatic ketones) and polar narcosis (anilines and phenols) mechanisms of toxic action. molar volume (MV) and 1-octanol/water partition coefficient (log K _ow) data were generated for each chemical. High-quality, log K _ow dependent quantitative structure-activity relationships were observed for each chemical class and mechanism of action for both endpoints. The volume fraction (V _t) for each chemical in the target phase was determined from the toxicant concentration in the water (toxicity data), the MV, and the target/water partition coefficient (K _tw) with K _tw considered equal to K _ow ^(1-a). Analyses of target sites, by way of “a” revealed that “a” was constant for a mechanism of action regardless of chemical class, but distinct for a given test system. Mean V _t was constant for each mechanism of action regardless of chemical class or test system. These results suggest, at least for reversible physical mechanisms, that volume fraction analyses are significant in determining the mechanism of toxic action of a chemical.     

l-DOPA production by immobilized tyrosinase

The production of l-DOPA using l-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and l-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinyl pyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U _C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U _IMO). The optimal conditions were t=24 h, G=2% (v/v), and U _C=163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U _IMO) was 23.3 U and the rate of l-DOPA production rate was 53.97 mg/(L·h).     

Action of white-rot fungus Panus tigrinus on sugarcane bagasse

Biological pretreatments with three selected strains of Panus tigrinus were used for delignification of sugarcane bagasse. The fungi with potential for delignification were analyzed by determining the chemical composition of the decayed bagasse samples, and the selectivity in terms of weight loss of the different components was evaluated. All the strains grow abundantly on bagasse as unique carbon source. After determining the chemical composition of degraded bagasse, P. tigrinus FTPT-4745 was selected as the most efficient strain on a 6-g scale, since the carbohydrates were preserved. P. tigrinus FTPT-4741 and FTPT-4742 were the most efficient strains on a large scale (100 g).     

Chemometric Analysis with Near-Infrared Spectroscopy for Chemically Pretreated <Emphasis Type="Italic">Erianthus</Emphasis> toward Efficient Bioethanol Production

In this paper, we report the combination of a near-infrared (NIR) spectroscopic method with multivariate analysis in order to develop a calibration model of the saccharification ratio of chemically pretreated Erianthus. The regression models clearly depend on the NIR spectral regions, and the information of CH and aromatic framework vibrations contributed most effectively to the alkaline dataset. From interpretations of the regression coefficient, lignin and cellulose were negatively and positively correlated with the saccharification ratio, respectively, and this result was supported by the data from wet chemical analysis. A more complex dataset was obtained from varied chemical pretreatments; here, the saccharification ratio was either small or had no linear correlation with each structural monocomponent. These results enabled the successful construction of the PLS regression model. NIR spectroscopy can be a rapid screening method for the saccharification ratio, and furthermore, can provide information of the key factors influencing the realization of more efficient enzymatic accessibility.     

Chemo‐Enzymatic Synthesis and Determination of the Absolute Configuration of Both Enantiomers of Methyl trans‐5‐Oxo‐2‐pentylpyrrolidine‐3‐carboxylate Precursors of the Aza Analogues of (+)‐ and (−)‐Methylenolactocin

Enantiomerically pure methyl esters of (+)‐(2R,3S)‐ and (−)‐(2S,3R)‐5‐oxo‐2‐pentylpyrrolidine‐3‐carboxylic acid with 99% and 98% ee were obtained by enzymatic resolution of the corresponding racemic mixture using α‐chymotrypsin and pig‐liver acetone powder, respectively. Their absolute configurations were established by chemical methods, i.e., conversion of the trans‐γ‐lactam moiety to the corresponding γ‐lactone of known configuration. The favorable interactions between the trans‐γ‐lactam and α‐chymotrypsin were rationalized by molecular‐mechanics calculations, which suggest a different situation for the cis‐diastereoisomer.     

Thermal properties of nylons containing carbonyl groups

Nylons containing carbonyl groups in backbone chains were prepared and their response to heat was studied. The carbonyl groups were introduced using either HOOC(CH₂)₄CO(CH₂)₄COOH or H₂N(CH₂)₅CO(CH₂)₅NH₂ in the initial monomer compositions. In addition to pyrolysis of these polyamides, the progress of chemical and physical changes as a function of temperature was continuously monitored and analyzed by FT–IR spectroscopy on cast films placed in an environmental chamber. Introduction of the carbonyl groups into the polymer backbone resulted in a significant reduction of the thermal stability of the corresponding polyamide. Possible mechanisms for the thermal degradation entailing these carbonyl groups and N-acylamide compounds are discussed.