Screening of Arabidopsis thaliana stems for variation in cell wall polysaccharides

A high-throughput method is described by which Arabidopsis thaliana stems can be screened for variation in cell wall composition after hydrolysis with Driselase or trifluoroacetic acid (TFA). Driselase, a mixture of fungal enzymes, hydrolyses cellulose (to glucose) and all the major matrix polysaccharides (to monosaccharides and/or characteristic disaccharides); TFA hydrolyses the matrix polysaccharides, but not cellulose, to monosaccharides. Two different wild-type ecotypes, Columbia and Wassilewskija, showed only minor differences in wall carbohydrate composition. A small number of T-DNA-tagged populations that were screened contained individuals in which the proportion of cellulose, xyloglucan or xylan differed quantitatively from the wild-type. Differences from the wild-type were also observed in the susceptibility of the hemicelluloses to hydrolysis by Driselase, probably reflecting differences in wall architecture.     

Xylan deposition on secondary wall of Fagus crenata fiber

Summary. Delignified and/or xylanase-treated secondary walls of Fagus crenata fibers were examined by field emission scanning electron microscopy. Microfibrils with a smooth surface were visible in the innermost surface of the differentiating fiber secondary wall. There was no ultrastructural difference between control and delignified sections, indicating that lignin deposition had not started in the innermost surface of the cell wall. There was no ultrastructural difference between control and xylanase-treated sections. Microfibrils on the outer part of the differentiating secondary wall surface had globular substances in delignified sections. These globular substances disappeared following xylanase treatment, indicating that these globules are xylan. The globular substances were not visible near the inner part of the differentiating secondary wall but gradually increased toward the outer part of the secondary wall, indicating that xylan penetrated into the cell wall and continuously accumulated on the microfibrils. Mature-fiber secondary walls were also examined by field emission scanning electron microscopy. Microfibrils were not apparent in the secondary wall in control specimens. Microfibrils with many globular substances were observed in the delignified specimens. Following xylanase treatment, the microfibrils had a smooth surface without any globules, indicating that the globular substance is xylan. These results suggest that cellulose microfibrils synthesized on the plasma membrane are released into the innermost surface of the secondary wall and coated with a thin layer of xylan. Successive deposition of xylan onto the cell wall increases the microfibril diameter. The large amounts of xylan that accumulated on microfibrils appear globular but are covered with lignin after they are deposited. Received February 20, 2001/Accepted September 1, 2001     

Effects of physical properties for starch acetate powders on tableting

The aim of the study was to investigate particle and powder properties of various starch acetate powders, to study the effect of these properties on direct compression characteristics, and to evaluate the modification opportunity of physical properties for starch acetate powders by using various drying methods. At the end of the production phase of starch acetate, the slurry of starch acetate was dried using various techniques. Particle, powder, and tableting properties of end products were investigated. Particle size, circularity, surface texture, water content and specific surface area varied according to the particular drying method of choice. However, all powders were freely flowing. Bulk and tapped densities of powders varied in the range of 0.29 to 0.44 g/cm³ and 0.39 to 0.56 g/cm³, respectively. Compaction characteristics revealed that all powders were easily deformed under compression, having yield pressure values of less than 66 MPa according to Heckel analysis. All powders possessed a significant interparticulate bond-forming capacity during compaction. The tensile strength values of tablets varied between 10 and 18 MPa. In conclusion, physical properties of starch acetate could be affected by various drying techniques. A large specific surface area and water content above 4% were favorable properties by direct compression, especially for small, irregular, and rough particles.     

Synthesis of oxidized methyl 4-O-methyl-beta-D-glucopyranoside and methyl beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside derivatives as substrates for fluorescence labeling reactions

The synthetic cellulose model compounds methyl 4-O-methyl-beta-D-glucopyranoside and methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside and related 6-O-protected intermediates were oxidized in good to fair yields using Swern-conditions or bromine/bis(tributyltin) oxide, respectively, to afford compounds containing 6-aldehyde, 3-keto, and 2,3-diketo groups. Cellobiose and oxidized monosaccharides were then labeled with the carbonyl-selective fluorescence marker 9-(7-amino-1,4,7-trioxaheptyl)-9H-carbazolecarboxamide (CCOA). The labeled derivatives serve as model compounds for the determination of minute amounts of carbonyl groups in cellulosic polysaccharides.     

Process control for enhanced L-phenylalanine production using different recombinant Escherichia coli strains

A novel fed-batch approach for the production of L-phenylalanine (L-Phe) with recombinant E. coli is presented concerning the on-line control of the key fermentation parameters glucose and tyrosine. Two different production strains possessing either the tyrosine feedback resistant aroF(fbr) (encoding tyrosine feedback resistant DAHP-synthase (3-desoxy-D-arabino-heptusonate-7-phosphate)) or the wild-type aroF(wt) were used as model systems to elucidate the necessity of finding an individual process optimum for each genotype. With the aid of tyrosine control, wild-type aroF(wt) could be used for L-Phe production achieving higher final L-Phe titers (34 g/L) than the aroF(fbr) strain (28 g/L) and providing higher DAHP-synthase activities. With on-line glucose control, an optimum glucose concentration of 5 g/L could be identified that allowed a sufficient carbon supply for L-Phe production while at the same time an overflow metabolism leading to acetate by-product formation was avoided. The process approach is suitable for other production strains not only in lab-scale but also in pilot-scale bioreactors.     

Effects of cellulase on the modification of cellulose

Multicomponent cellulases, purified endoglucanases and cellobiohydrolases were assayed and shown to modify pure natural cellulose (softwood pulp). Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme, and the treatment conditions. The reactivity of cellulose for some dissolving and derivatization processes may be improved by enzymatic hydrolysis. Endoglucanases decreased the average degrees of polymerization (DP) and improved the alkaline solubility of cellulose most efficiently. The variation in the supramolecular structure estimated from the infrared spectra of the cellulose samples was found to be correlated with the reactivity and might represent wide variations in conformation caused by the breakdown of the hydrogen bonds.     

Ethanol acetylation by mycelium-bound carboxylesterase of Aspergillus oryzae: estimation of thermodynamic parameters and integral productivity

The results collected at different temperatures for ethanol acetylation by cell-bound carboxylesterase from lyophilized cells of Aspergillus oryzae have been used to investigate the kinetics and thermodynamics of this esterification in n-heptane. The occurrence of reversible unfolding followed by irreversible denaturation of the enzyme has been proposed to explain the increase in the starting rate of ethyl acetate formation with temperature observed up to 55 °C and the consequent fall beyond this threshold. The Arrhenius model has been used to estimate the apparent activation enthalpies of both the acetylation reaction (H = 29–33 kJ mol^–1) and reversible enzyme unfolding (H _u = 56–63 kJ mol^–1). The results of residual activity tests performed with cells previously exposed at different temperatures for variable times enabled us also to estimate the first-order rate constant of irreversible denaturation (2.40 × 10^–3 h^–1 < k _d < 8.11 × 10^–3 h^–1) as well as the related thermodynamic parameters (H _d = 22 kJ mol^–1; S _d = –0.29 kJ mol^–1 K^–1). This last phenomenon proved particularly slow for the system under consideration, probably because the biocatalyst link to the mycelium was able to improve its thermostability. In view of future continuous application, the effects of operating time, starting substrate concentration and temperature on the theoretical integral productivity of a fixed-bed column filled with this biocatalyst have been investigated.     

Chiral recognition ability of curdlan triacetate: solvent and temperature effects

The chiral recognition ability of the chiral stationary phase (CSP) consisting of curdlan (beta-1,3-glucan) triacetate coated on silica gel was clearly changed by the contacting solvents and heat treatment. The chiral recognition ability significantly decreased, particularly at temperatures above 45 degrees C, depending on the racemates. The molecular weight of the curdlan triacetate slightly influenced its ability. The recognition abilities of curdlan tricetate that was lost by heat treatment were partially recovered by contact with methanol. However, when it was contacted with ethanol a different selectivity was observed. The labile chiral recognition ability of curdlan triacetate is in striking contrast to the very stable chiral recognition of cellulose (beta-1,4-glucan) triacetate (Chiralcel OA). This difference may be ascribed to the conformational stability of the acetates consisting of curdlan (beta-1,3-glucan) and cellulose (beta-1,4-glucan) with different sugar linkages.     

Inorganic compounds have dual effect on recombinant protein production: influence of anions and cations on serine alkaline protease production

AIMS: Investigation of concerted effects of cations, i.e. Mg2+ and Mn2+, in combination with their anions, i.e. sulphate, chloride and acetate (Ac), on the physiology of Bacillus licheniformis carrying pHV1431::subC to improve the fermentation medium for serine alkaline protease (SAP) production, whereupon, determination of the acid that can be used in pH control. METHODS AND RESULTS: The cell concentrations increased with the increase in MnSO4 and Mn(Ac)2 concentrations, and the highest values were obtained at Co(MnSO4) = 0.20 mmol l-1 and Co(Mn(CH3COO)2) = 4.0 mmol l-1, as 2.3 and 2.2 g l-1, respectively. However, Co(MnCl2) did not influence biomass concentration. SAP production was inhibited with MnCl2 after Co(MnCl2) = 0.60 mmol l-1, but with MnSO4 SAP production was inhibited drastically. Whereas, at high concentrations of Mn(Ac)2 SAP production increased and the highest activity was obtained as ASAP = 1285 U ml-1 at t = 65 h. With the Mg compounds, cell concentrations increased with the increase in the concentrations of MgSO4, MgCl2 and Mg(Ac)2; and the anions did not show any influence on the cell growth. Similar to the results of Mn compounds, the glucose consumption rate increased with the increase in MgSO4 and MgCl2 concentrations; contrariwise, decreased with the increase in Mg(Ac)2 concentrations, due to the use of acetate as the second carbon source. Co(MgSO4) = 0.40 mmol l-1, Co(MgCl2) = 1.60 mmol l-1 and Co(Mg(Ac)2) = 0.40 mmol l-1 were the optimum concentrations separately, and the highest SAP activity was obtained with Mg(Ac)2 as ASAP = 1338 U ml-1 at t = 47 h. Consequently, ion acetate and its acid HAc appear, respectively, as the superior anion for the essential cations and the control agent for pH control in the bioreactor. Finally, optimum initial concentrations and the concerted effects of Mg(Ac)2 and Mn(Ac)2 were investigated, and the optimum concentrations were found respectively as 0.40 and 0.80 mmol l-1, while the maximum activity was obtained as ASAP = 1010 U ml-1 at a shortened cultivation time of t = 39 h. CONCLUSIONS: Mn(Ac)2 and Mg(Ac)2 together enhanced the cell formation and SAP synthesis rates, moreover, SAP synthesis started at an earlier cultivation time. SIGNIFICANCE AND IMPACT OF THE STUDY: Each inorganic compound with its cation and anion has dual effect on the metabolism. Mg2+ and Mn2+ at their specific concentrations influence the regulation of the pathways that might cause better coupling of supply and demand for the amino acids on the basis of the amino acid composition of the enzyme molecule.     

Identification of chemically modified peptide from poly(D,L-lactide-co-glycolide) microspheres under in vitro release conditions

The purpose of this research was to study the chemical reactivity of a somatostatin analogue octreotide acetate, formulated in microspheres with polymers of varying molecular weight and co-monomer ratio under in vitro testing conditions. Poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA) microspheres were prepared by a solvent extraction/evaporation method. The microspheres were characterized for drug load, impurity content, and particle size. Further, the microspheres were subjected to in vitro release testing in acetate buffer (pH 4.0) and phosphate buffered saline (PBS) (pH 7.2). In acetate buffer, 3 microsphere batches composed of low molecular weight PLGA 50∶50, PLGA 85∶15, and PLA polymers (≤10 kDa) showed 100% release with minimal impurity formation (<10%). The high molecular weight PLGA 50∶50 microspheres (28 kDa) displayed only 70% cumulative release in acetate buffer with significant impurity formation (∼24%). In PBS (pH 7.4), on the other hand, only 50% release was observed with the same low molecular weight batches (PLGA 50∶50, PLGA 85∶15, and PLA) with higher percentages of hydrophobic impurity formation (ie, 40%, 26%, and 10%, respectively). In addition, in PBS, the high molecular weight PLGA 50∶50 microspheres showed only 20% drug release with ∼60% mean impurity content. The chemically modified peptide impurities inside microspheres were structurally confirmed through Fourier transform-mass spectrometry (FT-MS) and liquid chromatography/mass spectrometry (LC-MS/MS) analyses after extraction procedures. The adduct compounds were identified as covalently modified conjugates of octreotide with lactic and glycolic acid monomers within polymeric microspheres. The data suggest that due to steric hindrance factors, polymers with greater lactide content were less amenable to the formation of adduct impurities compared with PLGA 50∶50 copolymers.