Change of surface structure of poly(3-hydroxybutyrate) film upon enzymatic hydrolysis by PHB depolymerase

The change in the surface structure of poly[(R)-3-hydroxybutyrate] [PHB] films upon the enzymatic hydrolysis was analyzed by attenuated total reflection infrared [ATR/IR] spectrometry. As enzymes, PHB depolymerases isolated from Ralstonia pickettii T1 and Pseudomonas stutzeri were used. By curve decomposition of the carbonyl stretching band of ATR/IR spectra, the change in the surface crystallinity of PHB films by exposure to buffer containing 0, 1, and 4 microg of PHB depolymerases was estimated. It has been widely believed that the enzymatic hydrolysis first occurs in the amorphous phase, followed by the degradation in the crystalline phase, and extracellular PHB depolymerase can degrade only polymer chains in the surface layer of the film. Therefore, the surface crystallinity had been expected to increase upon the enzymatic degradation. However, the results were contrary to this expectation. The surface crystallinity was decreased by the enzymatic attack. Because ATR/IR spectrometry is sensitive to a small change in molecular structure of the sample surface, the decrease in the crystallinity shown by ATR/IR experiments probably does not indicate the complete loss of regularity of the crystalline phase. Because the chains at crystalline surface are more mobile than those inside the crystals, the C=O band for crystalline surface may appear at a position similar to those of the amorphous or interfacial phase in ATR/IR spectra of PHB. Only the chains inside the crystals may contribute to the C=O band of the crystalline phase. Thus, we rather suppose that the decrease in the crystalline peak of the ATR/IR spectra reflects the change in chain mobility or the increase of crystalline surface area by cracking of lamellas at the surface layers of PHB films or both.     

Effects of structural imperfection on gelatinization characteristics of amylopectin starches with A- and B-type crystallinity

The aim of the present work was to investigate the effect of physical structures on the properties of starch granules. Starches with a high amylopectin content possessing A- and B-type crystallinity were chosen for the study. The gelatinization temperature decreased in the following order: maize (A) > potato (B) > wheat (A) > barley (A), which did not reflect a correlation with the type of crystallinity. Low values of gelatinization temperature were accompanied with high free surface energy of the crystallites. It is proposed that these data are caused by different types of imperfections in starch crystals. Annealing resulted in an enhancement of the gelatinization temperature and a decrease of the free surface energy of the crystallites for all starches reflecting a partial improvement of crystalline perfection. A limited acid hydrolysis (lintnerization) of the starches decreased the gelatinization temperature because of a partial disruption of the crystalline lamellae and an increase of the amount of defects on the edges of the crystallites. Annealing of the lintnerized starches improved the structure of maize and potato starch, giving them similar structural and physicochemical parameters, which was opposite the behavior of the annealed sample from wheat. The possible nature of removable and nonremovable defects inside the crystalline region of the starch granules is discussed. It is concluded that, besides the allomorphic A- and B-types of crystal packing, physical defects in the crystals possess a major impact on starch gelatinization.     

A new strategy for recycling and preparation of poly(L-lactic acid): hydrolysis in the melt

Poly(L-lactide) [i.e., poly(L-lactic acid) (PLLA)] was hydrolyzed in the melt in high-temperature and high-pressure water at the temperature range of 180-350 degrees C for a period of 30 min, and formation, racemization, and decomposition of lactic acids and molecular weight change of PLLA were investigated. The highest maximum yield of l-lactic acid, ca. 90%, was attained at 250 degrees C in the hydrolysis periods of 10-20 min. Too-high hydrolysis temperatures such as 350 degrees C induce the dramatic racemization and decomposition of formed lactic acids, resulting in decreased maximum yield of L-lactic acid. The hydrolysis of PLLA proceeds homogeneously and randomly via a bulk erosion mechanism. The molecular weight of PLLA decreased exponentially without formation of low-molecular-weight specific peaks originating from crystalline residues. The activation energy for the hydrolysis (deltaE(h)) of PLLA in the melt (180-250 degrees C) was 12.2 kcal x mol(-1), which is lower than 20.0 kcal x mol(-1) for PLLA and 19.9 kcal x mol(-1) for poly(dl-lactide) [i.e., poly(DL-lactic acid)] as a solid in the temperature range below the glass-transition temperature (21-45 degrees C). This study reveals that hydrolysis of PLLA in the melt is an effective and simple method to obtain l-lactic acid and to prepare PLLA having different molecular weights without containing the specific low-molecular-weight chains, because of the removal of the effect caused by crystalline residues.     

Hypocrea jecorina (Trichoderma reesei) Cel7A as a molecular machine: A docking study

Hypocrea jecorina (formerly Trichoderma reesei) Cel7A has a catalytic domain (CD) and a cellulose-binding domain (CBD) separated by a highly glycosylated linker. Very little is known of how the 2 domains interact to degrade crystalline cellulose. Based on the interaction energies and forces on cello-oligosaccharides computationally docked to the CD and CBD, we propose a molecular machine model, where the CBD wedges itself under a free chain end on the crystalline cellulose surface and feeds it to the CD active site tunnel. Enzyme-substrate interactions produce the forces required to pull cellulose chains from the surface and also to help the enzyme move on the cellulose chain for processive hydrolysis. The energy to generate these forces is ultimately derived from the chemical energy of glycosidic bond breakage.     

Recrystallisation behaviour of native and processed waxy maize starch in relation to the molecular characteristics

Molecular characteristics were determined for native waxy maize starch and maize starch modified in different way (by mechanical treatment or/and acid hydrolysis). Recrystallisation behaviour was studied. Methods used in this study were MALLS, HPAEC-PAD, NMR, DSC, SEM, light microscopy.

Five starch materials were subjected to storage under the same conditions in the presence of water (70 w/w%). Molecular weight, radius of gyration, initial crystallinity, and degree of polymerisation, degree of branching, chain length distribution profiles, were related to nucleation rate during the recrystallisation process, rate of recrystallisation, thermal stability and amount of obtained crystallinity. This allowed the following connections between the molecular characteristics and kinetic of recrystallisation to be proposed: Amylopectin molecular weight appeared to affect the number of starch crystallites formed and amount of crystallinity but not the stability of the rebuilt crystallites. The stability of rebuilt crystallites can be controlled by degree of polymerization, degree of branching and unit chain length distribution, characteristics which were similar for the starches.

A mixture of two starches, with and without crystalline structure in initial state but with molecular weight in same range, were stored and scanned in order to understand possible cocrystallisation effects.     

Standard free energy for the hydrolysis of adenylylated T4 DNA ligase and the apparent pKa of lysine 159

Equilibrium constants for the adenylylation of T4 DNA ligase have been measured at 10 pH values. The values, when plotted against pH, fit a titration curve corresponding to a pKa of 8.4 +/- 0.1. The simplest interpretation is that the apparent pKa is that of the 6-amino group of the AMP-accepting residue Lys159. Based on the pH dependence of the equilibrium constants, the value at pH 7.0 is 0. 0213 at 25 degrees C, corresponding to DeltaG'o = +2.3 kcal mol-1. From this value and the standard free energy change of -10.9 kcal mol-1 for the hydrolysis of ATP to AMP and PPi, we calculate that DeltaG'o for the hydrolysis of the adenylyl-DNA ligase is -13.2 kcal mol-1. The presence of conserved basic amino acid residues in the catalytic domain, which are proximal to the active site in the homologous catalytic domain of T7 DNA ligase, suggests that the pKa of Lys159 is perturbed downward by the electrostatic effects of nearby positively charged amino acid side chains. The lower than normal pKa 8.4 compared with 10.5 for the 6-amino group of lysine and the high energy of the alpha,beta-phosphoanhydride linkage in ATP significantly facilitate adenylylation of the enzyme.     

Novel preparation method for poly(L-lactide)-based block copolymers: extended chain crystallites as a solid-state macro-coinitiator

A novel synthetic method for poly(L-lactide) (PLLA)-based diblock copolymers was developed by the use of PLLA extended chain crystallites (or crystalline residues) as a solid-state macro-coinitiator. In this study, we showed one example, i.e., a synthesis of diblock copolymer composed of a crystalline PLLA chain and an amorphous poly(DL-lactide) chain by ring-opening polymerization of DL-lactide initiated with stannous octoate (i.e., tin(II) 2-ethylhexanoate) in the presence of PLLA extended chain crystallites. The PLLA extended chain crystallites were prepared by hydrolytic degradation of crystallized PLLA films at 97 degrees C for 70 h. The chains inside the extended chain crystallites are expected to be protected from transesterfication reaction. Gel permeation chromatography, polarimetry, 1H NMR spectroscopy, wide-angle X-ray scattering, and differential scanning calorimetry revealed that the diblock copolymer poly(L-lactide-block-DL-lactide) was successfully prepared without significant transesterification.     

Enzymatic, alkaline, and autocatalytic degradation of poly(L-lactic acid): effects of biaxial orientation

The hydrolytic degradation of biaxially oriented and de-oriented (melt-crystallized) poly(l-lactic acid) (PLLA) films was investigated in Tris-HCl-buffered solution (pH 8.6) with proteinase K, alkaline solution, and phosphate-buffered solution (pH 7.4) by the use of gravimetry, gel permeation chromatography, differential scanning calorimetry, and scanning electron microscopy. Biaxial orientation disturbed the proteinase K-catalyzed enzymatic degradation of PLLA films and the effects of biaxial orientation overcame those of crystallinity. The former may be due to the fact the enzyme cannot attach to the extended (strained) chains in the amorphous regions of the biaxially oriented PLLA film or cannot catalyze the cleavage of the strained chains. Another probable cause is that the enzyme can act only at the film surface of the biaxially oriented PLLA film, in marked contrast with the case of the de-oriented PLLA films where enzymatic degradation can proceed beneath the spherulitic crystalline residues. The effects of biaxial orientation on the alkaline and autocatalytic degradation of the PLLA films were insignificant for the periods studied here. The crystallinity rather than the biaxial orientation seems to determine the alkaline and autocatalytic degradation rates of the PLLA films. The accumulation of crystalline residues formed as a result of selective cleavage and removal of the amorphous chains was observed for the de-oriented PLLA films, but not for the biaxially oriented PLLA film, when degraded in the presence of proteinase K. This means the facile release of formed crystalline residues from the surface of the biaxially oriented PLLA film during enzymatic degradation, due to the fact that the crystalline regions of the biaxially oriented PLLA film were oriented with their c axis parallel to the film surface.     

Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is an M-type homopolymer.

Ferritin from the spleen of the Antarctic teleost Trematomus bernacchii is composed of a single subunit that contains both the ferroxidase center residues, typical of mammalian H chains, and the carboxylate residues forming the micelle nucleation site, typical of mammalian L chains. Comparison of the amino-acid sequence with those available from lower vertebrates indicates that T. bernacchii ferritin can be classified as an M-type homopolymer. Interestingly, the T. bernacchii ferritin chain shows 85.7% identity with a cold-inducible ferritin chain of the rainbow trout Salmo gairdneri. The structural and functional properties indicate that cold acclimation and functional adaptation to low temperatures are achieved without significant modification of the protein stability. In fact, the stability of T. bernacchii ferritin to denaturation induced by acid or temperature closely resembles that of mesophilic mammalian ferritins. Moreover iron is taken up efficiently and the activation energy of the reaction is 74.9 kJ.mol(-1), a value slightly lower than that measured for the human recombinant H ferritin (80.8 kJ.mol(-1)).     

Structural studies of an arabinoxylan isolated from Litsea glutinosa (Lauraceae)

A water-soluble arabinoxylan (D-xylose and L-arabinose in the molar ratio 1.0:3.4) was isolated from the mucilaginous bark of Litsea glutinosa (Lauraceae). The results of methylation analysis, partial hydrolysis, and 1H- and 13C-n.m.r. spectroscopy indicated a backbone of (1----4)-linked beta-D-xylopranosyl residues substituted at both positions 2 and 3 with side chains composed of either single or (1----3)-linked arabinofuranosyl residues. Both alpha-L- and beta-L-arabinofuranosyl residues were present. It is possible that side chains composed of two beta-L-arabinofuranosyl residues are attached mainly at O-2 of some xylosyl residues.     

Correlating the effect of pretreatment on the enzymatic hydrolysis of straw

Avicell, Alkali-treated straw cellulose (ATSC), and wheat straw were ball-milled to reduce crystallinity; wheat straw was delignified by hot (120 degrees C) sodium hydroxide solutions of various concentrations. The physically and chemically pretreated cellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Enzymic hydrolysis data were fitted by the hyperbolic correlation of Holtzapple, which involves two kinetic parameters, the maximum conversion (X(max)), and the enzymic hydrolysis time corresponding to 50% of X(max) (t(1/2)). An empirical correlation between X(max) and cellulose crystallinity, lignin content, and degree of delignification has been found under our experimental conditions. Complete cellulose hydrolysis is shown to be possible at less than 60% crystallinity indices or less than 10% lignin content.     

Structural and thermodynamic properties of rice starches with different genetic background Part 1. Differentiation of amylopectin and amylose defects

A combined DSC - HPAEC-PAD approach, gel permeation chromatography and mild long-term acidic hydrolysis were employed to study the effects of amylopectin chain-length distributional and amylose defects on the assembly structures of amylopectin (crystalline lamellae, amylopectin clusters) in A-type polymorphic starches extracted from 11 Thai cultivars of rice with different amylose level. Joint analysis of the data allowed determining the contributions of different populations of amylopectin chains to the thermodynamic melting parameters of crystalline lamellae. It was shown that amylopectin chains with DP 6-12 and 25or=37 could be related to chains stabilizing these structures. The total effect of amylose and amylopectin defects can be described by means of Thomson-Gibbs' equation. The increase of defects in the assembly structures is accompanied by rise of the rates of acidic hydrolysis of both amorphous and crystalline parts in starches.     

Decomposition of phosphoserine and phosphothreonine during acid hydrolysis

The rate of decomposition of phosphoserine and phosphothreonine, both as free O-phosphoamino acids and in peptides, was studied under conditions of acid hydrolysis, using 1, 2, and 6 n HCl at 110°C. For the free O-phosphoamino acids, the decomposition follows first-order kinetics and is two- to fourfold faster for phosphoserine than phosphothreonine. The rate of destruction of these O-phosphoamino acids during hydrolysis of peptides is dependent on the neighboring amino acid residues, and thus the hydrolysis of a free O-phosphoamino acid generally is not a good model for the hydrolysis of that O-phosphoamino acid in a peptide. For the three peptides studied, maximal recoveries of O-phosphoamino acids are obtained after hydrolysis in 6 n HCl for 2 to 4 hr.     

Characterization of nascent DNA fragments produced by excision of uracil residues in DNA.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.     

Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions

Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to suggest that reversal of a negative charge at a pocket has a larger effect on stability than a similar reversal at a protrusion and that this difference arises largely from short-range interactions with polar groups within the pocket, rather than long-range interactions with solvent-exposed charged groups.     

Gelatinization of starch in excess water: beyond the melting of lamellar crystallites. A combined wide- and small-angle X-ray scattering study

The gelatinization of waxy rice, regular rice, and potato starch suspensions (66% w/w moisture) was investigated by real-time small-angle X-ray scattering (SAXS) and wide-angle X-ray diffraction (WAXD) during heating and by fast ramp differential scanning calorimetry (DSC). The high-angle tail of the SAXS patterns suggested the transition from surface to mass fractal structures in the DSC gelatinization range. Amylose plays a major role in determining the dimensions of the self-similar structures that develop during this process as the characteristic power-law scattering behavior extends to lower scattering angles for regular than for waxy starches. Crystallinity of A-type starches is lost in the temperature region roughly corresponding to the DSC gelatinization range. At the end of the gelatinization endotherm, the B-type potato starch showed residual crystallinity (WAXD), while SAXS-patterns exhibited features of remaining lamellar stacks. Results indicate that the melting of amylopectin crystallites during gelatinization is accompanied by the (exothermic) formation of amorphous networks.     

Influence of in vitro hydrolytic degradation on the morphology and crystallization behavior of poly(p-dioxanone)

We have studied the hydrolytic degradation of high molecular weight poly(p-dioxanone), PPDX, sutures. The samples were degraded either in distilled water or in a phosphate buffer at 37 degrees C, and the starting viscosity-average molecular weight was 130 kg/mol. The hydrolytic degradation of PPDX occurs in an approximate two stage process where the amorphous regions of the sample are attacked faster than the crystalline regions of the sample. The changes experienced by the samples as degradation proceeded were successfully monitored by viscosimetry, differential scanning calorimetry (DSC), weight loss, pH changes, and scanning electron microscopy (SEM). Polarized optical microscopy (POM) observations performed on PPDX films revealed that PPDX crystallizes in spherulites whose detailed morphology depends on the supercooling employed during isothermal crystallization. Changes in the spherulitic morphology as molecular weight is reduced are only pronounced when the molecular weight is equal or lower than 8 kg/mol. The dependence of lamellar thickness as a function of isothermal crystallization temperature was examined by atomic force microscopy (AFM) in thin films of PPDX together with melting point data obtained by DSC. Through the use of the Thomson-Gibbs equation, we obtained a value of 166 erg/cm2 for the fold surface free energy of PPDX. This value is in the same range as those obtained previously for similar linear polyesters. The lamellar thickness, as well as the melting point, was found to have a small decreasing dependence with the molecular weight of the samples.     

Bombyx mori silk fibroin liquid crystallinity and crystallization at aqueous fibroin-organic solvent interfaces.

A banded morphology has been observed for Bombyx mori silk fibroin films obtained from an aqueous hexane interface; the period of the banding is approximately 1 microm. Morphology and diffraction from different regions of the banded structure suggest that it is a free surface formed by a cholesteric liquid crystal. Truncated hexagonal lamellar crystallites of B. mori silk fibroin have been observed in films formed in the surface excess layer of fibroin at the interface between aqueous fibroin and hexane or chloroform. Based on initial crystallographic evidence, a three-fold helical conformation has been ascribed to the fibroin chains within the crystals. The chain conformation and crystalline habit appear to be similar to the silk III structure previously observed at the air-water interface (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Valluzzi R, Gido S, Zhang W, Muller W, Kaplan D. Macromolecules 1996;29:8606-8614) but the crystalline packing is different. Diffraction data obtained for the crystallites are similar to diffraction behavior for a collagen-like model peptide. Diffraction patterns obtained from crystallized regions of the banded morphology can be indexed using the same unit cell as the hexagonal lamellar crystallites. Surfactancy of fibroin and subsequent aggregation and mesophase formation may help to explain the liquid crystallinity reported for silk, which is long suspected to play a role in the biological silk spinning process (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Willcox, P. J.; Gido, SP, Muller W, Kaplan DL. Macromolecules 1996:29:5106-5110; Magoshi J, Magoshi Y, Nakamura S. In: Kaplan D, Adams W, Farmer B, Viney C, editors, Mechanism of Fiber Formation of Silkworm. Washington, DC: American Chemical Society 1994:292-310; Magoshi J, Magoshi Y, Nakamura S. J Appl Polym Sci Appl Polym Symp 1985;41:187-204; Magoshi J, Magoshi Y, Nakamura S. Polym Commun 1985;26:309.).     

Oligo(methionyl) proteins. Enzymatic hydrolysis of the model isopeptides N epsilon-oligo(L-methionyl)-L-lysine

A number of model isopeptides containing oligo(methionine) chains varying in length (2-5 residues) covalently linked to the epsilon-amino group of lysine were synthesized by solid-phase procedures. Hydrolysis of these peptides by pepsin, chymotrypsin, cathepsin C (dipeptidyl peptidase IV) and intestinal aminopeptidase N was investigated using high-performance liquid chromatography to identify and quantify the hydrolysis products. Methionine oligomers grafted onto lysine were cleaved to tripeptides by pepsin. Chymotrypsin preferentially hydrolyzed the methionyl-methionine bond preceding the isopeptide bond. Cathepsin C released dimethionyl units from the covalently attached polymers. Intestinal aminopeptidase caused efficient hydrolysis of both peptides and isopeptide bonds although free methionine decreased the cleavage of the latter bond. Hydrophobic characteristics of oligo(methionine) chains promoted enzyme-catalyzed transpeptidations resulting probably from acyl-transfer-type reactions. Complementary hydrolysis of the isopeptides by these digestive enzymes suggests that covalent attachment of oligo(amino acid)s to food proteins may improve their nutritional value.