Fungus β-glycosidases: immobilization and use in alkyl-β-glycoside synthesis

Production of β-glycosidases: β-xylosidase and β-glucosidase by the fungus Sclerotinia sclerotiorum was optimized in the presence of different carbon sources. Immobilization supports with different physico-chemical characteristics were evaluated for use in continuous reactors. Immobilization and activity yields were calculated. Among the adsorption on Duolite, Amberlite, Celite and DEAE-sepharose, and entrapment in polyacrylamide gel or reticulation using glutaraldehyde, highest yields were obtained when β-xylosidase was adsorbed on Duolite A 7 and when β-glucosidase was adsorbed on DEAE-sepharose.

Enzyme preparations from S. sclerotiorum cultures were used in a biphasic (alcohol/aqueous) medium for the synthesis of alkyl-glycosides by trans-glycosylation of sugars and long-chain alcohols. The synthesis was studied under different conditions with primary and secondary alcohols as substrates, in the presence of free or immobilized enzyme. Xylan and cellobiose were used for the synthesis of alkyl-xylosides and alkyl-glucosides, respectively. The majority of the immobilized preparations were unable to catalyze the synthesis of alkyl-glycosides.

Highest yields were obtained when using xylan and C₄–C₆-alcohols. The reaction produced alkyl-β-xyloside and alkyl-β-xylobioside, as confirmed by MS/MS. Up to 22 mM iso-amyl-xyloside and 14 mM iso-amyl-xylobioside were produced from iso-amyl alcohol and xylan.     

Synthesis of pentopyranosyl-containing thiodisaccharides. Inhibitory activity against β-glycosidases

β-(1→4)-Thiodisaccharides formed by a pentopyranose unit as reducing or non reducing end have been synthesized using a sugar enone derived from a hexose or pentose as Michael acceptor of a 1-thiopentopyranose or 1-thiohexopyranose derivatives. Thus, 2-propyl per-O-acetyl-3-deoxy-4-S-(β-d-Xylp)-4-thiohexopyranosid-2-ulose (3) and benzyl per-O-acetyl-3-deoxy-4-S-(β-d-Galp)-4-thiopentopyranosid-2-ulose (11) were obtained in almost quantitative yields. The carbonyl function of these uloses was reduced with NaBH₄ or K-Selectride, and the stereochemical course of the reduction was highly dependent on the reaction temperature, reducing agent and solvent. Unexpectedly, reduction of 3 with NaBH₄–THF at 0 °C gave a 3-deoxy-4-S-(β-d-Xylp)-4-thio-α-d-ribo-hexopyranoside derivative (6) as major product (74% yield), with isomerization of the sulfur-substituted C-4 stereocenter of the pyranone. Reduction of 11 gave always as major product the benzyl 3-deoxy-4-S-(Galp)-4-thio-β-d-threo-pentopyranoside derivative 14, which was the only product isolated (80% yield) in the reduction with K-Selectride in THF at −78 °C. Deprotection of 14 and its epimer at C-2 (13) afforded, respectively the free thiodisaccharides 19 and 18. They displayed strong inhibitory activity against the β-galactosidase from Escherichia coli. Thus, compound 18 proved to be a non-competitive inhibitor of the enzyme (K_i = 0.80 mM), whereas 19 was a mixed-type inhibitor (K_i = 32 μM).     

Spoilage yeasts from Patagonian cellars: characterization and potential biocontrol based on killer interactions

Indigenous yeasts associated with surfaces in three North Patagonian cellars were isolated by means of selective media developed for the isolation of Dekkera/Brettanomyces yeasts; 81 isolates were identified as belonging to Candida boidinii (16%), Hanseniaspora uvarum (38%), Pichia guilliermondii (3%), Saccharomyces cerevisiae (1%), Geotrichum silvicola (16%) and the new yeast species Candida patagonica (26%). No Dekkera/Brettanomyces isolate was obtained, however, 41 isolates (51% of the total isolates) produced some enologically undesirable features under laboratory conditions including the production of 4-ethylphenol and 4-vinylphenol, observed in the Candida boidinii and Pichia guilliermondii isolates. The sensitivity of the 41 spoilage isolates and seven Brettanomyces bruxellensis collection strains was evaluated against a panel of 55 indigenous and ten reference killer yeasts. Killer cultures belonging to Pichia anomala and Kluyveromyces lactis species showed the broadest killer spectrum against spoilage yeasts, including Dekkera bruxellensis collection strains. These killer isolates could be good candidates for use in biocontrol of regionally relevant spoilage yeasts.     

Sensitive fluorimetric assays for α‐glucosidase activity and inhibitor screening based on β‐cyclodextrin‐coated quantum dots

A fluorescence method was established for a α‐glucosidase activity assay and inhibitor screening based on β‐cyclodextrin‐coated quantum dots. p‐Nitrophenol, the hydrolysis product of the α‐glucosidase reaction, could quench the fluorescence of β‐cyclodextrin‐coated quantum dots via an electron transfer process, leading to fluorescence turn‐off, whereas the fluorescence of the system turned on in the presence of α‐glucosidase inhibitors. Taking advantage of the excellent properties of quantum dots, this method provided a very simple, rapid and sensitive screening method for α‐glucosidase inhibitors. Two α‐glucosidase inhibitors, 2,4,6‐tribromophenol and acarbose, were used to evaluate the feasibility of this screening model, and IC₅₀ values of 24 μM and 0.55 mM were obtained respectively, which were lower than those previously reported. The method may have potential application in screening α‐glucosidase inhibitors. Copyright © 2015 John Wiley & Sons, Ltd.     

Patagonian wines: implantation of an indigenous strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in fermentations conducted in traditional and modern cellars

In this work we evaluate the implantation capacity of the selected S. cerevisiae indigenous strain MMf9 and the quality of the produced wines in a traditional (T) and a modern (M) cellar with different ecological and technological characteristics in North Patagonia (Argentina). Red musts were fermented in 10,000 l vats using the indigenous strain MMf9 as well as the respective controls: a fermentation conducted with a foreign starter culture (BC strain) in M cellar and a natural fermentation in T cellar. Since commercial S. cerevisiae starters are always used for winemaking in M cellar and in order to compare the results, natural fermentations and fermentations conducted by the indigenous strain MMf9 were performed at pilot (200 l) scale in this cellar, concomitantly. Thirty indigenous yeasts were isolated at three stages of fermentation: initial, middle and end. The identification of the yeast biota associated to vinifications was carried out using ITS1-5.8S-ITS2 PCR-RFLP. The intra-specific variability of the S. cerevisiae populations was evaluated using mtDNA-RFLP analysis. Wines obtained from all fermentations were evaluated for their chemical and volatile composition and for their sensory characteristics. A higher capacity of implantation of the indigenous MMf9 strain was evidenced in the fermentation carried out in M cellar (80% at end stage) than the one carried out in T cellar (40%). This behaviour could indicate that each cellar differs in the diversity of S. cerevisiae strains associated to wine fermentations. Moreover a higher capacity of implantation of the native starter MMf9 with regard to the foreign (BC) one was also found in M cellar. The selected indigenous strain MMf9 was able to compete with the yeast biota naturally present in the must. Additionally, a higher rate of sugar consumption and a lower fermentation temperature were observed in vinifications conducted by MMf9 strain with regard to control fermentations, producing wines with favourable characteristics. Even when its implantation in T fermentation was lower than that observed in M one, we can conclude that the wine features from MMf9 fermentations were better than those from their respective controls. Therefore, MMf9 selected indigenous strain could be an interesting yeast starter culture in North Patagonian wines.     

Isolation and characterization of ethanol-producing yeasts from fruits and tree barks

Aims:  Isolation and identification of yeasts converting xylose to ethanol.
Methods and Results:  A total of 374 yeasts were isolated from a variety of rotten fruits and barks of trees. Out of these, 27 yeast strains were able to assimilate xylose and produce 0·12–0·38 g of ethanol per gram of xylose. Based on phylogenetic analysis of D1/D2 domain sequence of LSU (Large Subunit) rRNA gene and phenotypic characteristics the ethanol-producing strains were identified as member(s) of the genera Pichia, Candida , Kluyveromyces, Issatchenkia, Zygosacchraomyces , Clavispora, Debaryomyces , Metschnikowia , Rhodotorula and Cryptococcus.
Conclusion:  Yeast strains producing ethanol from xylose have been isolated from a variety of rotten fruits and barks of trees and identified.
Significance and Impact of the Study:  Environmental isolates of yeasts which could convert xylose to ethanol could form the basis for bio-fuel production and proper utilization of xylan rich agricultural and forest wastes.     

Molecular characterization and secreted production of basidiomycetous cell-bound β-glycosidases applicable to production of galactooligosaccharides

Cell-bound β-glycosidases of basidiomycetous yeasts show promise as biocatalysts in galactooligosaccharide (GOS) production. Using degenerated primers designed from Hamamotoa singularis (Hs) bglA gene, we newly identified three genes that encode cell-bound β-glycosidase from Sirobasidium magnum (Sm), Rhodotorula minuta (Rm), and Sterigmatomyces elviae (Se). These three genes, also named bglA, encoded family 1 glycosyl hydrolases with molecular masses of 67‒77 kDa. The BglA enzymes were approximately 44% identical to the Hs-BglA enzyme and possessed a unique domain at the N-terminus comprising 110 or 210 amino acids. The Sm-, Rm-, and Se-BglA enzymes as well as the Hs-BglA enzyme were successfully produced by recombinant Aspergillus oryzae, and all enzymes were entirely secreted to the supernatants. Furthermore, addition of some nonionic detergents (e.g. 0.4% [v/v] Triton-X) increased the production, especially of the Hs- or Se-BglA enzyme. Out of the BglA enzymes, the Se-BglA enzyme showed remarkable thermostability (∼70°C). Additionally, the Sm- and Se-BglA enzymes had better GOS yields, so there was less residual lactose than in others. Accordingly, the basidiomycetous BglA enzymes produced by recombinant A. oryzae would be applicable to GOS production, and the Se-BglA enzyme appeared to be the most promising enzyme for industrial uses.     

Design and characterization of a model αβ peptide

CD and nmr spectroscopy were used to compare the conformational properties of two related peptides. One of the peptides, Model AB, was designed to adopt a helix-turn-extended strand (αβ) tertiary structure in water that might be stabilized by hydrophobic interactions between two leucine residues in the amino-terminal segment and two methionine residues in the carboxyl terminal segment. The other peptide, AB Helix, has the same amino acid sequence as Model AB except that it lacks the-Pro-Met-Thr-Met-Thr-Gly segment at the carboxyl-terminus. Although the carboxyl-terminal segment of Model AB was found to be unstructured, its presence increases the number of residues in a helical conformation, shifts the pKas of three ionizable side chains by 1 pH unit or more compared to an unstructured peptide, stabilizes the peptide as a monomer in high concentrations of ammonium sulfate, increases the conformational stability of residues at the terminal ends of the helix, and results in many slowly exchanging amide protons throughout the entire backbone of the peptide. These results suggest that interactions between adjacent segments in a small peptide can have significant structure organizing effects. Similar kinds of interactions may be important in determining the structure of early intermediates in protein folding and may be useful in the de novo design of independently folding peptides. © 1995 John Wiley & Sons, Inc.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Chiral Piperazine‐2,5‐dione Derivatives as Effective α‐Glucosidase Inhibitors. Part 4

The potential to inhibit α‐ and β‐glucosidases of a series of chiral piperazine‐2,5‐dione derivatives was investigated. Three of the seven compounds tested, viz., 1, 5b , and 5c , showed to be non competitive inhibitors of α‐glucosidase, whereas they exhibited very low inhibitory activity towards β‐glucosidase. The most active compound, 5c (K_I of α‐glucosidase=5 μm), had a 100‐fold α‐glucosidase/β‐glucosidase inhibitor selectivity.     

β-carrageenan: Isolation and characterization

β-Carrageenan, essentially devoid of ester sulfate, was isolated from the hot aqueous extracts of alkali-modified Eucheuma gelatinae, Eucheuma speciosa, and Endocladia muricatum by precipitating the more anionic moieties with a quaternary ammonium salt, isolating the fractions that did not precipitate, then treating these with an anion-exchange cellulose. The β-carrageenan was characterized by chemical analysis, optical rotation, and NMR. Gelling was found to be ion-independent, with T_g = 31–33°C and T_m = 63–70°C. Specific optical rotations of the isolated β-carrageenan samples were more positive than the κ-, λ-, and ι-carrageenans with which they were compared, while agarose, its stereoisomer, exhibited a negative specific rotation. Electrophoresis gels made from β-carrageenan were used to separate DNA fragments which exhibited faster migration than on an agarose gel of comparable concentration, indicating that β-carrageenan has a less restrictive pore structure.     

α‐Glucosidase Inhibitors from the Fungus Aspergillus terreus 3.05358

One new diketopiperazine alkaloid amauromine B ( 1 ), along with three known meroterpenoids, austalide B ( 2 ), austalides N and O ( 3 and 4 ), and two known steroids ( 5 and 6 ), was isolated and identified from the culture broth of the fungus Aspergillus terreus 3.05358. Their structures were elucidated by extensive spectroscopic techniques, including 2D‐NMR and MS analysis, the absolute configuration of 1 was unambiguously established by single crystal X‐ray diffraction analysis. All the isolates were evaluated for their inhibitory effects on α‐glucosidase. Amauromine B ( 1 ) and austalide N ( 3 ) exhibited more potent α‐glucosidase inhibitory activities than the positive control acarbose.     

Selection and structural analysis of de novo proteins from an α3β3 genetic library

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.     

New Alkaloids and α‐Glucosidase Inhibitory Flavonoids from Ficus hispida

Two new pyrrolidine alkaloids, ficushispimines A ( 1 ) and B ( 2 ), a new ω‐(dimethylamino)caprophenone alkaloid, ficushispimine C ( 3 ), and a new indolizidine alkaloid, ficushispidine ( 4 ), together with the known alkaloid 5 and 11 known isoprenylated flavonoids 6  –  16 , were isolated from the twigs of Ficus hispida. Their structures were elucidated by spectroscopic methods. Isoderrone ( 8 ), 3′‐(3‐methylbut‐2‐en‐1‐yl)biochanin A ( 11 ), myrsininone A ( 12 ), ficusin A ( 13 ), and 4′,5,7‐trihydroxy‐6‐[(1R*,6R*)‐3‐methyl‐6‐(1‐methylethenyl)cyclohex‐2‐en‐1‐yl]isoflavone ( 14 ) showed inhibitory effects on α‐glucosidase in vitro.     

Enzymatic Synthesis of Hexyl Glycosides from Lactose At Low Water Activity and High Temperature Using Hyperthermostable β-glycosidases

Optimization of hexyl-g-glycoside synthesis from lactose in hexanol at low water activity and high temperature was investigated using g-glycosidases from hyperthermophilic organisms: Sulfolobus solfataricus (LacS) and Pyrococcus furiosus (CelB). The method for water activity adjustment by equilibration with saturated salt solutions was adapted for use at high temperature. The influence of enzyme immobilization (on XAD-4, XAD-16, or Celite), addition of surfactants (AOT or SDS), substrate concentration, water activity, and temperature (60-90°C) on enzymatic activity and hexyl-g-glycoside yield were examined. Compared to other g-glycosidases in lactose conversion into alkyl glycoside, these enzymes showed high activity in a hexanol one-phase system and synthesized high yields of both hexyl-g-galactoside and hexyl-g-glucoside. Using 32 λg/l lactose (93 λmM), LacS synthesized yields of 41% galactoside (38.1 λmM) and 29% glucoside (27.0 λmM), and CelB synthesized yields of 63% galactoside (58.6 λmM) and 28% glucoside (26.1 λmM). With the addition of SDS to the reaction it was possible to increase the initial reaction rate of LacS and hexyl-g-galactoside yield (from 41 to 51%). The activity of the lyophilized enzyme was more influenced by the water content in the reaction than the enzyme on solid support. In addition, it was concluded that for the lyophilized enzyme preparation the enzymatic activity was much more influenced by the temperature when the water activity was increased. A variety of different glycosides were prepared using different alcohols as acceptors.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Syntheses and anti‐tubercular activity of β‐substituted and α,β‐disubstituted peptidyl β‐aminoboronates and boronic acids

Tuberculosis is still affecting millions of people worldwide, and new resistant strains of Mycobacterium tuberculosis are being found. It is therefore necessary to find new compounds for treatment. In this paper, we report the synthesis and in vitro testing of peptidyl β‐aminoboronic acids and β‐aminoboronates with anti‐tubercular activity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Vibrational and chiroptical spectroscopic characterization of γ‐turn model cyclic tetrapeptides containing two β‐Ala residues

The optical spectroscopic characterization of γ‐turns in solution is uncertain and their distinction from β‐turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on γ‐turn model cyclic tetrapeptides cyclo(Ala‐β‐Ala‐Pro‐β‐Ala) ( 1 ), cyclo(Pro‐β‐Ala‐Pro‐β‐Ala) ( 2 ) and cyclo(Ala‐β‐Ala‐Ala‐β‐Ala) ( 3 ). Conformational analysis performed at the 6‐31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1‐3 , featuring two inverse γ‐turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n→π* band near 230 nm and a positive π→π* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1‐3 show similar spectra with blue‐shifted bands. The VCD spectra in ACN‐d₃ of 1 and 2 show a +/?/+/? amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2 . A ?/+/+/? amide I VCD pattern was measured for 3 in TFE‐d₂. All three peptides give a positive couplet or couplet‐like feature (+/?) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at ～1630 cm^?1 or below) are indicative of a C₇ H‐bonded inverse γ‐turns with Pro in position 2, while γ‐turns encompassing Ala absorb at higher frequency (above 1645 cm^?1). Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Piezoelectric properties of poly-β-hydroxybutyrate and copolymers of β-hydroxybutyrate and β-hydroxyvalerate

The shear piezoelectricity was observed in oriented films of poly-β-hydroxybutyrate (PHB) and copolymers of β-hydroxybutyrate (HB) and β-hydroxyvalerate (HV). The piezoelectric stress constant 3₁₄ = e′₁₄ − ie″₁₄ (polarization/strain), the piezoelectric strain constant d₁₄ = d′₁₄ − id″₁₄ (polarization/stress), the elastic constant c = c′ + ic″ and the dielectric constant = ′ − i″ were determined at a frequency of 10 Hz over a temperature range from −150° to +150°C. Piezoelectric relaxations as well as elastic and dielectric relaxations were clearly observed at the glass transition temperature of about 15°C. In order to evaluate the piezoelectric constants (e₂ and d₂) for the piezoelectric phase which consists of the crystalline region and the oriented non-crystalline region, a spherical dispersion two phase model was utilized. Assuming the appropriate fixed values for the elastic and dielectric constants in the piezoelectric phase, d₂ and d₂ were calculated as a function of temperature. For a PHB and a copolymer (17 HV/83 HB), e₂ and d₂ showed relaxations, leading to a conclusion that the instantaneous piezoelectric constant in the crystalline phase is constant independent of temperature but the piezoelectric constant in the oriented non-crystalline phase is relaxational and has the opposite sign. For a copolymer (25 HV/75 HB) and a chloroform treated copolymer (17 HV/83 HB), e₂ and d₂ were constant independent of temperature, indicating that the oriented non-crystalline phase has disappeared owing to the increased molecular flexibility due to copolymerization or annealing in chloroform vapour.