Biomedical subjects

Yoshiharu Doi

Publications and source records attributed to Yoshiharu Doi.

At least 55 records · Page 3Linked to original sources

PubMed2002

Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase.

By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC(Ac), was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC(Ac) gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) [by LS5218 [fadR601 atoC(Con)] strain]. The expression vector for the phaC(Ac) gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%).

Acyltransferases↗

PubMed2002

A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA.

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.

Bacterial Proteins↗

PubMed2002

Enzyme inhibitors to increase poly-3-hydroxybutyrate production by transgenic tobacco.

Chemical regulation of secondary-metabolite synthesis was investigated through the improvement of poly-3-hydroxybutyrate (PHB) production in transgenic tobacco plants by the use of enzyme inhibitors. Two tobacco lines, BC3 and rCAB8, that produce PHB in both the cytosol and plastids were used. An acetyl-CoA carboxylase inhibitor, D-(+)-Quizalofop-ethyl, increased PHB accumulation in both lines 2-fold. The accumulation rate of plastidial PHB in the rCAB8 line was 2.5-fold higher than that of cytosolic PHB in the BC3 line. A specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, mevastatin, also increased PHB accumulation but only in the BC3 line. These results indicated that chemical regulation of the native metabolic flows by the specific enzyme inhibitors increased secondary-metabolite production in the transgenic tobacco plants we used.

Acetyl-CoA Carboxylase↗

PubMedDate not supplied

Side-chain effect of second monomer units on crystalline morphology, thermal properties, and enzymatic degradability for random copolyesters of (R)-3-hydroxybutyric acid with (R)-3-hydroxyalkanoic acids.

Three types of random copolymers with 94 mol % (R)-3-hydroxybutyric acid (3HB) and 6 mol % (R)-3-hydroxyalkanoic acids with different side-chain lengths, (R)-3-hydroxypentanoic acid (3HV), (R)-3-hydroxyhexanoic acid (3HHx), and medium-chain-length (R)-3-hydroxyalkanoic acids (mcl-3HA, C8-C12), were prepared by biological synthetic techniques. The solid-state structure and thermal properties of melt-crystallized films for copolymers were characterized by means of wide-angle X-ray diffraction, small-angle X-ray scattering, differential scanning calorimetry, and optical microscopy. The randomly distributed second monomer units, except for 3HV in copolyesters, act as defects of the P(3HB) crystal and are excluded from the P(3HB) crystalline lamellae. The lamellar thickness of copolymers decreased with an increase in the side-chain length of second monomer units. In addition, the growth rate of spherulites decreased with an increase in the carbon numbers of second monomer units at an identical crystallization temperature. These results indicate that a steric bulkiness of the second monomer unit affects the crystallization of (R)-3HB segments in random copolyesters. An enzymatic degradation test of melt-crystallized copolymer films was carried out in the presence of PHB depolymerase from Alcaligenes faecalis T1. Erosion rate of copolyesters was dependent on both the crystallinity and the lamellar thickness of samples. As the result, the rate of enzymatic degradation for copolymer films increased with an increase in the carbon numbers of second monomer units.

Carboxylic Ester Hydrolases↗

PubMedDate not supplied

Nonhydrolytic fragmentation of a poly[(R)-3-hydroxybutyrate] single crystal revealed by use of a mutant of polyhydroxybutyrate depolymerase.

This paper reports the initial process of the enzymatic degradation of solution-grown lamellar single crystals of bacterial poly[(R)-3-hydroxybutyrate] (P(3HB)) with an extracellular polyhydroxybutyrate (PHB) depolymerase purified from Alcaligenes faecalis T1. We used a hydrolytic-activity-disrupted mutant of the PHB depolymerase in order to avoid the influence of hydrolytic reaction in the system. The effect of addition of the mutant enzyme upon the P(3HB) single crystals was investigated by turbidimetric assay, high-performance liquid chromatography (HPLC), and atomic force microscopy (AFM). Suspension turbidity of the P(3HB) single crystals increased after addition of the mutant enzyme having no hydrolytic activity. No soluble product from the P(3HB) single crystals with the mutant enzyme was detected by HPLC. AFM observation of the P(3HB) single crystals adsorbed on highly ordered pyrolytic graphite revealed that the mutant enzyme yielded a lot of lengthwise crystal fragments from the P(3HB) single crystals. On the basis of these results, we concluded that the mutant enzyme disturbs the molecular packing of the P(3HB) polymer chain around the loose chain packing region in the single crystal, resulting in the fragmentation. Therefore, it is suggested that the enzymatic degradation of P(3HB) single crystals with a wild-type PHB depolymerase progresses via three steps: (1) adsorption of the enzyme onto the surface of the single crystal; (2) disturbance of the molecular packing of P(3HB) polymer chain in the single crystal by the adsorbed enzyme; and (3) hydrolysis of the disturbed polymer chain by the adsorbed enzyme.

Alcaligenes↗

PubMedDate not supplied

Crystallization, stability, and enzymatic degradation of poly(L-lactide) thin film.

Poly(L-lactide) (PLLA) thin film with 100 nm thickness was crystallized at 160 degreesC for 20 min from the melt obtained at 220 degreesC. Hexagonal crystals with three types of growth (derivative growth lamellae, overgrowth multistacked lamellae, and undergrowth multistacked lamellae) were simultaneously observed by atomic force microscopy (AFM). These phenomena are due to the differences of the formative points of secondary crystal nuclei against the basal lamella. Enzymatic degradation of PLLA thin film revealed two types of amorphous regions. These regions were identified as the free amorphous region around the crystals and the restricted amorphous region between the crystal and glass substrate. In situ observation of thermal behavior of lamellar crystals was performed to understand the correlation between the chain folding and stability of the crystal by using temperature-controlled AFM. The morphology of the sectors with [100] growth plane had changed to a comblike morphology despite the fact that the [110] growth plane remained unchanged, suggesting that the stability of the chain folding and the chain-packing state affected the thermal behavior.

Crystallization↗

PubMedDate not supplied

Crystalline/amorphous phase structure and molecular mobility of biodegradable poly(butylene adipate-co-butylene terephthalate) and related polyesters.

Differential scanning calorimetry (DSC), atomic force microscopy (AFM), wide-angle X-ray scattering (WAXD), and solid-state (13)C NMR have been used to investigate the crystalline/amorphous structure and molecular mobility of biodegradable poly(butylene adipate-co-44 mol % butylene terephthalate) [P(BA-co-44 mol % BT)] copolyester sample crystallized from the melt. The DSC endothermic peak, which is ascribed to the melting of the crystalline region, was broad relative to those reported for conventional partially crystalline polyesters. In AFM observation, spherulitic morphology was not observed while small particles with a size of about 100 nm were detected. The WAXD pattern of the sample was very broad. These results have indicated that a melt-crystallized P(BA-co-44 mol % BT) sample contains small crystals with a wide distribution in size. A solid-state (13)C NMR technique was also used to perform molecular-level and selective analyses for both butylene terephthalate and butylene adipate units. For the butylene terephthalate units, the existence of two components with different microstructure and molecular mobility was detected: one component was assigned to the alpha-form crystal of poly(butylene terephthalate) homopolymer (PBT) and the other was in amorphous regions. In contrast, all of butylene adipate units were located in amorphous regions. Solid-state NMR data have suggested that sizes of crystalline regions are less than 3 nm.

Biodegradation, Environmental↗

PubMedDate not supplied

Enzymatic hydrolysis of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4.

Enzymatic degradability has been investigated for a series of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s (P(3HB-co-3HP)s) with 3-hydroxypropionate (3HP) unit contents from 11 to 86 mol % as well as poly(3-hydroxybutyrate) (P(3HB)) and chemosynthesized poly(3-hydroxypropionate) (P(3HP)). The behavior of degradation by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pikettii T1 and Acidovorax Sp. TP4, defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were compared in relation to the thermal properties and crystalline structures of the PHA samples elucidated by differential scanning calorimetry and wide-angle X-ray diffraction. The degradation products were characterized by high-performance liquid chromatography and one- (1D) and two-dimension (2D) (1)H NMR spectroscopy. It was found that the PHA depolymerase of Acidovorax Sp. TP4 showed degradation behavior different from that shown by depolymerase of R. pikettii T1. PHA depolymerase from Acidovorax Sp. TP4 degraded the P(3HB-co-3HP) films with lower crystallinity in higher rates than those with higher crystallinity, no matter what kinds of crystalline structures they formed. In contrast, PHA depolymerase from R. pikettii T1 degraded P(3HB-co-3HP) films forming P(3HB) crystalline structure in higher rates than those forming P(3HP)s. The increase in amorphous nature of the P(3HB-co-3HP) films with P(3HB)-homopolymer-like crystalline structure increases and then decreases the rate of degradation by depolymerase from R. pikettii T1. The 3-hydroxybutyrate (3HB) monomer was produced as a major product by the hydrolysis of P(3HB) film by PHA depolymerase from Acidovorax Sp. TP4. The P(3HB-co-3HP) films could be degraded into 3HB and 3-hydroxypropionate (3HP) monomer at last, indicating that the catalytic domain of the enzyme recognized at least two monomeric units as substrates. While the PHA depolymerase from R. pikettii T1 hydrolyzed P(3HB) film into 3HB dimer as a major product, and the catalytic domain recognized at least three monomeric units. The degradation behavior of P(3HB-co-3HP) films by the PHA depolymerase of Acidovorax Sp. TP4 could be distinguished from that by the depolymerase of R. pikettii T1.

Betaproteobacteria↗

PubMedDate not supplied

Compatibilization effect of poly(epsilon-caprolactone)-b-poly(ethylene glycol) block copolymers and phase morphology analysis in immiscible poly(lactide)/poly(epsilon-caprolactone) blends.

The miscibility and phase behavior of two stereoisomer forms of poly(lactide) (PLA: poly (L-lactide) (PLLA) and poly(DL-lactide) (PDLLA)) blends with poly(epsilon-caprolactone)-b-poly(ethylene glycol) (PCL-b-PEG) and PCL-b-monomethoxy-PEG (PCL-b-MPEG) block copolymers have been investigated by differential scanning calorimetry (DSC). The DSC thermal behavior of both the blend systems revealed that PLA is miscible with the PEG segment phase of PCL-b-(M)PEG but is still immiscible with its PCL segment phase although PCL was block-copolymerized with PEG. On the basis of these results, PCL-b-PEG was added as a compatibilizer to PLA/PCL binary blends. The improvement in mechanical properties of PLA/PCL blends was achieved as anticipated upon the addition of PCL-b-PEG. In addition, atomic force microscopy (AFM) measurements have been performed in order to study the compositional synergism to be observed in mechanical tests. AFM observations of the morphological dependency on blend composition indicate that PLA/PCL blends are immiscible but compatible to some extent and that synergism of compatibilizing may be maximized in the compositional blend ratio before apparent phase separation and coarsening.

Calorimetry, Differential Scanning↗

PubMedDate not supplied

Enzymatic hydrolysis of chemosynthesized atactic poly(3-hydroxybutyrate) by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4 and Ralstonia pickettii T1.

The enzymatic degradability of chemosynthesized atactic poly([R,S]-3-hydroxybutyrate) [a-P(3HB)] by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pickettii T1 (PhaZ(ral)) and Acidovorax Sp. TP4 (PhaZ(aci)), defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were studied. The enzymatic degradation of a-P(3HB) by PhaZ(aci) depolymerase was confirmed from the results of weight loss and the scanning electron micrographs. The degradation products were characterized by one- and two-dimension (1)H NMR spectroscopy. It was found that a-P(3HB) could be degraded into monomer, dimer, and trimer by PhaZ(aci) depolymerase at temperatures ranging from 4 to 20 degrees C, while a-P(3HB) could hardly be hydrolyzed by PhaZ(ral) depolymerase in the same temperature range. These results suggested that the chemosynthesized a-P(3HB) could be degraded in the pure state by natural PHA depolymerase.

Biodegradation, Environmental↗

PubMedDate not supplied

Studies on comonomer compositional distribution of bacterial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)s and thermal characteristics of their factions.

The comonomer-unit compositional distributions have been investigated for bacterial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HH)] samples with 3HH unit content of 13.8, 18.0, 22.0, and 54.0 mol %. They were comonomer compositionally fractionated using chloroform/n-heptane mixed solvent at ambient temperature. The fractionation of P(3HB-co-18.0 mol %3HH) and P(3HB-co-22.0 mol % 3HH), which could not be carried out effectively at room temperature, were refractionated at 70 degrees C in the mixed solvent. Fractions with different 3HH unit content in a wide range (from 4.4 to 80.7 mol %) were obtained. By use of these fractions with narrow compositional distribution, the comonomer composition dependence of thermal properties was investigated by differential scanning calorimetry. The melting point (T(m)) and heat of fusion (DeltaH) decreased as the 3HH unit content increased in the range of low 3HH content (<40 mol %), while they increased as the 3HH unit content increased in the high 3HH content range (>70 mol %). The minimum T(m) and DeltaH values were found to exist at 3HH unit content of about 60 mol %. The glass transition temperature (T(g)) decreased linearly with the increase of 3HH unit content. The values of T(m), DeltaH, and T(g) of P(3HB-co-3HH)s were compared with those of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-3-hydroxypropionate), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate), and the effects of comonomer types on the thermal properties were revealed.

3-Hydroxybutyric Acid↗

PubMedDate not supplied

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.

Acyltransferases↗

PubMedDate not supplied

Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer.

Recombinant Ralstonia eutropha capable of producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer [P(3HB-co-3HHx)] from fructose was engineered by introduction of genes for crotonyl-CoA reductase (CCR) from Streptomyces cinnamonensis (ccrSc) and for PHA synthase and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (phaC-JAc). In this recombinant strain, C6-acyl-CoA intermediates were provided via beta-ketothiolase-mediated elongation of butyryl-CoA, which was generated from crotonyl-CoA by the function of CCR. The recombinant strain could accumulate the copolyester up to 48 wt % of dry cell weight with 1.5 mol % of 3HHx fraction from fructose, when the expression of ccrSc under the control of the PBAD promoter was induced with 0.01% L-arabinose. The absence of L-arabinose or the deletion of ccrSc from the plasmid resulted in accumulation of poly(3-hydroxybutyrate) homopolymer, indicating the critical role of CCR in the formation of the 3-hydroxyhexanoate unit. Higher CCR activity obtained by the addition of a larger amount of L-arabinose did not affect the composition but reduced the intracellular content of the copolyester. The P(3HB-co-1.5 mol % 3HHx) copolyester produced from fructose by the recombinant R. eutropha showed relatively lower melting temperatures (150 degrees C and 161 degrees C) and lower crystallinity (48 +/- 5%) compared to those (175 degrees C and 60 +/- 5%) of P(3HB) homopolymer. It has been found that the incorporation of a small amount (1.5 mol %) of 3HHx units into P(3HB) sequences leads to a remarkable change in the solid-state properties of P(3HB) crystals. The present study demonstrates the potential of the engineered pathway for the production of copolyesters having favorable characteristics from inexpensive carbon resources.

3-Hydroxybutyric Acid↗

PubMedDate not supplied

Isolation and characterization of polyhydroxyalkanoates inclusions and their associated proteins in Pseudomonas sp. 61-3.

Two types of polyester inclusions of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] were isolated from crude extract of Pseudomonas sp. 61-3. Proteins associated with each inclusion were separated by SDS-PAGE. PHA synthase 1 (PhaC1(Ps)), PhaF(Ps), and PhaI(Ps) were identified from P(3HB-co-3HA) inclusions by N-terminal amino acid sequences analyses, as well as PHB synthase (PhbC(Ps)) and 24-kDa unknown protein were identified from P(3HB) inclusions. The structural genes of PhaF(Ps) and PhaI(Ps) were located downstream of the pha locus. The relative PHA/PHB synthase activities of each inclusion were measured for various 3-hydroxyacyl-coenzyme As of 4-12 carbon atoms. Direct atomic force microscopy observation of P(3HB) and P(3HB-co-3HA) inclusions demonstrated that the two types of inclusions had different morphologies.

Acyltransferases↗

PubMedDate not supplied

Molecular mobility and phase structure of biodegradable poly(butylene succinate) and poly(butylene succinate-co-butylene adipate).

Molecular mobility and phase structure of biodegradable poly(butylene succinate) (PBS) and poly(butylene succinate-co-20 mol % butylene adipate) [P(BS-co-20 mol % BA)] have been investigated by high-resolution solid-state (13)C NMR. For both samples, two components with different (13)C spin-lattice relaxation time (T(1C)) values have been observed in the crystalline region. The crystalline component with shorter T(1C) value is assignable to the interface near amorphous phase. The crystalline component with longer T(1C) value is ascribed to the inside of the crystalline region. On the basis of T(1C), it has been concluded that the BA units are not included in the crystalline region of P(BS-co-20 mol % BA). Molecular mobility and higher-ordered structure of amorphous phase have been also compared between the melt and solid state. Variable-temperature high-resolution (13)C NMR measurements for the amorphous phase have revealed the remarkable difference in dynamics and structure between the melt and solid state.

Adipates↗

PubMedDate not supplied

Genetic analysis and characterization of poly(aspartic acid) hydrolase-1 from Sphingomonas sp. KT-1.

Sphingomonas sp. KT-1 hydrolyzes poly(aspartic acid) (PAA) containing alpha- and beta-amide units and has at least two different types of PAA hydrolases. The PAA hydrolase-1 hydrolyzes selectively beta-beta amide units in PAA. Molecular cloning of PAA hydrolase-1 from Sphingomonas sp. KT-1 has been carried out to characterize its gene products. Genetic analysis shows that the deduced amino acid sequence of PAA hydrolase-1 has a similarity with those of the catalytic domain of poly(3-hydroxybutyric acid) (PHB) depolymerases from Alcaligenes faecalis AE122 and Pseudomonas lemoignei. Site-specific mutation analysis indicates that (176)Ser is a part of a strictly conserved pentapeptide sequence (Gly-Xaa-Ser-Xaa-Gly), which is the lipase box, and plays as an active residue.

Amino Acid Sequence↗

PubMedDate not supplied

Oxidative degradation of cis- and trans-1,4-polyisoprenes and vulcanized natural rubber with enzyme-mediator systems.

Oxidative degradation of cis- and trans-1,4-polyisoprenes by two types of enzyme-mediator systems, lipoxygenase/linoleic acid and horseradish peroxidase/1-hydroxybenzotriazole, was investigated at 37 degrees C in aqueous media and analyzed by gel permeation chromatography. Lipoxygenase and horseradish peroxidase activate their substrates, linoleic acid and 1-hydroxybenzotriazole, respectively, for scission of main chains of both 1,4-polyisoprenes. Molecular weights of 1,4-polyisoprenes decreased during the treatment under both enzyme-mediator systems, and the depolymerization was completely inhibited by the addition of butylated hydroxytoluene. When the enzyme or the mediator from a reaction system was omitted, depolymerization did not progress, indicating that the scission of polymer chain is induced by the radicals generated only in the presence of both enzyme and mediator. Fenton reagent with linoleic acid was also effective against the degradation of both 1,4-polyisoprenes. Vulcanized natural rubber latex gloves were treated under these three methods, and surface degradation with hole formation was observed with a scanning electron micrograph.

Chromatography, Gel↗

PubMedDate not supplied

Correlation between structure of the lactones and substrate specificity in enzyme-catalyzed polymerization for the synthesis of polyesters.

Small-size (4-membered) and medium-size (5-, 6-, and 7-membered) unsubstituted lactones as well as unsubstituted macrolides (12 and 13 membered) were subjected to the ring-opening polymerization using the extracellular PHB depolymerase from Alcaligenes faecalis T1 (PhaZ(Afa)). The characteristic reactivities of the lactones were discussed based on a tertiary structure model of the active site of the PhaZ(Afa). With respect to the ring-size of the lactones, the 4-membered beta-propiolactone and 6-membered delta-valerolactone (delta-VL) showed the highest polymerization activity, and delta-VL seemed to be the upper size limit for the molecular recognition of the narrow active site cleft of PhaZ(Afa). On the other hand, epsilon-caprolactone, 11-undecanolide, and 12-dodecanolide, which showed excellent polymerization activities by lipases, were scarcely polymerized by PhaZ(Afa). This was ascribed to the difference in the recognition sites between PhaZ(Afa) and lipase. In addition, the effect of the substrate-binding domain of PhaZ(Afa) and the enantioselective ring-opening polymerization of (R,S)-beta-butyrolactone ((R,S)-beta-BL) were studied. The substrate-binding domain lacking PhaZ(Afa) showed higher reactivities than PhaZ(Afa) for the polymerization of the lactones and that a significant enantioselectivity was observed at the early stage of the polymerization of (R,S)-beta-BL to produce the (R)-enriched optically active poly(3-hydroxybutyrate).

Acyltransferases↗