Yasuyuki Tanaka

Microwave technique for efficient deacetylation of chitin nanowhiskers to a chitosan nanoscaffold.

A chitosan nanoscaffold in the form of a colloidal solution was obtained from the deacetylation of chitin whiskers under alkaline conditions by using a microwave technique in only 1/7 of the treatment time of the conventional method. Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance ((1)H NMR) techniques confirm the degree of deacetylation to be above 90% within 3 h. The wide-angle X-ray diffraction (WAXD) pattern clearly shows that the highly crystalline chitin whiskers are changed to amorphous chitosan. SEM micrographs show the aggregation of branched nanofibers, whereas the TEM micrographs reveal the scaffold morphology.

Cloning and characterization of farnesyl diphosphate synthase from the rubber-producing mushroom Lactarius chrysorrheus.

Farnesyl diphosphate is involved in rubber biosynthesis as an initiating substrate for both polyprenol and mushroom rubber. So far, we have isolated the cDNA of a farnesyl diphosphate synthase (FPS) for the first time from a rare rubber-producing mushroom, Lactarius chrysorrheus, by the degenerate RT-PCR technique based on sequence information of FPS genes from fungi and yeasts. The open reading frame was clarified to encode a protein of 381 amino acid residues with a calculated molecular weight of 42.9 kDa. The deduced amino acid sequence of L. chrysorrheus FPS showed about 50% identity with those of other fungi and yeasts as well as plants. We expressed the cDNA of L. chrysorrheus FPS in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein. The purified obtained protein showed FPS activity in which geranyl diphosphate (GPP) served as primary substrate, with a 2.4-fold higher kcat⁄Km value for GPP than for dimethylallyl diphosphate (DMAPP).

In vitro synthesis of high molecular weight rubber by Hevea small rubber particles

Hevea brasiliensis is one of few higher plants producing the commercial natural rubber used in many significant applications. The biosynthesis of high molecular weight rubber molecules by the higher plants has not been clarified yet. Here, the in vitro rubber biosynthesis was performed by using enzymatically active small rubber particles (SRP) from Hevea. The mechanism of the in vitro rubber synthesis was investigated by the molecular weight distribution (MWD). The highly purified SRP prepared by gel filtration and centrifugation in the presence of Triton((R)) X-100 showed the low isopentenyl diphosphate (IPP) incorporation for the chain extension mechanism of pre-existing rubber. The MWD of in vitro rubber elongated from the pre-existing rubber chains in SRP was analyzed for the first time in the case of H. brasiliensis by incubating without the addition of any initiator. The rubber transferase activity of 70% incorporation of the added IPP (w/w) was obtained when farnesyl diphosphate was present as the allylic diphosphate initiator. The in vitro synthesized rubber showed a typical bimodal MWD of high and low molecular weight fractions in GPC analysis, which was similar to that of the in vivo rubber with peaks at around 10(6) and 10(5) Da or lower. The reaction time independence and dependence of molecular weight of high and low molecular weight fractions, respectively, indicated that the high molecular weight rubber was synthesized from the chain extension of pre-existing rubber molecules whereas the lower one was from the chain elongation of rubber molecules newly synthesized from the added allylic substrates.

Gel Formation in Natural Rubber Latex: 1. Effect of (NH4)2HPO4 and TMTD/ZnO Additives

Abstract The rubber from commercially obtained high-ammonia latex (commercial HA-latex) increased in gel content significantly after long preservation of the latex with 1.0% w/v tetramethylthiuram disulfide (TMTD) and zinc oxide (ZnO). Deproteinization of the HA-latex did not decrease the gel content. The gel fraction of deproteinized commercial HA-latex (DPHA-latex) was not solubilized by toluene containing 1.0% ethanol, showing that the gel fraction is composed of chemically crosslinked rubber. The addition of (NH4)2HPO4, which is usually added to fresh latex (FL-latex) to remove excess amounts of Mg2+ ions by centrifugation, decreased the gel formation in FL-latex preserved with 0.6 % v/v NH4OH due to the removal of Mg2+ ions. The excess amounts of (NH4)2HPO4 accelerated the gel formation in preserved FL-latex and commercial HA-latex. The addition of 0.1% w/v TMTD/ZnO to preserved FL-latex treated with 5% w/v (NH4)2HPO4 caused an increase of gel content during storage. The gel formation in the commerci...

Gel formation in natural rubber latex: 2. Effect of magnesium ion

Abstract The effect of Mg2+ ions on the gel formation in fresh and commercial high ammonia natural rubber latices (FL-latex and commercial HA-latex) was analyzed from the gel content and 2+ content after treatment with (NH4)2SO4. The gel content of rubber from commercial HA-latex decreased significantly after (NH4)2SO4 treatment comparable to that of FL-latex. Long-storage commercial HA-latex containing 50% gel fraction showed no decrease in 2+ content after (NH4)2SO4 treatment. This gel fraction was not solubilized in toluene by the treatment of a proteolytic enzyme in latex or ethanol/toluene mixed solvent extraction of rubber. The 2+ content of rubber in long-storage commercial HA-latex, 0.005% (w/w rubber), decreased after treatment with (NH4)2SO4, while the same treatment showed little change on FL-latex, 0.035%. The toluene soluble fraction of these latices showed a decrease in the Mn value with an increase in the (NH4)2SO4 concentration. The gel content of FL- and HA-lattices increased with an incr...

Characterization of Hevea brasiliensis rubber from virgin trees : A possible role of cis-polyisoprene in unexploited tree

Abstract The role of rubber in Hevea brasiliensis was analyzed based on the structural analysis of rubber in rubber trees as latex. The rubber obtained from a mature-tree which has never tapped before, so-called “virgin mature-tree” (Vir-NR), contained the gel fraction higher than 80%, while that from a regularly tapped mature-tree (Reg-NR) was less than 3%. The gel showed almost the same structure as a crosslinked rubber prepared from fresh latex in the presence of peroxide, with the molecular weight between crosslinks (Mc) of 3×103. This value is extremely low comparing with that of 7×105 observed for the soft-gel in rubber from high-ammonia latex and Pale crepe. The sol fraction from Vir-NR was an oxidative degraded product containing aldehyde and epoxide groups. Aging properties of Vir-NR are much worse than those of Reg-NR. This indicates that rubber accepts free-radicals to form C-C crosslinking and partly oxidative scission of main-chains during storage in laticiferous tubes of Hevea trees. This su...

Effects of Nonrubber Components on the Crystallization Behavior of Natural Rubber

The crystallization behavior of highly purified natural rubber (NR) was investigated by polarized light microscopy. NR was purified by enzymatic deproteinization followed by transesterification to remove nonrubber components present in the rubber. Nucleation rate and growth rate were estimated from a number of spherulites of different sizes observed in the course of crystallization for the purified NR. The slope of a linear line drawn in a plot of the logarithmic growth rate vs. 1/TΔT for the purified rubber was identical to that for the untreated rubber, suggesting that the lateral surface free energy was not a function of the nonrubber components. In contrast, nucleation was found to be promoted in the presence of nonrubber components, such as the fatty acids. This demonstrated that the crystallization of NR was significantly promoted by the nucleating effect of the fatty acids.