Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli

Polyhydroxyalkanoate (PHA) synthase (PhaC) from Wautersia eutropha was expressed in a wide range of production level in Escherichia coli XL1-Blue cells and its effects on PhaC activity, poly[(R)-3-hydroxybutyrate] [P(3HB)] production and its molecular weights were investigated. The production level of PhaC was controlled both by the amount of chemical inducer (isopropyl-β-d-thiogalactopyranoside, IPTG) added into the medium and the use of different copy number of plasmids. In a flask experiment, as PhaC production level in the cells increased, the PhaC activity also increased in the range of low PhaC concentration. However, PhaC activity did not further increase in the range of high PhaC concentration, probably due to the formation of inclusion body in the cells. The molecular weight of P(3HB) was found to decrease with increasing PhaC activity. This trend was also verified in high cell density cultivation using 10-l jar fermentor. Furthermore, we demonstrated that the use of low copy number plasmid and appropriate induction of PhaC expression were effective in achieving both high productivity and high molecular weight of P(3HB).     

A simple and sensitive method for the determination of hydroxylamine in fresh-water samples using hypochlorite followed by gas chromatography.

A new and simple method for the determination of hydroxylamine in environmental water, such as fresh rivers and lakes using hypochlorite, followed by its gas choromatographic detection, has been developed. A glass vial filled with sample water was sealed by a butyl-rubber stopper and aluminum cap without head-space, and then sodium hypochlorite solution was injected into the vial through a syringe to convert hydroxylamine to nitrous oxide. The head-space in the glass vial was prepared with 99.9% grade N2 using a gas-tight syringe. After the glass vial was shaken for a few minutes, nitrous oxide in the gas-phase was measured by a gas chromatograph with an electron-capture detector. The dissolved nitrous oxide in the liquid-phase was calculated according to the solubility formula. The proposed method was applied to the analysis of fresh-water samples taken from Iu river and Hii river, flowing into brackish Lakes Nakaumi and Shinji, respectively.     

Selective capture of 1α,25-(OH)2-previtamin D3 utilizing polymer-supported trialkylsilyl triflate in the synthesis of 1α,25-(OH)2-vitamin D3

Catch and release method utilizing polymer-support was investigated in the separation of 1α,25-(OH)₂ pre- and provitamin D₃. Polymer-supported alkyldiisopropylsilyl triflate selectively captured the previtamin D₃ from a 26:74 mixture of pre- and provitamin D₃ produced by photoisomerization of provitamin D₃.     

Friction coefficient and structural transition in a poly(acrylamide) gel

The friction coefficient between the polymer network of an opaque poly(acrylamide) gel and water is measured as a function of the mole fraction of cross linker. The friction coefficients of opaque gels are 4 to 5 orders of magnitude smaller than those of the transparent gels. This drastic decrease in friction occurs when the mole fraction of cross linker is 0.2. In opaque gels, the friction coefficient of gels and the mole fraction of cross linker are related by a power law. The network structure of the opaque gels used in the friction measurements is examined with a confocal laser scanning microscope. The opaque gel network consists of a fractal aggregate of colloidal particles. The radius of particles and the volume occupied by the particles depend on the mole fraction of cross linker. Both relationships are well described by the power laws. The power law of the friction coefficient is well explained in terms of the power laws of the structural parameters and the Stokes equation of the hydrodynamic friction for the spherical particle. It indicates that the friction of the opaque gel is determined simply by the structure of the polymer network.     

A total synthesis of spiruchostatin A

We achieved the total synthesis of the histone deacetylase inhibitor spiruchostatin A, as the prelude to the preparation of a combinatorial library of its analogues. Two key reactions were an asymmetric acetate aldol reaction using a Zr-enolate and macrolactonization using the Shiina method.     

Microstructure and Viscosity of Aggregating Colloids under Strong Shearing Force

Disaggregation under strong shearing force is simulated for an aggregating colloid based on a sticky particle model which can describe the disaggregating and aggregating kinetics, the deformation, and the rupture of clusters with a minimum number of parameters. For a 2-dimensional system, the viscosity and coordination number of the model colloid are calculated at each time step, and the changes of microstructure with shear flow are observed directly by displaying the configuration of particles onto a monitor. The viscosity depends on both area fraction and shear rate, but coordination number depends only on shear rate. Furthermore, the viscosity and coordination number at steady state are independent of the initial state of particles, which indicates that the disaggregation and aggregation are mutually reversible. Copyright 1999 Academic Press.     

Chiral ligand-controlled asymmetric conjugate addition of lithium amides to enoates

The external chiral ligand-controlled asymmetric conjugate addition reaction of lithium amides with alpha,beta-unsaturated esters provided beta-amino esters in high yields and high enantioselectivities.     

Chemo-enzymatic Synthesis of Novel Oligo-N-acetyllactosamine Derivatives having a β(1-4)–β(1-6) Repeating Unit by Using Transition State Analogue Substrate

Chitinase-catalyzed hydrolytic and transglycosylating behavior of 1,2-oxazoline derivative of N-acetyllactosamine (LacNAc-oxa) 1 has been investigated. An extremely rapid hydrolysis (ring-opening of the oxazoline moiety) could be observed, suggesting that 1 behaves as a transition state analogue substrate for chitinase A1 (Bacillus circulans WL-12). This disaccharide monomer 1 was found to polymerize under basic conditions, giving rise to novel oligosaccharides having a β(1-4)–β(1-6) repeating unit in the main chain. The degree of polymerization of the resulting oligosaccharides was up to 5. This is the first example of enzymatic glycosylation reaction forming a β(1-6) bond catalyzed by chitinase.