Yoshiro Hatanaka

Enzyme-Free Quinone Crosslinking Reaction for Proteins: A Macromolecular Characterization Study Using Gelatin

To mimic the quinone hardening of extracellular proteins in invertebrates, we investigated an enzyme-free crosslinking of gelatin by HQ in a neutral aqueous phase. The mixture was rapidly transformed to a yellowish brown, thermally and mechanically stable hydrogel in the presence of a simple copper(II) salt. A dehydrated thin film made of the mixture was flexible, tough, and showed a large ultimate breaking force. Physicochemical examination of the gel suggested that the basic amino acid residues (lysine, hydroxylysine, and histidine) of the protein were modified by the quinone ring to form 2-6 crosslinks per protein. The enzyme-free crosslinking reaction is discussed with consideration of a copper(II) ion-catalyzed oxidation of HQ and the hydroquinone/protein adducts.

Asymmetric Reduction of Hydroxyacetone to Propanediol in Immobilized Halotolerant Microalga Dunaliella parva

Cells of the halotolerant microalga Dunaliella parva immobilized in calcium-alginate gel showed morphological characteristics and photosynthetic activity similar to those of free cells. The photosynthetic activity in immobilized cells fell only about 20% during 2 weeks incubation at 5 degrees C in the light. The asymmetric reduction of hydroxyacetone to (R)-propanediol was first examined in batch reactions with both free and immobilized cells. Since the activity of immobilized cells was as high as that of free cells, the optimum conditions for the column reactor were investigated using immobilized cells. The productivity of propanediol in immobilized cells increased in proportion to increases in the light intensity, suggesting that NADPH regenerated through photosynthesis was used for the asymmetric reduction. Electron microscopic observation of thin sections of immobilized cells after the reaction suggested that NADPH used for asymmetric reduction in the dark was produced through the metabolism of starch granules. The use of a CaCO3-free medium in the column reactor markedly prolonged the period of reductive activity and photosynthesis.

Sensitivity of the surface coat of the halotolerant green alga Dunaliella parva (Volvocales, Chlorophyceae) to lysozyme

The surface coat of Dunaliella parva Lerche was investigated using several techniques. Degradation by several cell lytic enzymes and ultrastructural observation revealed that D. parva has a specialized cell surface structure containing a glycoprotein that is sensitive not only to proteinases but also to lysozyme. This sensitivity was also demonstrated by electrophoresis of the cells and measurement of released glycerol after enzyme treatment. Immunochemical labeling indicated that the surface glycoprotein of D. parva is analogous to pepti‐doglycan.

Isolation and Characterization of Carboxylesterase from Vinyl Acetate-Assimilating Bacterium Isolated from Soil

Abstract A bacterium Pseudomonas species which is able to assimilate vinyl acetate and other esters as a sole source of carbon was isolated from soil. The bacterium contained three kinds of esterases (esterases I, II and III) which were separable on DEAE-cellulose column chromatography. Esterase II was purified and characterized. The enzyme is a monomer with the molecular weight of 27,000 and it hydrolyzed various esters most efficiently at pH 8.0. The activity of the enzyme was inhibited by diisopropylfluorophosphate. The purified enzyme was different from other esterases so far found in Pseudomonas sp. with respect to molecular structure and substrate specificity.

Reduction of hydroxyacetone with NADPH regenerated through photosynthetic pathway of the halotolerant alga Dunaliella parva

Abstract Regeneration of NADPH via photosynthetic pathway was applied to the development of a novel bioreactor in which NADPH is required for the desired reaction. In a preliminary experiment using dihydroxyacetone reductase (DHAR) from Dunaliella parva (D. parva) , hydroxyacetone (HA, Acetol) was reduced to yield ( R )-(−)-1,2-propanediol (( R )-PD) with an optical purity of 75%ee. The bioconversion using D. parva whole cells in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) showed that the NADPH required for this reaction can be generated by photosynthesis. The optimum reactor conditions for propanediol (PD) production were 50–100 mM HA at 25°C. The production rate of PD increased proportionally with increasing irradiation intensity up to 220 μE·s −1 ·m −2 . Although changes in cell density did not affect the productivity, agitation of the reaction mixture at 200 rpm raised the productivity. Since the amount of PD produced was almost the same as the amount of HA consumed, it was confirmed that more than 95% of the HA was converted to PD without turning into any by-products or being degraded. The PD productivity in the photobioreactor system using Dunaliella cells was comparable to that in the yeast cell system.

Isolation of disproportionating enzyme from halotolerant microalga Dunaliella parva (Volvocales, Chlorophyceae)

Starch metabolism in Dunaliella parva Lerche is regulated by the osmolarity of the surrounding solute. Two isozymes showing amylolytic activity were obtained after purification by gel filtration chromatography. The isozymes show disproportionating activity (D‐enzyme) that is specific for malto‐oligosaccharides as substrate. Properties of the D‐enzyme in D. parva are similar to those in higher plants. The activity of the D‐enzyme is also found in various Dunaliella and Chlamydomonas, indicating that the D‐enzyme is also important in the starch metabolism in algae.