The time profile of an enzymatic reaction at an early stage is generally considered to be linear. We observed that the time profile of the hydrolysis of 1 mM p-nitrophenyl-β-D-glucoside (pNP-Glc) by a β-glucosidase obtained from Agrobacterium tumefaciens (Cbg1) was not linear before 5% of the substrate was consumed, even though the initial concentration of the substrate was much higher than its Km value. The time profiles obtained with higher concentrations of pNP-Glc suggested that the time profile was the function of the absolute concentration of p-nitrophenol (pNP). The addition of various alcohols made the time profile linear. A self-transferring product inhibition model was constructed in which the pNP generated during the hydrolysis acts as an acceptor substrate to inhibit the hydrolysis. The theoretical curve agreed well with the experimental data.
Family 1 carbohydrate-binding module (CBM1) is widely distributed in fungal cellulases and proteins related to cellulose degradation. It plays an important role in adsorbing proteins and enhancing enzymatic degradation at liquid-solid interfaces. In this study, we constructed a fusion protein (CBM1-GFP) consisting of the CBM1 and a green fluorescent protein, and used this to develop a simple method of regulating CBM1 adsorption onto cellulose, which is available for enzyme recycling. We were able to control the amount of adsorbed CBM1-GFP within the range of 5-65% by increasing the concentration of acetate buffer. The same adsorption behavior was also seen in the recombinant Trametes hirsuta endo-glucanase (rEG) containing the CBM1, but not observed in cellobiohydrolase I (rEx-1). The amount of rEG adsorbed was significantly correlated to its hydrolytic activity against crystalline cellulose. After adsorption in 100 mM acetate buffer, the proteins were desorbed with water at 45°C. At this stage, the amount of adsorbed CBM1-GFP, rEx-1 and rEG decreased to 35, 20 and 7%, respectively. This regulation system can be easily combined with existing saccharification and enzyme recycling processes.
Potato cultivars originating from the Andes region of South America, France, Japan and North America are becoming increasingly popular with consumers in Japan. The objective of this study was to investigate the physicochemical and structural properties of the starches isolated from twelve potato cultivars from various regions of the world. In addition, the correlation among the starch properties was examined in order to gain a better understanding of this research. The average granule size ranged from 30.7 to 39.3 μm, and was negatively correlated with the peak gelatinization temperature by DSC and the pasting temperature by RVA. The phosphorus content ranged from 614 μg/g starch (dry weight) to 833 μg/g starch (dry weight), and the phosphorus content in four starch samples originating from France was lower than in all of the other samples. The swelling power (70°C) was negatively correlated with the pasting temperature by RVA and apparent amylose content, and positively correlated with the breakdown by RVA. The solubility (70°C) was negatively correlated with the peak viscosity and the breakdown by RVA. The digestibility after 24 h exposure to pancreatin was negatively correlated with the gelatinization temperature (the peak gelatinization temperature by DSC and pasting temperature by RVA), and positively correlated with the swelling power (70°C). It was observed many digested starch granules with rough surfaces and fine wrinkles by FE-SEM. The endo-corrosion on the surface of the starch sunk into the granules and made up of layered structures.
In a cellulose-producing bacterium Gluconacetobacter xylinus, endo-β-1,4-glucanase and β-glucosidase are suggested to have an important role in cellulose biosynthesis, and genes encoding these enzymes are present adjacent to the cellulose synthesis operon. However, in other cellulose-producing bacteria including Asaia bogorensis, β-glucosidase gene is not found next to the cellulose synthesis operon, and their cellulose productivities are lower than that of G. xylinus. To investigate whether A. bogorensis produces a similar type of β-glucosidase, crude enzyme fractions were prepared from G. xylinus and A. bogorensis. The hydrolytic activities toward p-nitrophenyl-β-D-glycosides were compared. In A. bogorensis, the hydrolytic activities were found in the cytosolic fraction, but the fraction did not indicate an ability to produce gentiobiose that is suggested to be an inducer for expression of the endo-β-1,4-glucanase gene in G. xylinus. We conclude that the mechanism of cellulose biosynthesis regulated by endo-β-1,4-glucanase may be different in A. bogorensis and G. xylinus.
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