Yoshitsune Shin-ya

Effects of N-acetylation degree on N-acetylated chitosan hydrolysis with commercially available and modified pectinases

Three types of N-acetylated chitosans (NACs) with different degrees of acetylation (DA) were prepared and used as a substrate for enzymatic hydrolysis with a commercially available pectinase and a modified one. Pectinase modification was conducted using polyalkyleneoxide-maleic anhydride copolymer (PEO-MA copolymer). The effects of DA on enzymatic reaction with native and modified pectinases were investigated experimentally. Initial hydrolysis rate and Michaelis-Menten kinetic parameters were measured by analysis of reducing sugars. DA of NAC strongly affected the hydrolytic characteristics of native and modified pectinases. N-acetylation of chitosan increased the initial hydrolysis rate and the enzyme-substrate affinity with respect to both pectinases: NACs with DA over 0.3 showed high initial hydrolysis rate and strong affinity between enzyme and substrate. Especially, when NAC with DA over 0.3 was treated with modified pectinase, the affinity became much stronger than the native pectinase.

Study on the Recovery of Phosphorus from Waste-Activated Sludge Incinerator Ash

Abstract Several batch studies that were made up of the acid extraction and the solvent extraction were performed to recover phosphorus from the waste-activated sludge (WAS) incinerator ash. In the acid extraction, the extraction efficiency of phosphorus relied on the acid type, liquid(acid)-to-solid (LacidS) ratio, and acid concentration. Phosphorus in the WAS incinerator ash was completely extracted by 1 M HCl at the LacidS ratio of 6.4:1. Subsequently, the solvent extraction was conducted to separate and concentrate phosphorus further from the acid extract. The efficiency of solvent extraction was affected mainly by the solvent type, liquid (solvent)-to-liquid (the acid extract) (Lsolv Lacid) ratio, and hydrogen ion concentration. Under the appropriate condition, 76% of phosphorus in the acid extract was extracted to 1-butanol phase, which corresponded to 80.1% as the mass fraction of phosphorus to total elements. Prior to the solvent extraction, the addition of bis (2-ethylhexyl) phosphoric acid (D2EHPA), which was available for removing aluminum from the acid extract, led to an additional increase in the term of the mass fraction of phosphorus to total elements. Overall results indicated that phosphorus in the WAS incinerator ash could be efficiently recovered and be a potential renewable resource.

Optimum conditions for the precipitation of chitosan oligomers with DP 5-7 in concentrated hydrochloric acid at low temperature

Chitosan was partially hydrolysed with 35% hydrochloric acid for 2 h at 80°C and the hydrolysate stored at 20°C after dilution with water to precipitate higher molecular weight (MW) chitosan oligomers. When the hydrolysate was not diluted with water, no precipitate was formed but 7.3% chitosan oligomers were precipitated at a dilution ratio of 1.0 (ml water:ml hydrolysate). The time for precipitation was not significantly changed after storing the hydrolysate at 20°C for 1 day. In addition, the precipitation yield was not significantly influenced by the concentration of HCl used for the hydrolysis except at less than 5.0 (ml HCl:g chitosan). However, the yield of precipitated oligomers changed with partial hydrolysis time. For 0.5 and 2 h hydrolysis, 10.1 and 7.3% of the oligomers were precipitated, respectively, but only 3.1% of the oligomers were obtained after a 4 h reaction. When methanol was added to the hydrolysate, the precipitation yield increased up to 70% but the amounts of lower MW chitosan oligomers in the precipitated oligomers also increased with the increase of higher MW. The precipitated oligomers were mainly composed of pentamers and hexamers.

Enzymatic activity of PEOMA-modified lysozyme obtained from degradation of Micrococcus luteus cell and N-acetylated chitosan

A chicken egg white lysozyme was modified with a polyethyleneoxide-maleic anhydride (PEOMA) copolymer and two types of substrates, Micrococcus luteus (ML) cell walls and N-acetylated chitosan (NAC), which were used to determine the enzymatic activity of the PEOMA-modified lysozymes with a different degree of modification (DM). The ML cell walls and NAC were used as the substrates in their heterogeneous and homogeneous states, respectively. Modified lysozymes whose DM values ranged from 0 to 70% were used for both substrates. The enzymatic activity of the PEOMA-modified lysozyme drastically decreased at a DM from 40 to 50% in both cases, and an identical relationship between the enzymatic activity and the DM were observed. From these results, it is obvious that the enzymatic activity of PEOMA-modified lysozymes can be determined by using the two substrates, without considering the state of the substrate.

Peroxidase chemically attached on polymeric micelle and its reaction with phenolic compounds

Horseradish peroxidase was chemically modified with comb-shaped polymaleic anhydride-alt-1-tetradecene (PMA-TD) in microemulsion systems to produce surface-active peroxidase that has capability to form micellar structures in aqueous solutions and can be concentrated at liquid/liquid interfaces without unfolding of the enzyme. For chemical modification oil-in-water (O/W) and water-in-oil (W/O) microemulsion systems composed of n-butyl acetate and a buffer solution were prepared because n-butyl acetate turned out to be less detrimental to the activity of peroxidase at high degree of modification compared to other organic solvents. The modification degree of amine groups on the surface of peroxidase by maleic anhydride groups on PMA-TD was reached at equilibrium after 1h reaction at 0°C, and 42% of amine groups were modified with 7-fold amount of PMA-TD to peroxidase (wt/wt). The activity of the PMA-TD-modified peroxidase measured with 2,4-dichlorophenol at pH 7.0 was increased by approximately 2-fold compared to native peroxidase. There was no significant shift in optimum pH after modification, and optimum pH measured with 2,4-dichlorophenol was observed at pH 7.0. For all six phenolic compounds tested, there was a significant increase in the reaction efficiency with the PMA-TD-modified peroxidase. The remarkable enhancement of the reaction efficiency by the modification was presumably because of micellar structures of PMA-TD that could concentrate hydrophobic phenolic oligomers into the core of the micelles. Overall, horseradish peroxidase chemically attached to the surface of PMA-TD micelles was found to be significantly effective for the oxidative polymerization of phenolic compounds.