Toshiaki Komaki

Pretreatment with Ammonia Water for Enzymatic Hydrolysis of Corn Husk, Bagasse, and Switchgrass

Bagasse, corn husk, and switchgrass were pretreated with ammonia water to enhance enzymatic hydrolysis. The sample (2 g) was mixed with 1–6 mL ammonia water (25–28% ammonia) and autoclaved at 120°C for 20 min. After treatment, the product was vacuum-dried to remove ammonia gas. The dried solid could be used immediately in the enzymatic hydrolysis without washing. The enzymatic hydrolysis was effectively improved with more than 0.5 and 1 mL ammonia water/g for corn husk and bagasse, respectively. In bagasse, glucose, xylose, and xylobiose were the main products. The adsorption of CMCase and xylanase was related to the initial rate of enzymatic hydrolysis. In corn husks, arabinoxylan extracted by pretreatment was substantially unhydrolyzed because of the high ratio of arabinose to xylose (0.6). The carbohydrate yields from cellulose and hemicellulose were 72.9% and 82.4% in bagasse, and 86.2% and 91.9% in corn husk, respectively. The ammonia/water pretreatment also benefited from switchgrass (Miscanthus sinensis and Solidago altissima L.) hydrolysis.

Properties of chitosanase from Bacillus cereus S1

Abstract. Chitosanase from Bacillus cereus S1 was purified, and the enzymatic properties were investigated. The molecular weight was estimated to 45,000 on SDS-PAGE. Optimum pH was about 6, and stable pH in the incubation at 40°C for 60 min was 6–11. This chitosanase was stable in alkaline side. Optimum temperature was around 60°C, and enzyme activity was relatively stable below 60°C. The degradations of colloidal chitosan and carboxymethyl cellulose (CMC) were about 30 and 20% relative to the value of soluble chitosan, respectively, but colloidal chitin and crystalline cellulose were not almost hydrolyzed. On the other hand, S1 chitosanase adsorbed on colloidal chitin completely and by about 50% also on crystalline cellulose, in contrast to colloidal chitosan, which it did not adsorb. S1 chitosanase finally hydrolyzed 100% N-deacetylated chitosan (soluble state) to chitobiose (27.2%), chitotriose (40.6%), and chitotetraose (32.2%). In the hydrolysis of various chitooligosaccharides, chitobiose and chitotriose were not hydrolyzed, and chitotetraose was hydrolyzed to chitobiose. Chitobiose and chitotriose were released from chitopentaose and chitohexaose. From this specificity, it was hypothesized that the active site of S1 chitosanase recognized more than two glucosamine residues posited in both sides against splitting point for glucosamine polymer.

Production of β-Mannanase and β-Mannosidase from Aspergillus awamori K4 and Their Properties

Abstractβ-Mannanase and β-mannosidase from Aspergillus awamori K4 was produced by solid culture with coffee waste and wheat bran. The optimum composition for enzyme production was 40% coffee waste–60% wheat bran. Two enzymes were partially purified. Optimum pH was about 5 for both enzymes, and optimum temperature was around 80°C for β-mannanase and 60–70°C for β-mannosidase. These enzymes produced some oligosaccharides from glucomannan and galactomannan by their hydrolyzing and transferring activities. β-Mannanase hydrolyzed konjak and locust bean gum 39.1% and 15.8%, respectively. Oligosaccharides of various molecular size were released from glucomannan of konjak, but on the addition of cellulase, mannobiose was released selectively. In locust bean gum, tetra-, tri-, and disaccharides (mannobiose) were mainly released by K4 β-mannanase. Tetra- and trisaccharides were heterooligosaccharides consisting of galactose and mannose residues. K4 β-mannosidase had a transglycosylation action, transferring mannose residue to alcohols and sugars like fructose.

Biological Pretreatment with Two Bacterial Strains for Enzymatic Hydrolysis of Office Paper

The cellulose-hydrolyzing strains, Sphingomonas paucimobilis MK1 and Bacillus circulans MK2, were separated from soil and were grown together in a single culture plate. Growth B. circulans MK2 in liquid culture required symbiosis with S. paucimobilis MK1. Biological pretreatment with the combined strain suspension after the liquid culture improved enzymatic hydrolysis of office paper from municipal wastes. Sugar recovery by S. paucimobilis MK1 (51%) was 1.4 times higher than that of the untreated sample (30%) and in the strain combination with B. circulans MK2, recovery was further improved by 2.5 times (75%). The sugar recovery in maximum condition was enhanced up to 94% for office paper. Furthermore, biological pretreatment effects were confirmed for more than 1 day less time. In X-ray diffraction patterns for the crystallinity of cellulose in office paper changed after biological pretreatment, the crystallinity was increased in comparison to that in untreated paper. The mechanism of biological pretreatment effect was explained by the fact that the strain acted as an endoglucanase, which hydrolyzes amorphous areas randomly.

Production of Fructooligosaccharides by β-Fructofuranosidases from Aspergillus oryzae KB

Aspergillus oryzae KB produces two types of beta-fructofuranosidases: F1 and F2. F1 produces the fructooligosaccharides (FOSs) 1-kestose, nystose, and fructosyl nystose from sucrose through a transfructosylation action, whereas F2 mainly hydrolyzes sucrose to glucose and fructose. F1 and F2 enzymes were more selectively produced from the KB strain in liquid media with a sucrose concentration>2% and <2%, respectively. Immobilization using an anion-exchange resin (WA-30; polystyrene with tertiary amine) and cross-linking with glutaraldehyde depressed the hydrolysis reaction of F2 (high hydrolyzing enzyme) alone and enhanced the thermal stability of F1 (high transferring enzyme). F1 enzyme produced in the high sucrose medium was immobilized, cross-linked, and packed in a tubular reactor for continuous production of FOSs (24.6% 1-kestose, 21.6% nystose, 5.7% and fructosyl nystose). In a long-term operation in which 60% sucrose was imputed at 55 degrees C, the composition of FOSs produced was 51.9% (transfer ratio: 92%), and production by the immobilized enzyme was maintained for 984 h.

Purification and properties of extracellular β-mannanases produced by Enterococcus casseliflavus FL2121 isolated from decayed Konjac

Abstract Two β-mannanases (M-I and M-II) were purified from the culture filtrate of Enterococcus casseliflavus FL2121 by ammonium sulfate precipitation, column chromatography of DEAE-Toyopearl 650M and Phenyl-Toyopearl 650M and preparative polyacrylamide gel electrophoresis to homogenities. The molecular weights of M-I and M-II were estimated to be 142,000 and 137,000 by SDS-polyacrylamide gel electrophoresis and 127,000 and 113,000 by gel filtration, respectively. M-I and M-II exhibited an optimum pH at 6.0 and an optimum temperature at 50°C. The enzymes were activated slightly by CoCl 2 and MnCl 2 , and inhibited strongly by AgNO 3 , HgCl 2 , EDTA, and N -bromosuccinimide. The K m values of M-I and M-II for konjac β-1,4-glucomannan were 0.14 and 0.30 (mg/ml), and the maximum velocities were 1,110 and 1,700 (U/mg protein), respectively. Both enzymes were endo-type β-mannanases hydrolyzing mannosides larger than β-1,4- d -mannotetraose.

Effects of Various Surfactants on Rheological Properties of Maize Starch Granules

ABSTRACT In this study, the formation of complexes between surfactants and the helical chains of amylopectins was confirmed. Nonionic surfactants with hydrophobic and hydrophilic groups of appropriate size and chemical structure enhanced the swelling and gelatinization processes of starch granules. Hydrophobic groups form complexes with the amylose and linear chains of amylopectin by becoming inserted into the hydrophobic inner area of the helical structures. The hydrophilic groups help the approach of the hydrophobic groups into the hydrated molecular chains and thus aid the formation of the complex. Among the anionic surfactants tested, SDS and sodium n-decyl benzenesulfate caused maximum swelling and gelatinization peaks. The average length of the amylopectin exterior chains is almost the same as that of the hydrophobic chains of SDS (16.9 A) and of sodium decyl benzenesulfate (18.2 A). This suggests that these anionic surfactants form rigid complexes with the exterior of the amylopectin by fitting the...

Production of L-arabinose from corn hull arabinoxylan by Arthrobacter aurescens MK5 α-L-arabinofuranosidase.

Arabinoxylans, which are comprised of a xylan backbone to which are attached glycosyl units that are primarily L-arabinofuranosyl units, are ubiquitous among plant species where it is a constituent of the cell wall. Arabinoxylan has attracted much attention as a potential biomass resource and L-arabinose has recently been reported to possess functional properties that are effective in the treatment of diabetes. Here, we report an α-L-arabinofuranohydrolase, isolated from the soil microbe Arthrobacter aurescens strain MK5, effective in releasing L-arabinose from corn hull arabinoxylan. When A. aurescens strain MK5 was grown in a liquid medium, corn hull arabinoxylan, which has a higher arabinose content (Ara/Xyl = 0.6) than oat spelts xylan (Ara/Xyl = 0.12), induced more efficient arabinoxylan hydrolase production. Analysis of enzyme activity in the culture broth revealed that arabinoxylan hydrolase activity was high, and α-L-arabinofuranosidase and β-xylosidase activities were low. The optimum pH of the MK5 arabinoxylan hydrolase at 40 °C was around 7 and enzyme activity was relatively stable at an alkaline pH up to 9.5. The optimum temperature at pH 7 was around 50 °C and enzyme activity was stable under 50 °C. During the hydrolysis of corn hull arabinoxylan, only L-arabinose was released and 45.1% maximum sugar recovery was achieved. The A. aurescens MK5 enzyme was a typical arabinoxylan α-L-arabinofuranohydrolase and was most effective at releasing L-arabinose from corn hull arabinoxylan, which has a high arabinose content. This enzyme may have important industrial applications.

Pasting Characteristics of Maize Starch Heat-Treated with Different Water-Ethanol Mixtures

ABSTRACT Pasting characteristics of maize starch heat-treated with six different water-to-ethanol ratios (%wt base 0:100, 10:90, 20:80, 30:70, 40:60, 50:50) were investigated; treated starches were called EW 0, 10, 20, 30, 40, and 50, respectively. Endotherms in DSC analysis shifted to a higher temperature as the water content in water-ethanol mixture increased. The removed amount of fatty acids was much higher in treatments for EW 10, 20, and 30. The RVA peak viscosity of EW 10 and 20 were highest among the treated starches and setbacks were more than twice that of untreated starch. The characteristic change in the RVA viscogram corresponded to the amount of leached amylose from the granule. EW 30 displays similar properties as conventional heat-moisture-treated starch, but maintained a higher viscosity of ≈300 RVU throughout the heating process. In treatment with water-ethanol mixtures, heat-moisture treatment and defatting effects generated new types of modified starches. EW 40 and 50 had no clear past...

Studies on Enzymatic Liquefaction and Saccharification of Starch:Part VII. On the Content of Insoluble Starch Particles in Some Types of Starch and Increase of These Materials by Treatment under Several Conditions

Sweet potato starch, available commercially, was found to contain originally about 25 mg% of the insoluble starch particles (ISSP), which could not be liquefied with bacterial alpha-amylase under the best known liquefaction process and remained as the residue.Some of the sweet potato starch which is considered to be difficult to liquefy in dextrose manufacture, often contains about 60 mg% of this material. The different species of starch are arranged in the increasing order to the ISSP content as follows: potato starch < sweet potato starch < rice starch < corn and wheat starch. The average amount of ISSP content in corn starch is about 229 mg%.By treating the sweet potato starch slurry at 55°~60°C, the ISSP content in the slurry was remarkably increased, and when potato starch (16% moisture) was heated in a pressure vessel with live steam of 115°~127°C, the ISSP content was increased to about 3000 mg%. ISSP content of sweet potato starch was also increased by the liquefaction with bacterial alpha-amylase...