Three commercial enzyme preparations of glucose oxidase (GOD) showed an oxidizing ability of glucuronic acid and galacturonic acid to form sugar acids. The optimum conditions of GOD reaction were at pH 8.0 and 50°C, whereass those of uronic acid oxidase (UOD) reaction were at pH 3.5 and 40°C. In spite of extensive attempts to separate GOD from UOD, the isolation of each activity was unsuccessful, and UOD reaction resulted from the wide substrate specificity of GOD reaction. More than 6-fold higher Km values for glucuronic acid and galacturonic acid than for glucose might suggest that UOD reaction was the alternative reaction of GOD enzyme. However, a more definite conclusion on UOD activity should be drawn from further studies. The reaction products from uronic acids were analyzed by HPLC and paper chromatography, and some products were shown to be identical with sugar acids.
The apparent distribution coefficients of glucose and fructose to cation-exchange resin in lithium, sodium, potassium, or calcium form at 25° were measured at various concentrations in a single component system. Both coefficients in any ionic form depended on their concentrations and became large at higher concentrations. A method to estimate the binding constant of fructose to a counterion was proposed under the assumption of there being no complex formation between glucose and the counterion, and the constant of fructose to each counter-ion was evaluated. The constant for the calcium ion was larger than those for other counter-ions.
In this study we attempted to control some specified metal-ion concentrations in a liquid medium and found a highly raw starch-digesting glucoamylase (RSDG) producing culture with protease-less activity, which we have called a metal-ion regulated liquid medium . Rhizopus sp. MKU 40 could not grow and produce glucoamylase (GA) in the liquid medium without metal ions (SLS medium). The addition of iron, magnesium, and zinc ions in the liquid medium was essential to growth and GA production of Rhizopus sp. MKU 40. Calcium ions also stimulated its growth and GA production. To summarize, the addition of 75 ppm calcium ions, 2 ppm iron ions, 49 ppm magnesium ions, and 0.7 ppm zinc ion to the SLS liquid medium was the best condition for producing GA and resulted in a high specific activity of GA (21.9 U/mg protein). Specific RSDG activity of the crude enzyme was 5.7 U/mg protein, and the ratio of RSDG activity to GA activity was 0.26. Since the purification was easy, the purification of GA was done in only two steps (ammonium sulfate precipitation and column chromatography on CM-Sephadex C-50) . The enzyme was purified about 4 .8-fold based on specific GA activity with an 86.0% yield based on GA activity from culture supernatant . The enzyme was proved to be homogeneous as judged by SDS-PAGE, and it has a molecular mass of about 80.4 kDa by comparison of its relative mobility on SDS-PAGE with those of standard proteins . For purified enzymes, the ratio of RSDG activity to GA activity was 0.16.
The effect of amaranth flour substitution on some rheological properties of wheat flour dough and bread was studied by using a home baker. At 5% amaranth flour substitution, the loaf volume was significantly larger than the control; thereafter it decreased distinctly as the amount of amaranth flour substitution increased. The addition of more than 1250 U hemicellulase per kg of flour to the 10% amaranth flour-substituted wheat flour increased the loaf volume significantly . Farinograph data showed that the stability time of the wheat flour dough decreased with 10% amaranth flour substitution . Based on the viscoelastic parameters, such as compression stress, modulus of elasticity, and viscosity coefficients, the 10% amaranth flour-substituted wheat flour doughs were distinctly harder than the control dough. Scanning electron microscopic observations showed that the gluten of the amaranth flour-substituted wheat flour dough seemed to be slightly rigid . The addition of calcium stearoyl-2-lactylate (CSL) and/or hemicellulase to the 10% amaranth flour-substituted wheat flour clearly increased volume of the bread loaf.
We researched into the characteristic of retrogradation of cooked rice treated by pressure after soaking. By β-amylase pullulanase (BAP method), the degree of gelatinization of the nonpressure treated rice immediately after cooking was 95.7% and 79.1% after 5 days. On the other hand, the degree of gelatinization of the pressure-treated rice immediately after cooking was 98.4% and 92.9% after 5 days. Retrograded rice was scanned by a Differential Scanning Calorimeter (DSC) .ΔH was 14.3 mJ/mg to the cooked rice immediately after cooking, and 10.4 mJ/mg to the rice treated by pressure. Furthermore, with the storage time ΔH in the rice came closer to ΔH in the rice flour with water. But in this case, d H in the pressure-treated cooked rice was more lower than d H in the nonpressure-treated cooked rice. The results of Near Infrared Reflectance (NIR) analysis were as follows: The first principal component score (coefficient of determination 90.5%) correlated to its storage term (R2=0.775). A difference between the pressure-treated and the nonpressure-treated ricewas observed by plotting the first and the fourth (c.d. 0.5%) principal component scores. With a value calculated by d H and a value measured by the BAP method as a purpose variable and the first through the fourth principal component scores as explanation variables, the multiple correlation analysis was calculated by the addition and reduction method. The fourth principal component score (R2=0.513)was primarily obtained, then the first principal component score (R2=0.818), and then the third principal component score (R2=0.865) significantly appeared. When the retrograded rice was heated with a microwave, the degree of gelatinization of nonpressure-treated cooked rice was 93.1 to 96.8%, but that of pressure-treated cooked rice was 100%. Even after 30 days, the retrogradation pressure treated rice showed a high degree of gelatinization by micro-wave heating.
The physicochemical properties of starches obtained from various parts of sago palm trunks at different growth stages were studied. Experimental materials were prepared from trees grown on a field at Dalat Kampung Teh in Sarawak, Malaysia. Trees in 7 different growth stages at ages ranging from 9 to 14.5 years were selected according to Jong's classification. Each tree in the respective growth stage was divided into 5 equal fractions, from root part (No. 1) to top part (No. 5), and a total of 35 fractions were obtained. The starch samples were prepared by Jong from these fractions. Observations by scanning electron micrography and X-ray diffraction analysis were conducted. Granular size distribution, viscosity, and texture of samples were determined, and the following results were obtained. The starch fractions from the tree aged 14.5 years and the upper parts of its trunk consisted of largely fine particles. A sharper X-ray diffraction pattern was observed in starch from the upper part of the trunk than in starch from the lower part. An increased sharpness of the X-ray diffraction pattern of starch from the upper part was observed as the growth period increased. The starches from a 9-year-old palm reached maximum viscosity of 137-151 RVU at 88-92°C, whereas starch from a 14.5-year-old palm reached maximum viscosity as high as 142-163 RVU at 76-7VC. Moreover, the texture of the latter's starch gels was firmest among the samples tested.
During the past few years great progress has been made in the understanding of the function of starch-degrading enzymes. Combining mutational analysis, multiple sequence alignment, and three dimensional structures of enzyme substrate analogue complexes formed the basis for developing amylolytic and related enzymes through protein engineering. In the case of barley a-amylase this has involved rational site-directed mutagenesis, regional random mutagenesis and isozyme sequence exchange as well as generation of isozyme chimeras. The approach has enabled modulation of enzyme specificity and identification of the functional roles of distinct residues and regions. The mutational analysis included the complex between barley α-amylase 2 and a proteinaceous inhibitor from barley seeds, BASI (barley a-amylase/subtilisin inhibitor). The interplay between the catalytic and the starch binding domains in glucoamylase from Aspergillus niger was investigated. The two domains are connected by a highly O-glycosylated linker and bind with 1:1 stoichiometry double-headed heterobifunctional synthetic analogues targeted to each domain. Glucoamylase is an inverting hydrolase and replacement of the catalytic base, a glutamic acid, by a cysteinesulfinic acid, gave rise to activity surpassing that of the wild-type enzyme, indicating a most surprising flexibility for the catalytic base.
Starchy materials such as cereals, legumes, tubers, and rhizomes are served as foods through the appropriate processing of cooking, which causes the gelatinization of starch. The gelatinized starch is thermodynamically unstable and tends to retrograde during storage. The retrogradation generally decreases the quality of foods known as the staling of bread and the hardening of cooked rice. Therefore, how to control gelatinization and retrogradation is of prime concern of cereal chemists and technologists. I have been involved in this study for nearly 40 years. As early as 1961 we found that microwave heating was very efficient for the rapid cooking of rice, but the cooked rice rapidly hardened by the retrogradation of starch. This was improved by the addition of a small amount of Takaamylase in cooking. Root vegetables are well known for being unable to become soft through cooking even by continued heating after heating has been suspended for a while halfway through cooking. By a detailed analysis of the gelatinization of starch during cooking, this phenomenon was found to be caused by the enhancement of retrogradation of the partially gelatinized starch by cooling. Furthermore, it was found that the heat-stable materials, including various minerals, enhanced the retrogradation. The relationship between structures and functions was investigated on various kinds of starch such as nagaimo (yam), lotus, arrowhead, taro, bracken, and tapioca used in confectioneries. It was found that starches having a high tendency to retrograde had amylopectin with long chain-length and smaller amylose molecules. For example, tapioca starch showed the lowest retrogradation tendency, and this appeared to be due to the short chain length of amylopectin and large amylose molecules, besides thee low amylose content. The relatively higher retrogradation tendency of potato starch seemed to be due to the long chain length of amylopectin. A clear-cut relationship was found between the phosphate content and the pasting viscosity in potato starch that the higher level of phosphorus gave the higher-pasting viscosities. Kuzu amylopectin showed a relatively low retrogradation tendency because of the medium chain length of amylopectin, but kuzu amylose had a high retrograding tendency because of small molecules. Bracken amylopectin with a similar chain length showed a similar retrograding tendency to that of kuzu. Some taro starches showed similar structures and properties to tapioca starch, and they also had low levels of amylose. The starches found in vegetables used in cuisine on New Year's Day have low retrograding tendencies. Therefore they are suitable for use as preservable foods. Amylose molecules are slightly branched molecules on average, and the branches are mostly short chains, but the function of side chains has not been investigated. We attempted to clarify this function by means of a model experiment on molecules that had incorporated small amounts of maltosyl side chains into linear chains by the reverse action of isoamylase. The results clearly showed that these small amounts of maltose side chains affected a decrease in retrograding tendency.
Levan is a microbial polyfructan; constructed from β-2, 6-fructofuranosyl main chain and β-2, 1-fructof uranosyl linked side, chains. The enzymatic synthesis of levan has been well studied for several decades. No detailed report has been made, however, to describe the degradation of levan and levan degrading enzymes; most previous reports were on unpurified enzymes. This paper deals with screenings, enzymatic properties, and classification of newly obtained levanases.
A strain of Brevibacterium sp. No. 9605 was isolated from soil and produced cyclodextrin glucanotransferase (CGTase, EC. 18.104.22.168). The CGTase produced mainly γ-cyclodextrin (γ-CD) from sarch in the initial stage of reaction, but then, the proportion of β-CD was increased and also produced a small amount of α-CD. The CDs formation was affected by the presence of calcium ion and ethanol . In the presence of ethanol, the yield of γ-CD from soluble starch was increased . It was thought to be due to the repression of β-CD production from starch and the repression of γ-CD degradation by coupling reaction. Brevibacterium CGTase showed a broad acceptor specificity to various monosaccha rides and phenolic compounds and produced more transfer products of D-mannose, L-rhamnose, 1, 3-dihydroxybenzene, 1, 3, 5-trihydroxybenzene, 3-hydroxybenzyl alcohol, and (+) -catechin using as acceptors than Bacillus macerans and Bacillus stearothermophilus CGTases did. The transfer products of D-mannose and kojic acid were 4-O-α-D-glucosyl-D-mannose and kojic acid 7-O-α-D-glucoside, respectively.