Various kinds of partial hydrolysates of waxy maize amylopectin were prepared by controlling the debranching actions of immobilized and free isoamylase, and free pullulanase, and then frac tionated into three fractions by a gel chromatography using Toyopearl HW-65F . The smallest frac tions (fr. 3) were again debranched with free isoamylase and analyzed by HPAEC-PAD in order to obtain information of chain distributions existing in the outer portion of amylopectin molecules. About 10-15% of the partial hydrolyses were appropriate to collect the shortish chains released from amylopectin. The chain distribution of the smallest fraction consisted of many short-chains with DP 6-15, and it could be thought that most of the shortish chains might be A chains derived from outer portions of amylopectin. The comparative experiments among the debranching enzymes led us to confirm that partial hydrolyzed materials obtained by the low enzyme activity of free isoamylase and pullulanase could be used to study the outer chains of amylopectins like immobilized isoamylase.
Based on kinetic studies of the sulfite dehydrogenase-catalyzed reaction investigated using the subcellular fraction instead of the microbe Thiobacillus thiooxidans, it was confirmed that the initial velocity is very proportional on the concentration of the subcellular fraction and the optimal pH is 7.5, which is the same as that obtained in the microbe. The kinetic parameters, Michaelis constant (Km) and maximum velocity (V), were evaluated to be 1.7±0.2 mM and 0.083±0.035 mM/min/mg protein, respectively. The ferricyanide concentration examined was found to be proportional to the initial velocity within 1.5 mM. It was also found that cytochrome c can be absorbed as a second substrate in place of ferricyanide. Thus, a new device should be very possible using the subcellular fraction of T. thiooxidans as a highly sensitive and automatic sensor apparatus for sulfite etermination.
Banana pulp, irrespective of the degree of ripening, contained soluble and cell wall-bound forms of acid α-glucosidases (SAAG and BAAG). BAAG was released with a neutral buffer containing 0.2-2 M NaCI after extracting of SAAG with a salt-free buffer. From the same bunch of ripe yellow bananas, SAAG and BAAG were purified at 732-fold and 264-fold, respectively, using ConASepharose and Sephadex G-150 gel column chromatographies. The molecular weights of SAAG and BAAG were 70, 000 and 90, 000, respectively, by gel filtration. These enzymes were typical maltases that required maltose (G2) and malto-oligosaccharides (G3-G7) as substrates, but not isomaltose, treharose, sucrose, pullulan, glycogen, or soluble starch. The Vmax/Km ratios (apparent hydrolytic efficiencies) of SAAG and BAAG toward G5-G7 were 89-112 and 14-52%, respectively, with the ratio toward maltose being 100%. As for ripe yellow banana where cell wall or amy loplastic membrane has not been maintained, it is speculated that BAAG could access starch and its hydrolytic products and would share in starch degradation in collaboration with amylases and SAAG.
The effect of substituting polished-graded hard-type wheat flour for commonly milled hard-type wheat flour on the properties of dough and bread was studied. Commonly milled flour (CW) was also prepared from hard-type wheat cultivar 1 CW and breadmaking was carried out by the AACC method. Thirty (30 CW) or 50% (50 CW) graded-flour substitution for CW was tested for baking using the 70-40% layer fractions of the whole grain. The addition of pentosanase (PEN) or sucrose fatty acid ester (SE) to 30 CW distinctly increased the loaf volume or softness of the bread crumbs due to the small size of the gas cells and retarded retrogradation. Moreover, 30 CW with PEN or SE increased the enthalpy value of the starch. On farinogram, the addition of SE to 30 CW de creased water absorption, arrival and development times, while it increased the stability time as compared with that of CW alone. The viscoelasticity of 30 CW with SE showed larger values for all parameters than those of CW. The ratio between resistance and extensibility of the 30 CW and SE dough increased significantly (after 90 and 135 min) as compared with CW on the extensogram. The substitution with graded flour alone or its combination with PEN or SE increased the rate of gas generation. Microscopic observation of the 30 CW dough with PEN or SE distinctly accelerated formation of the gluten network. The extensible gluten material could entirely cover the starch granules. From these results, the addition of graded flours and SE to CW might increase maturity of the dough. Furthermore, these additions were considered to control dispersion of the water in the dough and improve the dough and baking properties when compared with those of CW alone.
We analyzed the amino- and carboxyl-terminal amino acid sequences of five .α-amylases, Ba-L, Ba-S, pUXAI Amy, pUXA0 Amy, and pBLXA1 Amy, which originate from an α-amylase gene of Bacillus subtilis X-23. Ba-L and Ba-S were produced by wild-type strain B. subtilis X-23, while pUXAI Amy, pUXA0 Amy and pBLXA1 Amy were produced by Escherichia coli harboring dif ferent kinds of recombinant plasmids coding the α-amylase gene. Amino-terminal amino acid se quence analyses indicated that the signal peptides of Ba-L and Ba-S were the first 45 amino acid residues and those of pUXA1 Amy and pUXA0 Amy were the first 31 amino acid residues, while that of pBLXA1 Amy was the first 37 amino acid residues. Carboxyl-terminal sequence analyses showed that Ba-L and pUXA1 Amy contained the full length of the carboxyl-terminal-region polypeptide of the enzyme while Ba-S lacked 186 amino acid residues, and that pUXA0 Amy and pBLXA1 Amy lacked 88 and 189 amino acid residues, respectively, but had an extra 8 and 64 amino acid residues, respectively, originating from continuous translation of the vector region. All five enzymes showed the same characteristics with regard to molar activity, optimum temperature, optimum pH, and transglycosylation activity. These findings suggest that the amino- and carboxyl terminal regions of α-amylase have no effect on enzyme activity, but rather are flexible with regard to the truncation and addition of polypeptides.
Three distinct endo-xylanase components derived from Acremonium cellulase, a commercial cellulase product from a filamentous fungus Acremonium cellulolyticus, were extensively purified by consecutive column chromatographies and designated as xylanaseI, xylanase II and xylanase III. XylanasesI, IIand III were each homogeneous on both Native- and SDS-PAGE . The molecular weight (SDS-PAGE) and p1 values of xylanases I, II and III were 30 kDa and 5.1, 25 .5 kDa and 5.2, and 33.5 kDa and 5.7, respectively. The N-terminal amino acid sequences from the 1st up to the 25th residue of three enzymes were identical as follows: NH2-Ala-Glu-Ala-Ile-Asn-Tyr-Asn-Gln-Asn-Tyr-Ile-Ala-Ser-Gly-Ala-Asn-Val-Gln-Tyr-Ser-Pro-Asn-Ile-Ala-Ala-. Xylanases I, II and III had high, specific soluble xylan saccharification activities of 112.1, 86.1 and 74.8 U/mg of protein, respectively. The optimum pH and temperature for xylanases I, II and III were pH 3.5 and 55°C, pH 3.8 and 55°C, and pH 3.5 and 50°C, respectively. Xylanases I, II and III were completely stable over the ranges of pH 3.5-9.5 at 25°C for 2 h and at temperatures below 55°C, pH 3.0-9.5 and below 55°C, and pH 2.5-9.5 and below 50°C, respectively. The enzymes were almost completely inhibited by 1 mM KMnO4 and partially by 1 mM SDS, PCMB, Zn2+and Ag+ Substrate specificities and kinetic parameters for these enzymes were precisely examined. The enzymes were characterized as endo-type xylanases on the basis of their action patterns on soluble xylan and xylooligosaccharides. Xylanases I, II and III seem to be isoforms judging from N-terminal amino acid sequences and basic physicochemical and enzymatic properties as well as substrate specificities.
An isomalto-dextranase from Arthrobacter globiformis T6 was kinetically elucidated to be capa ble of splitting α-1, 4-glucosidic linkage of panose as well as α-1, 6-glucosidic linkage of isomalt otriose. The kinetic features of the experiments with the mixed substrates of isomaltotriose and panose, the linearity of Lineweaver-Burk plots, the dependence of the apparent maximal velocities on the mole fraction (f) of isomaltotriose in the mixed substrates, f = [isomaltotriose]/([isomaltotriose]+ [panose]) were in good agreement with those expected for a single catalytic site mecha nism. The enzyme is accompanied by isopullulanase activity, by which pullulan is endolytically hy drolyzed to release isopanose mainly. The isomalto-dextranase expressed by the recombinant E. coli cells also produced isopanose from pullulan. It was for the first time confirmed genetically that the enzyme had inherently isopullulanase activity.
Graded flour was prepared from the hard-type wheat grain 1 CW by the polished-grading method and the effect of the flour quality on the dough and baking properties was studied. Thegraded flours contained large amounts of ash or dietary fiber as compared with a conventionally milled hard-type 1 CW flour (CW). These graded flours also had larger amounts of K, Ca and Fe than CW. All the graded flours, except for the outermost layer, distinctly increased in water-soluble pentosan as compared with CW. Moreover, they contained higher β-amylase or diastatic activities and also higher amounts of damaged starch than CW. The specific volumes of the bread made from the graded flours were distinctly smaller than that of CW . None of the graded flours alone had a favorable effect on the bread firmness. For the viscoelastic properties, all parameters of the graded flours tested distinctly higher than those of CW. Microscopic observations of the graded flour dough alone indicated that the co-existence of bran could not produce a favorable gluten ma trix to result in bread of a good quality. These results suggested that graded flour alone could not make dough with the desired baking properties, because of the high amounts of diastatic activity, damaged starch, dietary fiber or wheat bran.
The α-glucan phosphorylase gene from Thermus aquaticus was isolated using partial amino acid sequences of purified enzyme. The identity of the gene was confirmed by expression in Es cherichia coli resulting in thermostable glucan phosphorylase activity. The open reading frame of this gene consisted of 2460 by and encoded a polypeptide of 819 amino acids. The deduced amino acid sequence exhibits high identity (32-43%) to 7 putative and 2 characterized glucan phosphorylases, but showed weak similarity to other well characterized glucan phosphorylases from various sources. Due to its high expression level and thermal stability, the recombinant enzyme was easily purified from E. coli cell extracts, and employed to characterize its activity. The smallest primer molecule for a synthetic reaction was maltotriose and the smallest effective substrate for a degrada tion reaction was maltotetraose. These results suggest that T. aquaticus glucan phosphorylase, and at least 9 other enzymes, form a new group of glucan phosphorylases whose structure and substrate specificity differ from the traditional glucan phosphorylases. The purified enzyme was also em ployed to investigate the effect of temperature and pH on the activities in both directions. The ac tivity exhibited a pH optimum of 8.0 for phosphorolytic reaction but 7.0 for synthesis reaction. The optimum temperature for phosphorolytic reaction was 80-85°C, while the one for synthesis reaction was 50°C.
It is successively reported by organizations which investigates economic trends that Japanese business is looking up in 2000. Contrary to these announcements, however, the actual economic situation is still sluggish. In the food industry where we are involved, the over-all situation doesn't show any growing trends though there is some gap among companies. The business stagnation can be perceived according to the purchase trends in the distribution sector announced in September 2000. Under these circumstances, sugar alcohols are contributing to the development of consumer goods such as refreshing soft drinks, chewing gum, Japanese or Western confectionery, health foods and processed foods. In addition, new sugar alcohols, which have recently been developed, suggest new functions for the production of consumer goods. Typical ones are Erythritol and Xyli tol. Our duty is to discover how we can efficiently communicate the functions of these sugar alco hols to the makers of consumer goods and assist them with their development of consumer goods having new functions. This report is about the market strategy from this viewpoint.