Jun-ichi Abe

A periodic distribution of the chain length of amylopectin as revealed by high-performance anion-exchange chromatography

Abstract The chains of amylopectin over dp 80 were separated on baseline resolution by high-performance anion-exchange chromatography with pulsed amperometric detection under improved conditions, and the chain-length (cl) distributions of amylopectins from eleven plant sources were analyzed. The differences in amount of each chain between arrowhead (Sagittaria trifolia L. var. sinensis Makino) and other kinds of amylopectins exhibited periodic waves which divided the abscissa at intervals of dp 12, except those of edible canna and yam amylopectins, where the interval was of the order of dp 15. Accordingly, chain-length distributions were fractionated into fa, dp 6–12; fb1, dp 13–24; fb2, dp 25–36, and fb3, dp >37. Amylopectin with short and long cl n had large and small amounts of the fa fraction, respectively, and showed A and B type X-ray diffractions of starch granules. The amount of the fa fraction was suggested to play an important role in the determination of starch crystalline polymorphs.

Multiple forms of β-mannanase from Bacillus sp. KK01

Abstract Bacillus sp. KK01, which was isolated from soil, produced β-mannansse about 2 U ml−1 in culture medium. By DEAE-ion exchange column chromatography, four enzyme components F1, F2, F3, and F4 were separated. All components showed maximum activity at pH 7.1. F1 showed maximum activity at 60°C, which was 5–10°C higher than those of the rest. The four components F1, F2, F3, and F4 hydrolyzed copra mannan to the extent of 23–31%; yielded mannobiose, mannotriose, and mannotetraose as hydrolysis products; and also hydrolyzed locust bean gum to the extent of 19–22%, giving similar products.

Production of the raw-starch digesting amylase of Aspergillus sp. K-27

SummaryAspergillus sp. K-27, isolated from soil, produced extracellular glucoamylase and α-amylase using wheat starch as a carbon source, and its productivity was doubled by the addition of α-methyl-d-glucoside to the medium. The crude enzyme preparation, which was found to be a mixture of 70% glucoamylase and 30% α-amylase, well degraded not only cereal starches but also tuber and root starches, and the initial velocity for potato starch was 72% of that for corn starch.

Selection of microorganisms which produce raw-starch degrading enzymes

SummaryMicroorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, “α-amylase resistant starch (α-RS)”, which is insoluble in water and hardly digested with Bacillus amyloliquefaciens α-amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced α-amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced α-amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.

Synthesis of branched cyclomalto-oligosaccharides using Pseudomonas isoamylase

Branched cyclomalto-oligosaccharides (cyclodextrins) were synthesised from cyclomalto-oligosaccharides and maltose or maltotriose through the reverse action of Pseudomonas isoamylase. The reaction rate was greater with maltotriose than with maltose, and with increasing size of the cyclomalto-oligosaccharide (cG6 less than cG7 less than cG8). Maltotriose is effective as both a side-chain donor and acceptor, and three isomers of 6-O-alpha-maltotriosylmaltotriose (branched G6) were formed through mutual condensation, but maltose was effective only as a side-chain donor. Each branched cyclomalto-oligosaccharide and G6 was purified by liquid chromatography, and their structures were determined by chemical, enzymic, and 13C-n.m.r. spectroscopic analyses.

Action of glucoamylase from aspergillus niger on phosphorylated substrate

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger was purified to be free from alpha-amylase and phosphatase (glucose 6-phosphate as substrate). The phosphatase was well separated from the glucoamylase by phosphocellulose ion-exchange chromatography. The glucoamylase action was prevented by the esterified phosphate groups of the substrate. Thus, the extensive action of the glucoamylase on potato starch exposed the 6-posphorylglucosyl residue of the starch at the non-reducing terminal and large molecular weight limit dextrins remained. The concomitant action of the phosphatase was necessary for the complete degradation of the starch.

Retention behaviour of cyclodextrins and branched cyclodextrins on reversed-phase columns in high-performance liquid chromatography

Abstract The theoretical plate numbers and the asymmetry factors of eight columns packed with C18-bonded phases were measured to obtain a reliable indication of column performance and for column-to-column comparisons. Among those columns, including silicone-coated silica gel- and porous polymer gel-based new C18 columns, silica-based, endcapped and monomeric phase columns showed the best performance. Cyclodextrins (CDs) and branched CDs were separated satisfactorily on six C18 columns with 3–7% aqueous methanol. Furthermore, multi-branched CDs could be separated from their isomers having the same molecular size. The elution order of CDs and branched CDs on C18 columns with aqueous methanol may or may not coincide with their solubilities in the mobile phases.

Large-Scale Purification and Characterization of Barley Limit Dextrinase, a Member of the α-Amylase Structural Family

ABSTRACT Homogeneous barley limit dextrinase (LD) was isolated on a large scale in a yield of 9 mg/kg of 10-day germinated green malt. This represents a 9,400-fold purification and 29% recovery of the activity in a flour extract in 0.2M NaOAc (pH 5.0) containing 5 mM ascorbic acid. The purification protocol consists of precipitation from the extract at 20–70% saturated ammonium sulfate (AMS), followed by diethylaminoethyl (DEAE) 650S Fractogel anion-exchange chromatography, and affinity chromatography on β-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mMβ-cyclodextrin and contains a single polypeptide chain of 105 kDa (SDS-PAGE) and pI 4.3. Sequence analysis of tryptic fragments, prepared from 2-vinylpyridinylated LD and purified by RP-HPLC, identified short motifs recognized in β-strand 2, 3, and 5 characteristic of a catalytic (β/α)8-barrel domain of the α-amylase family of amylolytic enzymes. Barley LD has ≈50 and 85% sequence identity to bacterial pullulanases and rice starch de...

Enzymic syntheses of doubly branched cyclomaltoheptaoses through the reverse action of Pseudomonas isoamylase

Abstract Two and three new cyclomaltoheptaose (β-cyclodextrin, cG 7 ) derivatives, respectively, were identified among the products obtained by the action of Pseudomonas isoamylase on maltose and maltotriose, and cG 7 . They were 6 A ,6 D -di- O -α-maltosyl-cG 7 and 6- O -α-(6 2 - O -α-maltosyl)maltosyl-cG 7 , and 6 A ,6 D -di- O -α-maltotriosyl-cG 7 , 6- O -α-(6 3 - O -α-maltotriosyl)maltotriosyl-cG 7 , and 6- O -α-(6 2 - O -α-maltotriosyl)maltotriosyl-cG 7 . In addition, 6 1 - and 6 2 - O -α-maltosylmaltose were identified as mutual condensation products of maltose. Maltose was the smallest substrate to act as both an acceptor and a donor for the action of Pseudomonas isoamylase.

Expression of periplasmic α-amylase of Xanthomonas campestris K-11151 in Escherichia coli and its action on maltose

A gene encoding the periplasmic alpha-amylase of Xanthomonas campestris K-11151 was cloned into Escherichia coli using pUC19 as a vector. An ORF of 1578 bp was deduced to be the amylase structural gene. The primary structure of the enzyme had little identity with other alpha-amylases, except with the enzyme from Bacillus megaterium. The enzyme was expressed in E. coli from the lac promoter of pUC19 and was found to be transported to the periplasmic space. The expressed enzyme showed the same thermal stability, optimum temperature and substrate specificity as the enzyme from X. campestris. The enzyme formed maltotetraose, but not 6(1)- nor 6(2)-maltosyl-maltose, from maltose by the reverse reaction, and the tetraose was then hydrolysed to maltotriose and glucose. The addition of maltotriose enhanced the production of glucose from maltose. In addition, maltose was formed by the condensation of glucose by the enzyme. Thus, the periplasmic alpha-amylase of X. campestris was shown to produce glucose from maltose by hydrolysing maltotetraose and possibly higher maltooligosaccharides, which were the products of a condensation reaction, as a major pathway, and by direct hydrolysis of maltose as a minor pathway.