Atsuo Kimura

Reaction of enzymes with starch granules: kinetics and products of the reaction with glucoamylase

Abstract The reaction of glucoamylase with starch granules from seven botanical sources (waxy maize, maize, barley, tapioca, amylomaize-7, shoti, and potato) and with four potato starches modified with acid in four types of alcohols (methanol, ethanol, 2-propanol, and 1-butanol) were studied using three concentrations of enzyme (2, 20, and 200 units mL −1 ). The kinetics of the formation of d -glucose were followed over 32 h for the three enzyme concentrations. The starches showed a wide degree of variance in their susceptibility to enzyme hydrolysis. They divided into three groups: waxy maize starch was the most susceptible being converted into 50, 95, and 98% d -glucose in 32 h for the three concentrations of enzyme, respectively; an intermediate group (barley, maize, and tapioca starches) was converted into 10–15, 60, and 75–80% d -glucose in 32 h for the three concentrations of enzyme, respectively; and the third and least susceptible group (amylomaize-7, shoti, and potato starches) was converted into 2–8, 9–16, and 13–21% d -glucose in 32 h for the three concentrations of enzyme, respectively. The percent conversion for the modified potato starches was proportional to the amount of enzyme and the degree of modification. The 100X (200 U mL −1 ) amount of enzyme gave 13, 17, 21 and 27% d -glucose in 32 h for potato starch modified in the four alcohols, respectively. The number and size of the granules were determined over 32 h of reaction using 10X (20 U mL −1 ) enzyme for waxy maize starch and 100X enzyme for the other starches. The number of granules for waxy maize, barley, maize, and tapioca starches significantly increased in the first part of the reaction and then decreased; this was paralleled by a decrease of 40–50% in the size of the granules. The number and size of the potato and shoti starch granules did not change very significantly and the number and size of amylomaize-7 starch granules decreased slightly but steadily to about 25% of the number and size of the native granules. The morphologies of the granules during reaction were studied by scanning electron microscopy. Extensive conversion into 50% d -glucose for the starches in the first two groups showed the classical “Swiss cheese” shell structure with many deep holes into the granules. The least susceptible starches in the third group did not show much change in morphology, except for some minor surface pitting.

A sialic acid-binding lectin from the mushroom Hericium erinaceum

A lectin was isolated from the mushroom Hericium erinaceum. This lectin is composed of two different subunits of 15 and 16 kDa and the molecular mass of the intact lectin was estimated to be 54 kDa by gel filtration. It exhibits specificity towards sialic acids, especially N‐glycolylneuraminic acid.

A lectin from mycelia of the fungus Ganoderma lucidum

A lectin (GLL-M) was isolated from mycelia of Ganoderma lucidum using affinity chromatography on BSM-Toyopearl. GLL-M is a monomer in its native form with a M(r) of 18,000. Another lectin was also purified from fruiting bodies of the same fungus. The two lectins were partially compared with each other.

Cloning and sequencing of an α-glucosidase gene from Aspergillus niger and its expression in A. nidulans

We have cloned an extracellular alpha-glucosidase gene from Aspergillus niger with oligonucleotide probes synthesized on the basis of the determined peptide sequences. The nucleotide sequence revealed an open reading frame of 985 amino acids split with three introns, and the deduced amino acid sequence was nearly identical to that of the alpha-glucosidase previously determined. The cloned gene was introduced into Aspergillus nidulans, and its expression in the transformants was shown to be regulated by the carbon sources in the medium, suggesting that a common regulatory expression system is shared by these two species as is the case of other starch-degrading enzymes of Aspergillus species.

A novel cyclotetrapeptide produced by Lactobacillus helveticus as a tyrosinase inhibitor

Abstract A novel cyclotetrapeptide cyclo(- l -Pro- l -Tyr- l -Pro- l -Val-) was isolated from the lactic bacterium Lactobacillus helveticus . The structure was determined by spectroscopic means and confirmed by acid hydrolysis of the peptide and sequence analyses of the resulting linear peptides.

Two N-acetyl-d-galactosamine-specific lectins from Phaeolepiota aurea

Two lectins, PAL-I and PAL-II, were isolated from the mushroom Phaeolepiota aurea by affinity chromatography on acid-treated Sepharose CL-4B followed by reverse-phase FPLC on ProRPC. Both of the lectins were tetramers of 16 kDa subunits. The lectins had little agglutination activity against native erythrocytes but Pronase treatment of erythrocytes increased the sensitivity to agglutination by the lectins. Both lectins exhibited slight preferences for type A compared with type B and O erythrocytes. In haemagglutination inhibition assays, N-acetylgalactosamine and both anomers of methyl N-acetylgalactosaminide were the best inhibitors.

Cloning of Corticium rolfsii glucoamylase cDNA and its expression in Saccharomyces cerevisiae

A cDNA coding for the glucoamylase of Corticium rolfsii AHU 9627 was cloned using synthetic oligonucleotide probes that code for inner amino acid sequences of the purified enzyme. This clone (CG 15) is 1900 base pairs long and contains the entire coding region for a polypeptide of 579 residues. Comparison with amino acid sequences of other fungal glucoamylases showed homologies of 35%–56%, and most homology with that of Aspergillus niger. The expression plasmid pACG 115 was constructed by introduction of the coding region of CG 15 into a yeast expression vector pAAH 5, containing the promoter and terminator of alcohol dehydrogenase (ADH1). Saccharomyces cerevisiae AH 22, containing the recombinant plasmid pACG 115, acquired starch-saccharifying ability.