Kiyoshi Mura

Purification and Characterization of an α-Amylase of Pichia burtonii Isolated from the Traditional Starter “Murcha” in Nepal

Among more than 20 yeast strains isolated from the traditional starter “murcha” in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an α-amylase. The purified enzyme, named Pichia burtonii α-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 °C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 °C for 30 min. The activity was inhibited by metal ions such as Cd2+, Cu2+, Hg2+, Al3+, and Zn2+.

Effects of Peanut-Skin Procyanidin A1 on Degranulation of RBL-2H3 Cells

Peanut skin contains large amounts of polyphenols having antiallergic effects. We found that a peanut-skin extract (PSE) inhibits the degranulation induced by antigen stimulation of rat basophilic leukemia (RBL-2H3) cells. A low-molecular-weight fraction from PSE, PSEL, also had inhibitory activity against allergic degranulation. A main polyphenol in PSEL was purified by gel chromatography and fractionated by YMC-gel ODS-AQ 120S50 column. Electrospray ionization mass spectrometry (ESI-MS) analysis of the purified polyphenol gave m⁄z 599 [M+Na]+. Based on the results of 1H-NMR, 13C-NMR spectra, and optical rotation analysis, the polyphenol was identified as procyanidin A1. It inhibited the degranulation caused by antigen stimulation at the IC50 of 20.3 μM. Phorbol-12-myristate-13-acetate (PMA) and 2,5,-di(tert-butyl)-1,4-hydroquinone (DTBHQ)-induced processes of degranulation were also inhibited by procyanidin A1. These results indicate that peanut-skin procyanidin A1 inhibits degranulation downstream of protein kinase C activation or Ca2+ influx from an internal store in RBL-2H3 cells.

Hypocholesterolemic Effect of Peanut Skin and Its Fractions: A Case Record of Rats Fed on a High-Cholesterol Diet

Peanut skin (PS) is characterized by almost exclusively consisting of polyphenols and fiber. We fractionated PS into a water-soluble fraction (WSF) and water-insoluble fraction (WIF), and further fractionated WSF into a soluble dietary fiber fraction (DF) and dietary fiber-free, water-soluble fraction (DFF-WSF). Male Sprague-Dawley rats were fed on high-cholesterol diets supplemented with PS and its fractions. PS, WSF, and DFF-WSF decreased the serum lipid and cholesterol levels and increased those in feces. This effect was probably due to the polyphenols that inhibited intestinal cholesterol absorption.

Roles of Residues Cys69, Asn104, Phe160, Gly232, Ser237, and Asp240 in Extended-Spectrum β-Lactamase Toho-1

ABSTRACT Toho-1, which is also designated CTX-M-44, is an extended-spectrum class A β-lactamase that has high activity toward cefotaxime. In this study, we investigated the roles of residues suggested to be critical for the substrate specificity expansion of Toho-1 in previous structural analyses. Six amino acid residues were replaced one by one with amino acids that are often observed in the corresponding position of non-extended-spectrum β-lactamases. The mutants produced in Escherichia coli strains were analyzed both for their kinetic properties and their effect on drug susceptibilities. The results indicate that the substitutions of Asn104 and Ser237 have certain effects on expansion of substrate specificity, while those of Cys69 and Phe160 have less effect, and that of Asp240 has no effect on the hydrolysis of any substrates tested. Gly232, which had been assumed to increase the flexibility of the substrate binding site, was revealed not to be critical for the expansion of substrate specificity of this enzyme, although this substitution resulted in deleterious effects on expression and stability of the enzyme.

Molecular Cloning and Characterization of an α-Amylase from Pichia burtonii 15-1

An α-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii α-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis α-amylase, 58% with Saccharomycopsis sp. α-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the Km values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the kcat values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the kcat/Km values of PBA were higher.

Distribution of Prx-linked Hydroperoxide Reductase Activity among Microorganisms

Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670–27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

Serum cholesterol reduction by feeding a high-cholesterol diet containing a lower-molecular-weight polyphenol fraction from peanut skin.

Feeding a high-cholesterol diet with a water-soluble peanut skin polyphenol fraction to rats reduced their plasma cholesterol level, with an increase in fecal cholesterol excretion. The hypocholesterolemic effect was greater with the lower-molecular-weight rather than higher-molecular-weight polyphenol fraction. This effect was possibly due to some oligomeric polyphenols which reduced the solubility of dietary cholesterol in intestinal bile acid-emulsified micelles.

Gene cloning and characterization of a Bacillus vietnamensis metalloprotease.

A Bacillus vietnamensis metalloprotease (BVMP) with high affinity toward collagen was isolated and purified from the culture supernatant of Bacillus vietnamensis 11-4 occurring in Vietnamese fish sauces. The BVMP gene was cloned and its nucleotide and coded amino acid sequences determined. BVMP consists of 547 amino acid residues, with the zinc-binding sites conserved in common metalloproteases. It shares 57% amino acid identity with thermolysin originating from Bacillus thermoproteolyticus. The three-dimensional structure of BVMP was deduced by computer-aided modeling with the use of the known three-dimensional thermolysin structure as a template. Like thermolysin, BVMP cleaved the oxidized insulin B-chain at the peptide bonds involving the N-terminal sides of hydrophobic and aromatic amino acids. BVMP also showed high hydrolytic activity toward gelatin, collagen, casein, and elastin, especially toward the skeletal proteins at increased NaCl concentration. The high activity was found to be due to enhanced affinity to the substrates. Kinetical data on BVMP indicated that the Km values for the hydrolysis of Cbz-GPGGPA as a collagen model decreased as the concentration of added NaCl increased. Some contribution of this enzyme during the aging of fish sauces at high salt concentrations can thus be expected.

Novel Angiotensin I-Converting Enzyme Inhibitory Peptides Found in a Thermolysin-Treated Elastin with Antihypertensive Activity

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC50 value of 531.6 µg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 µg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC50 value of 244 µM against ACE and may have potential use as a functional food.

Characterization of the quality of imbibed soybean at an early stage of pre-germination for the development of a new protein food item.

This was a pilot study carried out to develop a new protein food item from imbibed soybean before germination. It identified the significance of a short stage after imbibition and before germination, and that vitamin C production was activated in as little as 16 h from the start of imbibition, without any influence on the soy protein quality or sensory acceptability, while longer imbibition caused the imbibed soybean to activate its phytophysiological metabolism for germination. DNA microarray analysis indicated that the genes for carbohydrate metabolism were up-regulated prior to 16 h, and that the expression rates of genes responsible for environmental factors were down-regulated. Thereafter, the expression rates of the genes associated with lipid metabolism and secondary metabolite production were changed. This information should contribute to a better understanding of how to develop a new soy protein item in pre-germination before active physiological processes begin.