Takashi Shin

A Novel Laccase Inhibitor, N-Hydroxyglycine, Produced by Penicillium citrinum YH-31

A Novel Laccase Inhibitor, N-Hydroxyglycine, Produced by Penicillium citrinum YH-31 Sawao Murao, Yuji Hinode, Eiko Matsumura, Atushi Numata, Kenzo Kawai, Hirofumi Ohishi, Hisanori Jin, Hiroshi Oyama & Takashi Shin a Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan b Osaka University of Pharmaceutical Sciences, Matsubara, Osaka 580, Japan Published online: 12 Jun 2014.

Comparative studies of suidatrestin, a specific inhibitor of trehalases

Suidatrestin, isolated from a Streptomyces strain, was characterized as a new trehalase inhibitor. Its inhibitory potential was 7 to 50-fold higher than that of validamycin when tested against insect, fungal and mammalian trehalases. The kinetic properties of suidatrestin were studied in vitro with trehalases from flight muscle mitochondria of the fly, Protophormia terraenovae, from larval midgut of the moth, Spodoptera littoralis, and from porcine kidney, as well as with maltase from yeast. Suidatrestin was inactive on maltase but inhibited all trehalases with IC50 values of 0.08-0.1 microM; Ki values ranged from 0.02 to 0.05 microM. The very low Ki/K(m) ratios (3.9 x 10(-6) -4.9 x 10(-6)) indicated excellent in vitro inhibitory action of suidatrestin. When injected into larvae of S. littoralis, suidatrestin required high and repetitive doses which lead to reversible inhibition of larval growth only. Consecutive omission of the inhibitor even stimulated weight increase above that of controls. Significant mortality was achieved at a rather high dose only. Injection of a growth-inhibiting dose of suidatrestin did not change hemolymph osmolality as a measure of sugar concentration. The discrepancy between in vitro and in vivo potency of suidatrestin may be understood once its chemical structure is fully known.

Pepstatin-Insensitive Carboxyl Proteinases

As reported previously [1, 2], we succeeded in isolating Scytalidium lignicolum in 1972 [3], which produced S-PI (Pepstatin Ac) [4]-insensitive carboxyl proteinases. This strain produced four distinct carboxyl proteinases: A-1, A-2, B, and C [5–7]. None of them were inactivated by S-PI, diazoacetyl-DL-norleucinemethylester (DAN) [8], and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) [9]. However, as an exception, the carboxyl proteinase B was inactivated by EPNP. They had unique substrate specificities [10–14] in addition to a unique behavior against inhibitors. The complete amino acid sequence of carboxyl proteinase B [15] was quite different from those of other enzymes. Furthermore, it was found that unlike other carboxyl proteinases, one of the catalytic residues of the enzyme is glutamic acid [16, 17]. To our knowledge, it was the first demonstration of a glutamic proteinase. We have further demonstrated that enzymes having properties similar to those of Scytalidium were widely distributed among fungi [18–22], bacteria [23, 24] and thermophilic bacteria [25]. On the basis of the results obtained so far, we proposed that carboxyl proteinases should be classified into two groups: pepstatin-sensitive carboxyl proteinases (aspartic proteinase) and pepstatin-insensitive carboxyl proteinases (Scytalidium type) [1, 2].

Purification and Characterization of Arctium lappa L. (Edible Burdock) Polyphenol Oxidase

Polyphenol oxidase of Arctium lappa L. (edible burdock) has been purified by chromatographies on DEAE-cellulose, Sephadex G-75, and phenyl-Cellulofine to a homogeneous state as judged by SDS polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 31,000 by SDS-PAGE and 25,000 by gel filtration on TSKgel G2000SW. The optimum pH was 7.0 and the enzyme was stable at pH 7.0-9.0. The enzyme oxidized triphenols such as pyrogallol and phloroglucinol, and was completely inhibited by sulfide and cyanide, while it was neither affected by kojic acid nor N-hydroxyglycine (laccase inhibor). These results indicated that the enzyme had properties different from those of polyphenol oxidases from other sources such as mandarin orange and soybean.

Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain

Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which was designated parasporin-5 (PS5). PS5 was purified from B. thuringiensis serovar tohokuensis strain A1100 based on its cytocidal activity against human leukemic T cells (MOLT-4). The 50% effective concentration (EC50) of PS5 to MOLT-4 cells was approximately 0.075 μg/mL. PS5 was expressed as a 33.8-kDa inactive precursor protein and exhibited cytocidal activity only when degraded by protease at the C-terminal into smaller molecules of 29.8 kDa. Although PS5 showed no significant homology with other known parasporins, a Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) search revealed that the protein showed slight homology to, not only some B. thuringiensis Cry toxins, but also to aerolysin-type β-pore-forming toxins (β-PFTs). The recombinant PS5 protein could be obtained as an active protein only when it was expressed in a precursor followed by processing with proteinase K. The cytotoxic activities of the protein against various mammalian cell lines were evaluated. PS5 showed strong cytocidal activity to seven of 18 mammalian cell lines tested, and low to no cytotoxicity to the others.

Isolation and Purification of Ascorbate Oxidase from Acremonium sp.HI-25

A screening test was undertaken to isolate microorganisms that produced ascorbate oxidase. The enzyme activity was found in a culture filtrate of a fungal strain (HI-25), newly isolated from a soil sample. Based on the morphological characteristics, this isolate was identified as Acremonium sp. From the examinations of cultural conditions, optimum conditions for enzyme production were found; strain HI-25 was aerobically cultured by a jar fermenter at 25°C in a medium containing 5% glycerol, 2% defatted soybeans, 0.1% monosodium L-glutamate, 0.1% KH2PO4, 0.02% MgSO4 ·7H2O, and 0.01% KCl, pH 6.0. After cultivation, an ascorbate oxidase was purified from the culture filtrate by an ammonium sulfate fractionation, column chromatographies on DEAE-cellulose and Butyl-Toyopearl, and gel filtration twice on Sephadex G-100. The purification was 850-fold with an activity yield of 8.8%. The purified enzyme gave a single band on SDS polyacrylamide gel electrophoresis, and had a molecular weight of 80,000 by SDS polyacrylamide gel electrophoresis and 76,000 by native gel filtration. This enzyme was most active at pH 4.0, 45°C, and was most stable between pH 6.0-10.0 and at temperatures below 60°C.

Cloning of a thermostable ascorbate oxidase gene from Acremonium sp. HI-25 and modification of the azide sensitivity of the enzyme by site-directed mutagenesis

A gene encoding a thermostable ascorbate oxidase (ASOM) was cloned from Acremonium sp. HI-25 and sequenced. The gene comprised 1709 bp and was interrupted by a single intron of 57 bp. ASOM consisted of 551 amino acids including a signal peptide with a molecular mass of 61200, and contained four histidine-rich regions with high sequence homology to the corresponding regions of other multicopper oxidases. The ASOM gene was expressed in Aspergillus nidulans under the Aspergillus oryzae Taka-amylase A gene promoter. The recombinant enzyme (An-ASOM) exhibited almost the same enzymatic properties as ASOM. The ASOM gene was mutated by site-directed mutagenesis with reference to the amino acid sequences of plant enzymes to generate enzymes with altered azide sensitivity. Site-directed mutagenesis at the trinuclear active copper site resulted in an increase in azide resistance; the Ala465Leu and Phe463Trp/Ala465Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively.

Structure of trehalostatin: a potent and specific inhibitor of trehalase

A structural study of trehalostatin 1, a specific inhibitor of blowfly trehalase, revealed that 1 contains an unusual five-membered pseudocyclitol.

Coriolus versicolor Laccase Catalyzes the Decarboxylation of 2-(4-Hydroxyphenyl)-glycine and 4-Hydroxymandelic Acid

Laccasc (benzenediol: oxygen oxidoreductase, EC 1.1O.3.2) has been found in some fungal strains belonging to various classes.i) The enzyme generally catalyzes the removal of hydrogen from phenolic hydroxyl groups or aromatic amino groups using molecular oxygcn as a primary electron acceptor to give radicals, which undergo free-radical additions and related reactions to give various products.2-s) During the investigation n a laccase from the fungus Coriolus versieotor, we found that the enzyme catalyzed the conversion of 2-(4-hydroxyphenyl)glycine (HPG) or 4hydroxyrnandelic acid (HMA) to 4-hydroxybenzaldehyde (HBA), which was a single product from either substrate and was not converted further. Laccase is known to remove

Molecular Cloning, Sequencing, and Expression in Escherichia coli of the Gene Encoding a Novel 5-Oxoprolinase without ATP-Hydrolyzing Activity from Alcaligenes faecalis N-38A

ABSTRACT The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-β-d-thiogalactopyranoside-inducibletac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited byp-chloromercuribenzoic acid, EDTA,o-phenanthroline, HgCl2, and CuSO4. The EDTA-inactivated enzyme was completely restored by the addition of Zn2+ or Co2+. In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme.