Yasuhiko Asada

Screening of basidiomycetes for lignin peroxidase genes using a DNA probe

SummaryBasidiomycetes were screened for lignin peroxidase (LPO) genes using a DNA probe prepared from the LPO restriction fragment ofPhanerochaete chrysosporium. Southern blot analysis showed restriction fragments of chromosomal DNA ofBjerkandera adusta andCoriolus consors hybridized with the probe.Bjerkandera adusta produced LPO in a glucose-peptone medium. Ion-exchange chromatography showed that this fungus produced multiple molecular forms of LPO. One of the enzymes, LPO-2, was purified and characterized. The molecular weight of LPO-2 was 41000 with a pI of 4.2. Spectral analysis demonstrated that LPO-2 is a haem protein. The enzyme cleaved lignin model dimers mainly at the Cα-Cβ position of the side chain. The LPO-2 exhibited close similarity to LPOs ofP. chrysosporium with respect to their basic properties.

Molecular breeding of the basidiomycete Coprinus cinereus strains with high lignin-decolorization and -degradation activities using novel heterologous protein expression vectors

Abstract Two chromosome-integrating vectors, pLC1 and pLC2, were used. The former is the pUC19-based vector carrying the Lentinus edodes ras gene promoter and priA gene terminator, and the latter is the pBR322-based vector carrying the promoter and terminator of the priA gene. The manganese (II) peroxidase (MnP) cDNA (mnpc) derived from Pleurotus ostreatus was fused between the promoter and terminator of pLC1 and pLC2, yielding the recombinant plasmids pLC1-mnp and pLC2-mnp. These plasmids were introduced into protoplasts of the Coprinus cinereus trp1 strain with the C. cinereus TRP1-containing plasmid pCc1001 by co-transformation. Two Trp+ transformants for each plasmid, showing clearly higher lignin-decolorization activities, were obtained through introduction of pLC1-mnp and pLC2-mnp. Southern-blot analysis revealed that the four transformants all possess the mnpc sequence on their chromosomes. One Trp+ MnP+ transformant (named TF2-7), which was derived from the introduction of pLC2-mnp and carried the highest number of copies (approx. 10) of mnpc, showed remarkably high lignin-decolorization and -degradation activities; at the time of cultivation when only 35%–40% of the lignin was decolored and degraded by the control Trp+ transformant obtained by the introduction of pCc1001 alone, almost all of the lignin was decolored and degraded by TF2-7.

Molecular analysis of a Bjerkandera adusta lignin peroxidase gene

SummaryA cDNA clone, λLPO-1, encoding a major lignin peroxidase from the basidiomycete Bjerkandera adusta was isolated and characterized. The nucleotide sequence of λLPO-1 predicts a mature protein consisting of 349 amino acids with a molecular weight of 37,225 preceded by a signal peptide of 23 amino acid residues. We have also cloned and sequenced the gene encoding lignin peroxidase from B. adusta. Comparison of these sequences reveals a lignin peroxidase gene structure consisting of 1,116 bp of protein-encoding DNA that is interrupted by four intervening sequences. The putative eukaryotic regulatory sequence, a TATA box, is present at position — 75 relative to the translational initiation codon. Amino acid sequence homology between the coding regions of λLPO-1 and of the lignin peroxidase cDNA clone λML-1 from Phanerochaete chrysosporium is 61%.

Cloning and sequencing of a ligninase gene from a lignindegrading basidiomycete, Phanerochaete chrysosporium

SummaryA ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3′ and 5′ intron boundaries. The putative eukaryotic regulatory sequences, i.e. “CAAT” and “TATA” box-like sequences, are present in the 5′ flanking region.

Singlet oxygen induced products of linoleates, 10- and 12-(Z,E)-hydroxyoctadecadienoic acids (HODE), can be potential biomarkers for early detection of type 2 diabetes.

Current diagnostic tests such as glycemic indicators have limitations for early detection of impaired glucose tolerance (IGT), which leads to diabetes. Oxidative stress induced by various oxidants in a random and destructive manner is considered to play an important role in the pathophysiology of a number of human disorders and diseases such as impaired glucose tolerance. We have developed an improved method for the measurement of in vivo lipid peroxidation, where the presence of 8-iso-prostaglandin F2α (8-iso-PGF2α), hydroxyoctadecadienoic acids (HODEs), hydroxyeicosatetraenoic acids (HETEs), and 7-hydroxycholesterol (7-OHCh), as well as their parent molecules, linoleic acid (LA) and cholesterol (Ch), was determined by performing LC-MS/MS (for 8-iso-PGF2α, HODE, and HETE) and GC-MS (for 7-OHCh, LA, and Ch) after reduction with triphenyl phosphine and saponification by potassium hydroxide. We then applied this method to volunteers (n = 57), including normal type (n = 43), “high-normal” (fasting plasma glucose, 100–109 mg/dL, n = 7), pre-diabetic type (IGT, n = 5), and diabetic type (n = 2) subjects who are diagnosed by performing oral glucose tolerance tests (OGTTs). Several biomarkers in plasma, such as insulin, leptin, adiponectin, interleukin-6, tumor necrosis factor-α, high sensitivity-C-reactive protein, HbA1c, and glucose levels were measured during OGTT. We found that the fasting levels of (10- and 12-(Z,E)- HODE)/LA increased significantly with increasing levels of HbA1c and glucose during OGTT and with insulin secretion and resistance index. In conclusion, 10- and 12-(Z,E)-HODE may be prominent biomarkers for the early detection of IGT and “high-normal” type without OGTT.

Skipping Breakfast is Correlated with Obesity

Objective: Despite the fact that the total energy intake of Japanese people has decreased, the percentage of obese people has increased. This suggests that the timing of meals is related to obesity. The purpose of the study was to investigate the relationship between the timing of meals and obesity, based on analyses of physical measurements, serum biochemical markers, nutrient intake, and lifestyle factors in the context of Chrononutrition. Participants and Methods: We analyzed data derived from 766 residents of Toon City (286 males and 480 females) aged 30 to 79 years who underwent detailed medical examinations between 2011 and 2013. These medical examinations included. (1) physical measurements (waist circumference, blood pressure, etc.); (2) serum biochemical markers (total cholesterol, etc.); (3) a detailed questionnaire concerning lifestyle factors such as family structure and daily habits (22 issues), exercise and eating habits (28 issues), alcohol intake and smoking habits; (4) a food frequency questionnaire based on food groups (FFQg); and (5) a questionnaire concerning the times at which meals and snacks are consumed. Results: The values for body mass index (BMI) and waist circumference were higher for participants who ate dinner less than three hours before bedtime (<3-h group) than those who ate more than three hours before bedtime (>3-h group). The Chi-square test showed that there was a significant difference in eating habits, e.g., eating snacks, eating snacks at night, having dinner after 8 p.m., and having dinner after 9 p.m., between the <3-h group and the >3-h group. Multiple linear regression analysis showed that skipping breakfast significantly influenced both waist circumference (β = 5.271) and BMI (β = 1.440) and that eating dinner <3-h before going to bed only influenced BMI (β = 0.581). Conclusion: Skipping breakfast had a greater influence on both waist circumference and BMI than eating dinner <3-h before going to bed.

Purification and characterization of the extracellular laccase produced by Trametes polyzona WR710-1 under solid-state fermentation

Laccase from Trametes polyzona WR710–1 was produced under solid‐state fermentation using the peel from the Tangerine orange (Citrus reticulata Blanco) as substrate, and purified to homogeneity. This laccase was found to be a monomeric protein with a molecular mass of about 71 kDa estimated by SDS–PAGE. The optimum pH was 2.0 for ABTS, 4.0 for L‐DOPA, guaiacol, and catechol, and 5.0 for 2,6‐DMP. The Km value of the enzyme for the substrate ABTS was 0.15 mM, its corresponding Vmax value was 1.84 mM min−1, and the kcat/Km value was about 3960 s−1 mM−1. The enzyme activity was stable between pH 6.0 and 8.0, at temperatures of up to 40 °C. The laccase was inhibited by more than 50% in the presence of 20 mM NaCl, by 95% at 5 mM of Fe2+, and it was completely inhibited by 0.1 mM NaN3. The N‐terminal amino acid sequence of this laccase is AVTPVADLQISNAGISPDTF, which is highly similar to those of laccases from other white‐rot basidiomycetes.

Catalytic properties and crystal structure of quinoprotein aldose sugar dehydrogenase from hyperthermophilic archaeon Pyrobaculum aerophilum

We identified a gene encoding a soluble quinoprotein glucose dehydrogenase homologue in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The enzyme was extremely thermostable, and the activity of the pyrroloquinoline quinone (PQQ)-bound holoenzyme was not lost after incubation at 100 degrees C for 10 min. The crystal structure of the enzyme was determined in both the apoform and as the PQQ-bound holoenzyme. The overall fold of the P. aerophilum enzyme showed significant similarity to that of soluble quinoprotein aldose sugar dehydrogenase (Asd) from E. coli. However, clear topological differences were observed in the two long loops around the PQQ-binding sites of the two enzymes. Structural comparison revealed that the hyperthermostability of the P. aerophilum enzyme is likely attributable to the presence of an extensive aromatic pair network located around a beta-sheet involving N- and C-terminal beta-strands.

An extracellular NADH-oxidizing peroxidase produced by a lignin-degrading basidiomycete, Phanerochaete chrysosporium

A lignin-degrading basidiomycete, Phanerochaete chrysosporium, produces an extracellular peroxidase which in turn produces H2O2 by catalyzing the oxidation of NADH and NADPH. The high enzyme activity was observed in the culture grown under nutrient nitrogen limitation (low-N) and high oxygen tension (high-O2). The enzyme activity was absent in non-ligninolytic agitated culture and in the cultures of non-ligninolytic mutant strains of this organism. The culture method using polyurethane foam cubes as a support for the growing mycelia showed the beneficial effect of producing a large amount of the enzyme. The enzyme is capable of catalyzing the oxidation of NADH and NADPH in the absence of added H2O2, and its activity was inhibited strongly by catalase and superoxide dismutase. It is suggested that this peroxidase participates in the ligninolytic system of Phanerochaete chrysosporium as a donor of H2O2, which is required for the lignin-peroxidase reaction, by oxidizing extracellular NADH and NADPH.

Extracellular Laccase Produced by an Edible Basidiomycetous Mushroom, Grifola frondosa: Purification and Characterization

A major laccase isozyme (Lac 1) was isolated from the culture fluid of an edible basidiomycetous mushroom, Grifola frondosa. Lac 1 was revealed to be a monomeric protein with a molecular mass of 71 kDa. The N-terminal amino acid sequence of Lac 1 was highly similar to those of laccases of some other white-rot basidiomycetes. Lac 1 showed the typical absorption spectrum of a copper-containing enzyme. The enzyme was stable in a wide pH range (4.0 to 10.0), and lost no activity up to 60 °C for 60 min. The optimal pH of the enzyme activity varied among substrates. The K m values of Lac 1 toward 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol, catechol, and 3,4-dihydroxy-L-phenylalanine were 0.0137 mM, 0.608 mM, 0.531 mM, 2.51 mM, and 0.149 mM respectively. Lac 1 activity was remarkably inhibited by the chloride ion, in a reversible manner. Lac 1 activity was also inhibited by thiol compounds.