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While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified the gene RvNaRL, which had sequence similarity to nitrate reductases found in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify its function of the protein product RvNaRL, we established a RNAi mediated knockdown and a CRISPR-Cas9-mediated knockout experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knockdown and knockout cells exhibited significant growth only when ammonium was supplied. However, in contrast to the wild-type cells, no substantial growth was observed when nitrate was supplied. Such arrested growth in absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we inferred that R. viridis achieved its advanced kleptoplasty for photoautotrophy, owing to acquisition of the nitrate assimilation by the horizontal gene transfer.

Purpose This prospective post-marketing surveillance (PMS) was designed to collect data on the safety and effectiveness of naldemedine in routine clinical practice in patients with opioid-induced constipation (OIC) and cancer pain in Japan and explore the characteristics of patients prone to diarrhea. Methods The enrolled patients received naldemedine (0.2 mg, once a day) orally for up to 12 weeks. In the safety analysis, adverse drug reactions (ADRs), including diarrhea as a special interest, were assessed. Effectiveness was evaluated, especially regarding the frequency and condition of bowel movement. Results In the safety analysis set (n = 1177), 145 ADRs occurred in 133 (11.30%) patients, and diarrhea was the most frequent event (n = 107, 9.09%). Most cases of diarrhea were non-serious (98.1%). Most ADRs were non-serious (93.8%), and they resolved within 2 weeks (75.9%). No patient characteristics influenced the risk of diarrhea development or aggravation. Both the frequency (75.0% and 83.2%) and condition of bowel movement (80.0% and 88.0%) were improved at 2 and 12 weeks, respectively in the effectiveness analysis set (n = 953). Frequency and condition of bowel movement were also improved in patients excluded (e.g., Eastern Cooperative Oncology Group performance status was ≥ 3) or with very small numbers (e.g., received weak opioid) in the clinical trials. Conclusions This PMS indicates that naldemedine is well tolerated and effective in patients of various backgrounds in routine clinical practice who have OIC and cancer pain. Trial registration UMIN000042851.

Euglena gracilis produces ATP in the anaerobic mitochondria with concomitant wax ester formation, and NADH is essential for ATP formation and fatty acid synthesis in the mitochondria. This study demonstrated that mitochondrial cofactor conversion by nicotinamide nucleotide transhydrogenase (NNT), converting NADPH/NAD+ to NADP+ /NADH, is indispensable for sustaining anaerobic metabolism. Silencing of NNT genes significantly decreased wax ester production and cellular viability during anaerobiosis but had no such marked effects under aerobic conditions. An analogous phenotype was observed in the silencing of the gene encoding a mitochondrial NADP+ -dependent malic enzyme. These results suggest that the reducing equivalents produced in glycolysis are shuttled to the mitochondria as malate, where cytosolic NAD+ regeneration is coupled with mitochondrial NADPH generation.

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here we focused on the nitrogen metabolism ofRapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates,R. viridisexploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data ofR. viridis, we identified the gene RvNaRL, which had sequence similarity to nitrate reductases found in plants. Phylogenetic analysis revealed that RvNaRLwas acquired by a horizontal gene transfer event. To verify its function of the protein productRvNaRL, we established a RNAi mediated knockdown and a CRISPR-Cas9-mediated knockout experiments for the first time inR. viridisand applied them to this gene. The RvNaRLknockdown and knockout cells exhibited significant growth only when ammonium was supplied but, in contrast to the wild-type cells, no substantial growth when nitrate was supplied. Such arrested growth in absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains as observed. These results indicate thatRvNaRL is certainly involved in nitrate assimilation byR. viridis. Thus, we infer thatR. viridisachieved its advanced kleptoplastic strategy owing to a posteriori acquisition of the nitrate assimilation pathway the horizontal gene transfer.

Baloxavir marboxil is an oral anti-influenza drug that inhibits the cap-dependent endonuclease of the virus polymerase acidic protein. In clinical trials, baloxavir reduced the time to alleviation of influenza symptoms and time to resolution of fever in adults, adolescents, and children. The purpose of this study is to collect data on the safety and effectiveness of baloxavir when used in clinical practice. This postmarketing surveillance (clinicaltrials.jp; JapicCTI-183882), conducted at 688 Japanese hospitals or clinics (March 2018 to March 2019), enrolled patients of any age with influenza A or B infection who received a single, weight-based dose of baloxavir. Adverse drug reactions (ADRs) were seen in 11.2% of 3094 patients during the 7-day observation period; the most common ADR was diarrhea (6.1%). ADRs were more common in children aged12 years (14.1%) than in adults (10.0%). Almost all ADRs were non-serious (98.9%) and were recovered or recovering (96.7%). Median time to alleviation of symptoms (N = 2884) was 2.5 days (overall, influenza A, and influenza B groups). Median time to resolution of fever (N = 2946) was 1.5 days (overall, influenza A, and influenza B groups). Biphasic fever (increased temperature after previous fever resolution) was seen in 6.7% of patients overall and 28.6% of patients6 years infected with influenza B, similar to rates published elsewhere with other influenza drugs and in untreated influenza. This postmarketing surveillance of3000 patients suggests that baloxavir is well tolerated and effective regardless of patient age or influenza virus type.

Konjac glucomannan (KGM) is a principal component of the gelatinous food Konjac. Konjac production through alkali treatment releases an undesirable amine-odor. Two acetylesterases (AME1 and AME2) active against konjac glucomannan (polymer or oligomer) were purified from the supernatant of Aspergillus oryzae RIB40 culture. We cloned the genes encoding AME1 and AME2 based on the genomic information of A. oryzae, constructed their expression systems in A. oryzae, and obtained the recombinant enzymes (rAME1 and rAME2). rAME1 did not act on the KGM polymer but only on the KGM oligomer, releasing approximately 60% of the acetic acid in the substrate. However, rAME2 was active against both KGM substrates, releasing approximately 80% and 100% of acetic acid from the polymer and oligomer, respectively. Both enzymes were active against xylan and exhibited a trace activity on ethyl ferulate. The acetyl group position specificities of both enzymes were analyzed via heteronuclear single quantum correlation NMR using oligosaccharides of glucomannan prepared from Aloe vera (AGM), which has a higher acetyl group content than KGM. rAME1 acted specifically on single-substituted acetyl groups and not on double-substituted ones. In contrast, rAME2 appeared to act on all the acetyl groups in AGM. Treatment of 3% KGM with rAME2 followed by heating to 90 °C resulted in gel formation under weakly acidic conditions. This is the first study to induce gelation of KGM under these conditions. A comparison of the breaking and brittleness properties of gels formed by alkaline and enzymatic treatments revealed similar texture of the two gels. Furthermore, scanning electron microscopy of the surface structure of both gels revealed that both formed a fine mesh structure. Our findings on enzymatic gelation of KGM should lead to the development of new applications in food manufacturing industry.

teaserEuglenoids show great promise to benefit our world; as biofuels, environmental remediators, anti-cancer agents, robotics design simulators and food nutritional agents, but the absence of reference genomes currently limit realizing these benefits. The Euglena International Network (EIN) (https://euglenanetwork.org/) aims to address these challenges, and is currently seeking formative phase support and funding.Body startOf the nearly 1000 known species of euglenoids (Triemer and Zakryś, 2015), including Euglena gracilis and Rhabdomonas costata, fewer than 2 % have been explored for any level of translational potential in the past 20 years. The absence of reference genomes currently limits biotechnology applications, including the development of efficient tools for genetic manipulation in euglenoids.EIN aims to advance euglenoid science through a creative amalgam of academic institutions, national research institutes and biotechnology industry, to translate and exploit euglenoids through genome sequencing. EIN has defined goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry professionals and is currently formulating policy and partnership principles, driven by EIN Executive and Science committees. However, for EIN’s activities to be maintained and durable, long-term support is vital. We call on national and continental funding agencies and research councils, protists and algae communities, and biotechnology and pharmaceutical industries, to embrace, support and fund translational exploitation of these highly valuable organisms.

Three recombinant β-galactosidases (BGALs; PcBGAL35A, PcBGAL35B, and PcGALX35C) belonging to the glycoside hydrolase (GH) family 35 derived from Penicillium chrysogenum 31B were expressed using Pichia pastoris and characterized. PcBGAL35A showed a unique substrate specificity that has not been reported so far. Based on the results of enzymological tests and 1H-nuclear magnetic resonance, PcBGAL35A was found to hydrolyze β-1,4-galactosyl residues linked to L-rhamnose in rhamnogalacturonan-I (RG-I) of pectin, as well as p-nitrophenyl-β-D-galactopyranoside and β-D-galactosyl oligosaccharides. PcBGAL35B was determined to be a common BGAL through molecular phylogenetic tree and substrate specificity analysis. PcGALX35C was found to have similar catalytic capacities for the β-1,4-galactosyl oligomer and polymer. Furthermore, PcGALX35C hydrolyzed RG-I-linked β-1,4-galactosyl oligosaccharide side chains with a degree of polymerization of 2 or higher in pectin. The amino acid sequence similarity of PcBGAL35A was approximately 30% with most GH35 BGALs, whose enzymatic properties have been characterized. The amino acid sequence of PcBGAL35B was approximately 80% identical to those of BGALs from Penicillium sp. The amino acid sequence of PcGALX35C was classified into the same phylogenetic group as PcBGAL35A. Pfam analysis revealed that the three BGALs had five domains including a catalytic domain. Our findings suggest that PcBGAL35A and PcGALX35C are enzymes involved in the degradation of galactosylated RG-I in pectin. The enzymes characterized in this study may be applied for products that require pectin processing and for the structural analysis of pectin.

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 13(2),17(3)-cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.

Aspergillus oryzae is a safe microorganism that is commonly used in food production. We constructed a self-cloning vector capable of high expression in A. oryzae. Using the vector, three putative pectin methylesterase (PME) genes belonging to Carbohydrate Esterase family 8 derived from A. oryzae were expressed, and several characteristics of the gene products were examined. The effects of temperature and pH on the three enzymes (AoPME1, 2, and 3) were similar, with optimal reaction temperatures of 50 - 60 °C and optimal reaction pH range of 5 - 6. The specific activities of AoPME1, 2, and 3 for apple pectin were significantly different (34, 7,601, and 2 U/mg, respectively). When the substrate specificity was examined, AoPME1 showed high activity towards pectin derived from soybean and pea. Although AoPME2 showed little activity towards these pectins, it showed very high activity towards apple- and citrus-derived pectins. AoPME3 showed low specific activity towards all substrates tested. Sugar composition analysis revealed that apple- and citrus-derived pectins were rich in homogalacturonan, while soybean- and pea-derived pectins were rich in xylogalacturonan. When pea pectin was treated with endo-polygalacturonase or endo-xylogalacturonase in the presence of each PME, specific synergistic actions were observed (endo-polygalacturonase with AoPME1 or AoPME2 and endo-xylogalacturonase with AoPME1 or AoPME3). Thus, AoPME1 and AoPME3 hydrolyzed the methoxy group in xylogalacturonan. This is the first report of this activity in microbial enzymes. Our findings on the substrate specificity of PMEs should lead to the determination of the distribution of methoxy groups in pectin and the development of new applications in the field of food manufacturing.

application/pdf, Article, Mycoscience. 2018, 59 (6), p.479-482

In Euglena gracilis, wax ester fermentation produces ATP during anaerobiosis. Here, we report that anaerobic wax ester production is suppressed when the mitochondrial electron transport chain complex I is inhibited by rotenone, whereas it is increased by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The ADP/ATP ratio in anaerobic cells is elevated by treatment with either rotenone or CCCP. Gene silencing experiments indicate that acyl-CoA dehydrogenase, electron transfer flavoprotein (ETF), and rhodoquinone (RQ) participate in wax ester production. These results suggest that fatty acids are synthesized in mitochondria by the reversal of β-oxidation, where trans-2-enoyl-CoA is reduced mainly by acyl-CoA dehydrogenase using the electrons provided by NADH via the electron transport chain complex I, RQ, and ETF, and that ATP production is highly supported by anaerobic respiration utilizing trans-2-enoyl-CoA as a terminal electron acceptor.

application/pdf, Article, Process Biochemistry, 2018, 74, p.28-34

Highlights • There have been few reports about gene cloning and expression of α-amylases from E. fetida. • Ef-Amy I and II were shown to 89% identity of amino acid sequences. • The catalytically important residues of α-amylase of GH family 13 were conserved in Ef-amy I and II. • The substrate specificities of rEf-Amy I and II were dissimilar. • It found that rEf-Amy I and II could be possible use for simultaneous saccharification and fermentation process., We identified the raw-starch-digesting α-amylase genes a earthworm Eisenia fetid α amylase I and II (Ef-Amy I and Ef-Amy II). Each gene consists of 1,530 base pairs (bp) that encode proteins of 510 amino acids, as indicated by the corresponding mRNA sequences. Ef-Amy I and II showed an 89% amino acid identity. The amino acid sequences of Ef-Amy I and II were similar to those of the α-amylases from porcine pancreas, human pancreas, Tenebrio molitor, Oryctolagus cuniculus, and Xenopus (Silurana) tropicalis. Each gene encoding mature Ef-Amy I and II was expressed in the GS115 strain of Pichia pastoris. The molecular masses of the recombinant Ef-Amy I and II were 57 kDa each, and catalytically important residues of α-amylases of the GH family 13 were conserved in both proteins. These amylases exhibited raw-starch-digesting activity at 4 °C. The substrate specificities of rEf-Amy I and II were dissimilar. rEf-Amy I and II were shown to be active even in 40% ethanol, 4 M NaCl, and 4 M KCl., Graphical abstract Image, graphical abstract

Chitin is the second most abundant biopolymer in nature and is an important resource. In this study, we identified a chitinase gene, named Eisenia fetida-Chitinase (EF-Chi) gene, of 1494 base pairs (bp) that encodes a protein of 498 amino acids as indicated by the corresponding mRNA sequence. The amino acid sequence of EF-Chi was similar to those of chitinases from Eisenia andrei (99%), Branchiostoma floridae (50%) and Oryzias latipes (49%), and a gene encoding mature EF-Chi was expressed in the GS115 strain of Pichia pastoris. The molecular mass of the purified recombinant EF-Chi (rEF-Chi) was estimated to be 60 kDa and catalytically important residues of chitinases of the glycoside hydrolase (GH) family 18 were conserved in EF-Chi. The optimal catalytic temperature of rEF-Chi was identified as 60 °C, and the hydrolytic product from colloidal chitin was N-acetyl-chitobiose, suggesting that EF-Chi is an exo-type enzyme.

In Euglena gracilis, pyruvate:NADP+ oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP+ oxidoreductase. In contrast, pyruvate:NADP+ oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.

The chemical structure of pea pectin was delineated using pectin-degrading enzymes and biochemical methods. The molecular weight of the pea pectin preparation was 488,000, with 50 % arabinose content, and neutral sugar side chains attached to approximately 60 % of the rhamnose residues in rhamnogalacturonan-I (RG-I). Arabinan, an RG-I side chain, was highly branched, and the main chain was comprised of α-1,5-l-arabinan. Galactose and galactooligosaccharides were attached to approximately 35 % of the rhamnose residues in RG-I. Long chain β-1,4-galactan was also present. The xylose substitution rate in xylogalacturonan (XGA) was 63 %. The molar ratio of RG-I/homogalacturonan (HG)/XGA in the backbone of the pea pectin was approximately 3:3:4. When considering neutral sugar side chain content (arabinose, galactose, and xylose), the molar ratio of RG-I/HG/XGA regions in the pea pectin was 7:1:2. These data will help understand the properties of pea pectin.

Naringin (Nar) has antioxidant and anti-inflammatory properties. It was recently reported that enzymatic modification of Nar enhanced its functions. Here, we acylated Nar with fatty acids of different sizes (C2–C18) using immobilized lipase from Rhizomucor miehei and investigated the anti-inflammatory effects of these molecules. Treatment of murine macrophage RAW264.7 cells with Nar alkyl esters inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, with Nar lauroyl ester (Nar-C12) showing the strongest effect. Furthermore, Nar-C12 suppressed the LPS-induced expression of inducible NO synthase by blocking the phosphorylation of inhibitor of nuclear factor (NF)-κB-α as well as the nuclear translocation of NF-κB subunit p65 in macrophage cells. Analysis of Nar-C12 uptake in macrophage cells revealed that Nar-C12 ester bond was partially degraded in the cell membrane and free Nar was translocated to the cytosol. These results indicate that Nar released from Nar-C12 exerts anti-inflammatory effects by suppressing NF-κB signaling pathway.

We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins.

application/pdf, Article, Enzyme and Microbial Technology, 2018, 117, p.15-22

application/pdf, Article, FEBS Letter, 2018, 592, p.1378-1388

In Euglena gracilis, pyruvate:NADP

Euglenoids are able to assimilate fatty acids and alcohols with various carbon-chain lengths, and ethanol is known to be one of the best carbon sources to support the growth of Euglena gracilis. Ethanol is first oxidized to acetate by the sequential reactions of alcohol dehydrogenase and acetaldehyde dehydrogenase in the mitochondria, and then converted to acetyl coenzyme A (acetyl-CoA). Acetyl-CoA is metabolized through the glyoxylate cycle which is a modified tricarboxylic acid (TCA) cycle in which isocitrate lyase (ICL) and malate synthase (MS) function to bypass the two decarboxylation steps of the TCA cycle, enabling the net synthesis of carbohydrates from C2 compounds. ICL and MS form a unique bifunctional enzyme localized in Euglena mitochondria, not in glyoxysome as in other eukaryotes. The unique glyoxylate and glycolate metabolism during photorespiration is also discussed in this chapter.

Lipids are being increasingly used as biodiesel feedstock, and several saturated wax esters from Euglena gracilis are candidates for outdoor bulk production. Wax ester fermentation in Euglena is strongly increased by hypoxia, but key events underlying the metabolic shift toward wax ester biosynthesis are poorly understood. Profiling of wax esters and other lipophilic compounds is the first step for research toward the clarification of wax ester fermentation molecular mechanisms, and thus, a simple and comprehensive platform for their profiling is required. In this study, we established a profiling method for wax esters and lipophilic compounds in Euglena using gas chromatography-mass spectrometry (GC–MS). Using this method, we compared accumulation profiles of wax esters and lipophilic compounds between a wild-type Euglena Z strain and a bleached SM-ZK strain. Both the wild-type and the bleached strains contained C14:0 fatty acid-C14:0 fatty alcohol as a dominant wax ester. Wax ester fermentation initiated 4 h after the cessation of oxygen supply by halting the culture agitation resulting in linear increase and proportional changes of wax ester amounts during 24 h. However, complete anoxia by nitrogen gas aeration inhibited wax ester production and the addition of bicarbonates reversed the inhibition, suggesting that there is a need for an additional carbon source for wax ester fermentation under anoxia. Our simple method enables the investigation of metabolic responses leading to wax ester fermentation in Euglena.

The crystal structure of endo-1,4-β-glucanase from the earthworm Eisenia fetida, which retained sufficient cellulase activity at low temperature, was determined at 1.5 Å resolution., The saccharification process is essential for bioethanol production from woody biomass including celluloses. Cold-adapted cellulase, which has sufficient activity at low temperature (

Chitinase C from Ralstonia sp. A-471 (Ra-ChiC) has a catalytic domain sequence similar to goose-type (G-type) lysozymes and, unlike other chitinases, belongs to glycohydrolase (GH) family 23. Using NMR spectroscopy, however, Ra-ChiC was found to interact only with the chitin dimer but not with the peptidoglycan fragment. Here we report the crystal structures of wild-type, E141Q, and E162Q of the catalytic domain of Ra-ChiC with or without chitin oligosaccharides. Ra-ChiC has a substrate-binding site including a tunnel-shaped cavity, which determines the substrate specificity. Mutation analyses based on this structural information indicated that a highly conserved Glu-141 acts as a catalytic acid, and that Asp-226 located at the roof of the tunnel activates a water molecule as a catalytic base. The unique arrangement of the catalytic residues makes a clear contrast to the other GH23 members and also to inverting GH19 chitinases.

Euglenoids are able to assimilate fatty acids and alcohols with various carbon-chain lengths, and ethanol is known to be one of the best carbon sources to support the growth of Euglena gracilis. Ethanol is first oxidized to acetate by the sequential reactions of alcohol dehydrogenase and acetaldehyde dehydrogenase in the mitochondria, and then converted to acetyl coenzyme A (acetyl-CoA). Acetyl-CoA is metabolized through the glyoxylate cycle which is a modified tricarboxylic acid (TCA) cycle in which isocitrate lyase (ICL) and malate synthase (MS) function to bypass the two decarboxylation steps of the TCA cycle, enabling the net synthesis of carbohydrates from C2 compounds. ICL and MS form a unique bifunctional enzyme localized in Euglena mitochondria, not in glyoxysome as in other eukaryotes. The unique glyoxylate and glycolate metabolism during photorespiration is also discussed in this chapter.

Titanium (Ti) implants are increasingly being used for dental parts. There is no problem with the attraction of a static magnetic field for Ti in magnetic resonance imaging (MRI), since Ti is paramagnetic. However, there is a risk of radio frequency (RF) heat generation within Ti. 3.0 T-MRI scanners are becoming increasingly common. The specific absorption rate (SAR) of 3.0 T-MRI is quadruple that of SAR compared with 1.5 T-MRI due to its being proportional to the square of the strength of a static magnetic field. The effect of heat generation in 3.0 T-MRI can thus be greater than in 1.5 T-MRI. So, using 1.5 T and 3.0 T-MRI scanners, we measured the temperature of several Ti implants using the same scanning parameters during MRI scanning. Our measurements showed the rise in temperature of the Ti implants to be a maximum of 0.4 degrees C. In this study, however, Ti in a human mouth was not directly measured, so we need to attempt to perform MRI carefully on patients with Ti implants.

A new protease was purified from the culture filtrate of a plant worm, Nomuraea atypicola . The activity of the protease was suppressed by metalloprotease inhibitors such as EDTA and 1,10-phenanthroline, suggesting that it might be a metalloprotease. Its molecular mass was estimated to be 48 kDa by SDS-PAGE, and its optimal pH and temperature were pH 8.5–9.0 and 40 °C, respectively. The N-terminal amino acid sequence of the metalloprotease was similar to those of fungalysin metallopeptidases of the M36 family from fungi such as Coccidioides posadasii , Pyrenophora tritici-repentis , and Arthroderma gypseum , supporting the idea that it is a fungalysin-like metallopeptidase.

We previously reported that Fusarium oxysporum 12S produces two bifunctional proteins, FoAP1 and FoAP2, with α-d-galactopyranosidase (GPase) and β-l-arabinopyranosidase (APase) activities. The aim of this paper was to purify a third GPase, FoGP1, from culture supernatant of F. oxysporum 12S, to characterize it, and to determine its mode of action towards gum arabic. A cDNA encoding FoGP1 was cloned and the protein was overexpressed in Escherichia coli. Module sequence analysis revealed the presence of a GH27 domain in FoGP1. The recombinant enzyme (rFoGP1) showed a GPase/APase activity ratio of 330, which was quite different from that of FoAP1 (1.7) and FoAP2 (0.2). Among the natural substrates tested, rFoGP1 showed the highest activity towards gum arabic. In contrast to other well-characterized GPases, rFoGP1 released a small amount of galactose from α-galactosyl oligosaccharides such as raffinose and exhibited no activity toward galactomannans, which are highly substituted with α-galactosyl side chains. This indicated that FoGP1 is an unusual type of GPase. rFoGP1 released 30% of the total galactose from gum arabic, suggesting the existence of a large number of α-galactosyl residues at the non-reducing ends of gum arabic side chains. Together, rFoGP1 and α-l-arabinofuranosidase released four times more arabinose than α-l-arabinofuranosidase acting alone. This suggested that a large number of α-l-arabinofuranosyl residues is capped by α-galactosyl residues. 1H NMR experiments revealed that rFoGP1 hydrolyzed the α-1,3-galactosidic linkage within the side chain structure of [α-d-Galp-(1→3)-α-l-Araf-(1→] in gum arabic. In conclusion, rFoGP1 is highly active toward α-1,3-galactosyl linkages but negligibly or not active toward α-1,6-galactosyl linkages. The novel FoGP1 might be used to modify the physical properties of gum arabic, which is an industrially important polysaccharide used as an emulsion stabilizer and coating agent.

Accumulation profiles of wax esters in Euglena gracilis Z were studied under several environmental conditions. The highest amount of total wax esters accumulated under hypoxia in the dark, and C28 (myristyl-myristate, C14:0-C14:0) was prevalent among all conditions investigated. The wax ester production was almost completely suppressed under anoxia in the light, and supplying exogenous inorganic carbon sources restored wax ester fermentation, indicating the need for external carbon sources for the wax ester fermentation. 13C-labeling experiments revealed specific isotopic enrichment in the odd-numbered fatty acids derived from wax esters, indicating that the exogenously-supplied CO2 was incorporated into wax esters via the propionyl-CoA pathway through the reverse tricarboxylic acid (TCA) cycle. The addition of 3-mercaptopicolinic acid, a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, significantly affected the incorporation of 13C into citrate and malate as the biosynthetic intermediates of the odd-numbered fatty acids, suggesting the involvement of PEPCK reaction to drive wax ester fermentation. Additionally, the 13C-enrichment pattern of succinate suggested that the CO2 assimilation might proceed through alternative pathways in addition to the PEPCK reaction. The current results indicate that the mechanisms of anoxic CO2 assimilation are an important target to reinforce wax ester fermentation in Euglena.

Rhamnogalacturonan (RG) I is one of the main components of pectins in the plant cell wall. We recently reported two RG I-degrading enzymes, endo-RG and exo-RG lyases, secreted by Penicillium chrysogenum 31B. Here, our aims were to purify a RG rhamnohydrolase (PcRGRH78A) from the culture filtrate of this strain and to characterize this enzyme. On the basis of the internal amino acid sequences, the encoding gene, Pcrgrh78A, was cloned and overexpressed in Aspergillus oryzae. The deduced amino acid sequence of PcRGRH78A is highly similar to an uncharacterized protein belonging to glycoside hydrolase family 78. Pfam analysis revealed that PcRGRH78A contains a bacterial α-l-rhamnosidase (PF05592) domain. PcRGRH78A shows optimal activity at 45°C and pH 5. The specificity of PcRGRH78A toward rhamnose (Rha)-containing substrates was compared with that of a P. chrysogenum α-l-rhamnosidase (PcRHA78B) belonging to glycoside hydrolase family 78. PcRGRH78A specifically hydrolyzes RG oligosaccharides that contain Rha at their nonreducing ends, releasing the Rha, but has no activity toward naringin, hesperidin, or rutin. In contrast, PcRHA78B effectively degrades p-nitrophenyl α-l-rhamnopyranoside and the three flavonoids, but not RG oligosaccharides. When galactosyl RG oligosaccharides were used as the substrate, PcRGRH78A released Rha in 3.5-fold greater amounts in the presence of β-galactosidase than in its absence, indicating that PcRGRH78A preferentially acts on Rha residues without the galactose moiety at nonreducing ends. To our knowledge, this is the first report of a gene encoding a RG rhamnohydrolase.

In magnetic resonance imaging (MRI), when radiofrequency (RF) is irradiated to a subject with metallic implant, it can generate heat by RF irradiation. Recently 3 T MRI scanner has spread widely and imaging for any regions of whole body has been conducted. However specific absorption rate (SAR) of 3 T MRI becomes approximately four times as much as the 1.5 T, which can significantly affect the heat generation of metallic implants. So, we evaluated RF heating of artificial hip joints in different shapes and materials in 1.5 T and 3 T MRI. Three types of artificial hip joints made of stainless alloy, titanium alloy and cobalt chrome alloy were embedded in the human body-equivalent phantom respectively and their temperature change were measured for twenty minutes by 1.5 T and 3 T MRI. The maximum temperature rise was observed at the bottom head in all of three types of artificial hip joints, the rise being 12°C for stainless alloy, 11.9°C for titanium alloy and 6.1°C for cobalt chrome alloy in 1.5 T. The temperature rise depended on SAR and the increase of SAR had a good linear relationship with the temperature rise. It was found from the result that the RF heating of metallic implants can take place in various kinds of material and the increase of SAR has a good linear relationship with the temperature rise. This experience shows that reduction of SAR can decrease temperature of metallic implants.

An endo-1,4-β-glucanase from Bellamya chinensis laeta was purified to electrophoretically homogeneous state. The molecular weight of the purified enzyme was estimated 70,000 by SDS-PAGE. The enzyme was most active at pH 5.5 and 50 °C, and stable at around pH 10 and 50 °C. The enzyme exhibited the significant activity at 20 °C (30 % of the activity at optimal 50 °C). The enzyme was hydrolyzed cellohexaose into cellobiose, cellotriose, and cellotetraose as main products. Three cDNAs (BC-EG70a, BC-EG70b, and BC-EG70c) encoding the endo-1,4-β-glucanase were cloned by PCR-based method. Three endo-1,4-β-glucanases consisted of 1758 bp encoding 586 amino acids. The three genes were almost the same nucleotide sequences. The deduced proteins were consisted of a signal sequence, cellulose binding domain, linker, and catalytic domain. The amino acid sequence of BC-EG70a shares sequence identity degree with the endo-1,4-β-glucanases of Haliotis discus hannai (61 %), Ampullaria crossean (52 %), and Mizuhopecten yessoensis (51 %) which all belong to glycoside hydrolase family 9.

A protein with strong removal activity against the natural estrogen estriol was purified from a culture supernatant of Pleurotus eryngii var. tuoliensis C.J. Mou. The protein was characterized as a laccase and had a molecular mass of 60kDa on SDS-PAGE. The enzyme was most active at pH 7.0 and 50°C. The partial N-terminal amino acid sequence of the enzyme showed homology with laccases from mushrooms, such as Pleurotus ostreatus, Coriolus versicolor (current name: Trametes versicolor), Pycnoporus cinnabarinus, and P. eryngii. A recombinant yeast assay confirmed that laccase treatment was very efficient for removing the estrogenic activity of steroid estrogens. Our results suggest that the enzyme may be applicable as a potential factor for removing natural steroid hormones.

Thermal injuries have been sometimes reported due to a closed conducting loop formed in a part of the patient's body during magnetic resonance imaging (MRI). In recent years, 3.0 T-MRI scanner has been widely used. However, it is considered that the specific absorption rate (SAR) of 3.0 T-MRI can affect the heat of the loop because its own SAR becomes approximately 4 times as much as that of the1.5 T-MRI scanner. With this, the change in temperature was measured with human body-equivalent loop phantom in both 1.5 T-MRI and 3.0 T-MRI. In the two scanners, the temperature during 20 min of scanning time was measured with three types of sequences such as field echo (FE), spin echo (SE), and turbo SE (TSE) set up with the same scanning condition. It was found from the result that rise in temperature depended on SAR of the scanning condition irrespective of static magnetic field intensity and any pulse sequences. Furthermore, the increase of SAR and rise in temperature were not only in proportion to each other but also were indicated to have good correlation. However, even low SAR can occasionally induce serious thermal injuries. It was found from result that we had to attempt not to form a closed conducting loop with in a part of the patient's body during MRI.

Previously, we have reported that purification and characterization of an anti-plant viral serine protease (EF-SP1) from Eisenia foetida . In this study, a new serine protease (EF-SP2) showing strong antiviral activities against cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) was purified from the coelomic fluid of the earthworm E. foetida . The activity of EF-SP2 was suppressed by various known serine protease inhibitors, suggesting that the EF-SP2 is a serine protease. Its molecular weight was estimated to be 26,000 by SDS–PAGE, and its optimal pH and temperature were pH 9.5 and 60 °C, respectively. N-terminal amino acid sequence of EF-SP2 was the same as those of E. foetida serine proteases (EFE-d and EFE-e) with fibrinolytic activity, but different from that of EF-SP1. The enzymatic properties of anti-plant serine proteases (EF-SP1 and EF-SP2) and fibrinolytic enzymes (EFE-d and EFE-e) were similar to each other, e.g., substrate specificity, molecular weight, and effect of inhibitors. Our results suggest that EF-SP2 as well as EF-SP1 can be also applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.

1,3-β- d -glucanase from the earthworm Eisenia foetida was purified to electrophoretically homogeneous state. The molecular weight of the purified enzyme was estimated 42,000 by SDS-PAGE. The N-terminal amino acid sequence of the enzyme was very similar to those of CCF-I from E. foetida and CCF-like protein from Aprorrectodea caliginosa . The enzyme was most active at pH 6.0 and 60 °C, and stable at pH 6.0–10.5 and 60 °C. The enzyme was inhibited by metal ions Mn 2+ , Cu 2+ , Fe 2+ , Al 3+ , and hydrolyzed 1,3-β- d -linked oligosaccharides (triose, tetraose, and pentose) into glucose, and biose as end products.

The glyoxylate cycle is a modified form of the tricarboxylic acid cycle, which enables organisms to synthesize carbohydrates from C2 compounds. In the protozoan Euglena gracilis, the key enzyme activities of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), are conferred by a single bifunctional protein named glyoxylate cycle enzyme (Euglena gracilis glyoxylate cycle enzyme [EgGCE]). We analyzed the enzymatic properties of recombinant EgGCE to determine the functions of its different domains. The 62-kDa N-terminal domain of EgGCE was sufficient to provide the MS activity as expected from an analysis of the deduced amino acid sequence. In contrast, expression of the 67-kDa C-terminal domain of EgGCE failed to yield ICL activity even though this domain was structurally similar to ICL family enzymes. Analyses of truncation mutants suggested that the N-terminal residues of EgGCE are critical for both the ICL and MS activities. The ICL activity of EgGCE increased in the presence of micro-molar concentrations of acetyl-coenzyme A (CoA). Acetyl-CoA also increased the activity in a mutant type EgGCE with a mutation at the acetyl-CoA binding site in the MS domain of EgGCE. This suggests that acetyl-CoA regulates the ICL reaction by binding to a site other than the catalytic center of the MS reaction.

In this study, we cloned the gene encoding goose-type (G-type) lysozyme with chitinase (Ra-ChiC) activity from Ralstonia sp. A-471 genomic DNA library. This is the first report of another type of chitinase after the previously reported chitinases ChiA (Ra-ChiA) and ChiB (Ra-ChiB) in the chitinase system of the moderately thermophilic bacterium, Ralstonia sp. A-471 and also the first such data in Ralstonia sp. G-type lysozyme gene. It consisted of 753 bp nucleotides, which encodes 251 amino acids including a putative signal peptide. This ORF was modular enzyme composed of a signal sequence, chitin-binding domain, linker, and catalytic domain. The catalytic domain of Ra-ChiC showed homologies to those of G-type lysozyme (glycoside hydrolases (GH) family 23, 16.8%) and lysozyme-like enzyme from Clostridium beijerincki (76.1%). Ra-ChiC had activities against ethylene glycol chitin, carboxyl methyl chitin, and soluble chitin but not against the cell wall of Micrococcus lysodeikticus. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the enzyme, the second and third glycosidic linkage from the non-reducing end were split producing (GlcNAc)2 + (GlcNAc)4 and (GlcNAc)3 + (GlcNAc)3 of almost the same concentration in the early stage of the reaction. The G-type lysozyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3 + (GlcNAc)3 predominating over that to (GlcNAc)2 + (GlcNAc)4. Thus, Ra-ChiC was found to be a novel enzyme in its structural and functional properties.

A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40-50 degrees C. The protease activity at 4 degrees C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.

We previously described four α- l -arabinofuranosidases (ABFs) secreted by Penicillium chrysogenum 31B. Here, we cloned the fifth and sixth genes (Pcabf43B and Pcabf51C) encoding the ABFs PcABF43B and PcABF51C in this strain and overexpressed these genes in Escherichia coli. The deduced amino acid sequences of PcABF43B and PcABF51C were highly similar to putative ABFs belonging to glycoside hydrolase families 43 and 51, respectively. Semiquantitative reverse transcription polymerase chain reaction indicated that both genes were induced by arabinose, arabinitol, arabinan, and arabinoxylan; however, the Pcabf51C gene was constitutively expressed at low levels in P. chrysogenum 31B. PcABF43B had optimal activity at 20 °C and pH 5–6, indicating that this enzyme was psychrophilic and had the lowest optimal temperature reported for ABFs. PcABF51C had optimal activity at 45 °C and pH 6–7. Both recombinant enzymes showed high activity on arabino-oligosaccharides, but little activity on arabinose-containing polysaccharides, such as l -arabinan. Next, we compared the substrate specificities of PcABF43B, PcABF51C, and AFQ1, a P. chrysogenum ABF that preferentially degraded oligosaccharides over polysaccharides. PcABF43B was found to preferentially hydrolyze (1→3)-linkages in branched arabino-oligosaccharides and released only a small amount of arabinose from linear α-1,5-arabino-oligosaccharides. In contrast, AFQ1 and PcABF51C showed higher activities on linear arabino-oligosaccharides than on branched arabino-oligosaccharides. AFQ1 showed high catalytic efficiencies for α-1,5- l -arabinofuranobiose (α-1,5-Ara2) and α-1,5- l -arabinofuranotriose (α-1,5-Ara3) at the same level. In contrast, intracellular PcABF51C showed much higher catalytic efficiency for α-1,5-Ara2 than for α-1,5-Ara3.

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca2+ and Mn2+ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.