Authorship：Lead author,　Corresponding author   Language：Japanese  

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Publisher：Oxford University Press (OUP)  

DOI： 10.1093/pcp/pcz014

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Authorship：Corresponding author   Publisher：OMICS Publishing Group  

DOI： 10.4172/2329-9029.1000220

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：OMICS Publishing Group  

DOI： 10.4172/2329-9029.1000226

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Language：English   Publisher：Elsevier GmbH.  

DOI： 10.1016/j.reach.2017.07.001

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DOI： 10.4172/2329-9029.1000199

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

Rice seeds were exposed outside of the international space station to assess the risk of space environment exposure on gene expression associated with seed germination. The germination percentages of the space-stored and ground-stored seeds exposed for 13 months were 48 and 96% respectively. Those for 20 months were 7 and 76%, respectively. Germination was defined 3 days after imbibition, except for the space-stored seeds exposed for 20 months, which germinated 5 days after imbibition. Subsequent RNA-seq analyses of the dry seeds, germinated seeds, and roots and shoots of seedlings revealed that the mutation rates of mRNA sequences were not significantly different between space-stored and ground-stored samples exposed for 13 months and 20 months. In all, 4 and 16 transcripts of glycolysis-related genes were increased in the germinated seeds after 13-month and 20-month exposure, respectively. Also, 2 and 39 transcripts of long-lived mRNA required for germination were decreased more than 2-fold in the dry seeds after 13-month and 20-month exposure, respectively. These results suggest that damage to long-lived mRNA in seeds by a space environment delays and reduces germination.

DOI： 10.1016/j.lssr.2016.10.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Putative nudix hydrolase (NUDX) genes, which encode amino acid sequences showing homology with those of Arabidopsis NUDXs and conserve nudix motif, were identified from barley. The 14 deduced barley NUDXs (HvNUDX1-14) were classified into established subfamilies, except for 8-oxo-deoxyguanosine 5 '-triphosphate (8-oxo-dGTP) pyrophosphohydrolase and mRNA decapping enzyme subfamilies, and three substrate-unknown subfamilies. Drought and UV-C stresses, respectively, up-regulated 7 and 4 HvNUDX genes, but some homologs of Arabidopsis NUDXs showed different responses to abiotic stress. HvNUDX12 gene, belonging to diadenosine tetraphosphates (Ap(4)A) pyrophosphohydrolase subfamily gene and up-regulated by UV-C, was expressed in Escherichia coli cells. The recombinant protein showed 8-oxo-dGTP, Ap(4)A, and guanosine-3 ',5 '-tetraphosphate (ppGpp) pyrophosphohydrolase activities, and the suppression of the lacZ amber mutation in a mutT-deficient E. coli cells caused by the incorporation of 8-oxo-GTP into mRNA was prevented to a significant degree. These results suggest that barley NUDXs have unique constitution and response of NUDX to abiotic stress.

DOI： 10.1080/09168451.2014.978259

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Spaceflight environment have been shown to generate reactive oxygen species (ROS) and induce oxidative stress in plants, but little is known about the gene expression of the ROS gene network in plants grown in long-term spaceflight. The molecular response and adaptation to the spaceflight environment of Mizuna plants harvested after 27 days of cultivation onboard the International Space Station (ISS) were measured using genome-wide mRNA expression analysis (mRNA-Seq).
Results: Total reads of transcripts from the Mizuna grown in the ISS as well as on the ground by mRNA-Seq showed 8,258 and 14,170 transcripts up-regulated and down-regulated, respectively, in the space-grown Mizuna when compared with those from the ground-grown Mizuna. A total of 20 in 32 ROS oxidative marker genes were up-regulated, including high expression of four hallmarks, and preferentially expressed genes associated with ROS-scavenging including thioredoxin, glutaredoxin, and alternative oxidase genes. In the transcription factors of the ROS gene network, MEKK1-MKK4-MPK3, OXI1-MKK4-MPK3, and OXI1-MPK3 of MAP cascades, induction of WRKY22 by MEKK1-MKK4-MPK3 cascade, induction of WRKY25 and repression of Zat7 by Zat12 were suggested. RbohD and RbohF genes were up-regulated preferentially in NADPH oxidase genes, which produce ROS.
Conclusions: This large-scale transcriptome analysis revealed that the spaceflight environment induced oxidative stress and the ROS gene network activation in the space-grown Mizuna. Among transcripts altered in expression by space conditions, some were common genes response to abiotic and biotic stress. Furthermore, certain genes were exclusively up-regulated in Mizuna grown on the ISS. Surprisingly, Mizuna grew in space normally, as well as on the ground, demonstrating that plants can acclimate to long-term exposure in the spaceflight environment by reprogramming the expression of the ROS gene network.

DOI： 10.1186/1471-2229-14-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Barley seeds were exposed to outer space for 13 months in a vented metal container without a climate control system to assess the risk of physiological and genetic mutation during long-term storage in space. The space-stored seeds (SO generation), with an 82% germination rate in 50 seeds, lost about 20% of their weight after the exposure. The germinated seeds showed normal growth, heading, and ripening. The harvested seeds (SI generation) also germinated and reproduced (S2 generation) as did the ground-stored seeds. The culm length, ear length, number of seed, grain weight, and fertility of the plants from the space-stored seeds were not significantly different from those of the ground-stored seeds in each of the SO and SI generation. Furthermore, the SI and S2 space-stored seeds respectively showed similar beta-glucan content to those of the ground-stored seeds. Amplified fragment length polymorphism analysis with 16 primer combinations showed no specific fragment that appears or disappears significantly in the DNA isolated from the barley grown from the space-stored seeds. Though these data are derived from nine SO space-stored seeds in a single exposure experiment, the results demonstrate the preservation of barley seeds in outer space for 13 months without phenotypic or genotypic changes and with healthy and vigorous growth in space. (C) 2011 COSPAR. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.asr.2011.05.017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The RF SRC-Institute of Biomedical Problems, Russian Academy of Sciences, developed Biorisk hardware to study the effects of long-term exposure of dormant forms of various organisms to outer space and used it to complete a series of experiments on the Russian Module (RM) of the International Space Station (ISS).
The experiments were performed using prokaryotes (Bacillus bacteria) and eukaryotes (Penicillium, Aspergillus, and Cladosporium fungi), as well as spores, dormant forms of higher plants, insects, lower crustaceans, and vertebrates. The biological samples were housed in two containers that were exposed to outer space for 13 or 18 months. The results of the 18-month experiment showed that, in spite of harsher temperature than in the first study, most specimens remained viable.
These experiments provided evidence that not only bacterial and fungal spores but also dormant forms of organisms that reached higher levels of evolutionary development had the capability to survive a long-term exposure to outer space. This observation suggests that they can be transferred on outer walls of space platforms during interplanetary missions. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.actaastro.2010.05.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MAIK NAUKA/INTERPERIODICA/SPRINGER  

The transcriptome of barley Hordeum vulgare grown aboard the International Space Station was studied using microarray analysis. In the spaceflight environment, mRNA levels of over 500 genes were changed more than twofold; among them, genes of stress response proteins, in particular, heat shock proteins, pathogenesis-related proteins, and antioxidant proteins. Further analysis by real-time PCR confirmed enhanced transcription of reactive oxygen species scavenging genes. The superoxide dismutase (sod) mRNA level in the space environment was 6-fold higher than in earth conditions. The transcript levels of glutamyl transferase (gst), catalase (cat), and ascorbate peroxidase (apx) were increased in spaceflight 24, 18, and 3 times in comparison to ground control, respectively. For the first time, it has been shown that spaceflight environment can induce oxidative stress in plants.

DOI： 10.1134/S0026893310050080

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The gene expression and enzyme activity of superoxide dismutase, catalase, and ascorbate peroxidase in the space-grown barley were not significantly different from those of the ground-grown barley. Cu2+ reducing and radical scavenging activities in an extract of the space-grown barley were lower than those of the ground-grown barley by 0.7 fold, suggesting that the space environment does not induce oxidative stress, and reduces antioxidant capacity in plants.

DOI： 10.1271/bbb.100139

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Comparative two-dimensional electrophoresis showed six proteins, which were significantly produced in the root of salt-tolerant barley. These proteins were identified as stress/defense-related proteins that do not scavenge reactive oxygen species directly, suggesting that salt-tolerant barley develops not only an antioxidative system, but also physical and biochemical changes to cope with salt stress.

DOI： 10.1271/bbb.90456

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1134/S0012496609030223

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Arabidopsis plants transformed by promoter of A. thaliana serine racemase fused with beta-glucuronidase (GUS) reporter gene showed strong GUS staining in elongating and developing cells such as tip regions of primary and lateral roots, developing leaves, and shoot meristems. RT-PCR and digital northern hybridization showed that expression of the serine racemase gene was not induced by l- and d-serine, light irradiation, biotic and abiotic stresses.

DOI： 10.1007/s00726-008-0112-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER GMBH, URBAN & FISCHER VERLAG  

The growth kinetics and variations in cell wait matrix polysaccharides and glycoside hydrolases during seedling development of the drought-tolerant wheat cultivar (cv. Hong Mang Mai) were compared with the drought-sensitive cultivar (cv. Shirasagi-komugi). After 15d of culture in water at 22 degrees C under constant irradiance of 98 mu mol m(-2) s(-1), the length of the coleoptile and leaf sheath of Hong Mang Mai seedlings was 1.7 times longer than those of Shirasagikomugi seedlings. In the cell watts isolated from coleoptiles and Leaf sheaths of the seedling of the two cultivars, the contents of arabinose, xylose, and glucose changed during development. The cell walls were fractionated progressively with 50 mM CDTA, 50 mM Na(2)CO(3), 1M KOH and 4M KOH, and sugar composition was determined. The amount of CDTA-soluble fraction from the Hong Mang Mai cell walls was 2.4-fold higher than that from the Shirasagikomugi cell walls at 6d of culture, and a considerable decrease was observed during development. The ratio of arabinose to xylose in 1M KOH-soluble fraction from the two cultivars decreased. The amount of 4M KOH-soluble fraction from the Shirasagikomugi cell watts was affected much more than those of the Hong Mang Mai cell watts. Many glycoside hydrolase activities were detected in the protein fractions from coleoptiles and leaf sheaths of the two cultivars, and the activities of licheninase, 1,3-1,4-beta-glucanase, and 1,3-beta-glucanase in the LiCl-soluble protein fraction increased drastically during development of the Shirasagikomugi seedlings. These findings suggest that the metabolism of the cell wall matrix polysaccharides of the drought-tolerant wheat cultivar is far different from that of the drought-sensitive wheat cultivar during seedling development. (c) 2007 Elsevier GmbH. All rights reserved.

DOI： 10.1016/j.jplph.2007.07.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Several D-amino acids have been identified in plants. However, the biosynthetic pathway to them is unclear. In this study, we cloned and sequenced a cDNA encoding a serine racemase from barley which contained an open reading frame encoding 337 amino acid residues. The deduced amino acid sequence showed significant identity to plant and mammalian serine racemases and contained conserved pyridoxal 5-phosphate (PLP)-binding lysine and PLP-interacting amino acid residues. The purified gene product catalyzed not only racemization of serine but also dehydration of serine to pyruvate. The enzyme requires PLP and divalent cations such as Ca2+, Mg2+, or Mn2+, but not ATP, whereas mammalian serine racemase activity is increased by ATP. In addition to the results regarding the effect of ATP on enzyme activity and the phylogenetic analysis of eukaryotic serine racemases, the antiserum against Arabidopsis serine racemase did not form a precipitate with barley and rice serine racemases. This suggests that plant serine racemases represent a distinct group in the eukaryotic serine racemase family and can be clustered into monocot and dicot types. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2007.03.040

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5'-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca2+, Mg2+, Mn2+, Fe2+, or Ni2+, at alkaline pH for both activities. The racemization process is highly specific toward L-serine, whereas L-alanine, L-arginine, and L-glutamine were poor substrates. The V-max/K-m values for racemase activity of L- and D-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for L- and D-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward L-serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2006.01.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

An enzyme catalyzing the hydrolysis of triacylglycerol was purified from an earthworm. The N-terminal amino acid sequence and the catalytic function of the purified enzyme were identical to those of Isozyme C, an isozyme of the earthworm-serine proteases. No other lipase proteins were found in the earthworm cells. The isozyme might act on the hydrolysis of triacylglycerol as well as the protein decomposition.

DOI： 10.1271/bbb.69.2009

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Language：Japanese   Publisher：水産育種研究会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

cDNAs expressed preferentially in an Al-tolerant microorganism were isolated by subtraction hybridization with cDNAs of Al-sensitive Penicillium chrysogenum IFO4626 as driver cDNA and cDNAs of the Al-tolerant mutant derived from the wild cells by UV irradiation as tester cDNA. Northern blot analysis revealed that mRNA levels of six genes were increased significantly in the Al-tolerant mutant after exposure to Al stress when compared with the wild cells. Two genes accumulated in both the presence and absence of Al stress and four genes were induced by Al stress in the Al-tolerant mutant. cDNA fragments were amplified by rapid amplification of cDNA ends and sequenced to obtain full-length cDNAs of the six genes. Two genes were novel or predicted ones and the others showed significant homology to known genes, ADP/ATP translocase, enolase, cysteine synthase, and glucoamylase, which are induced by environmental stresses in prokaryotic and eukaryotic cells. These enzyme activities increased in the Al-tolerant mutant when compared to those in the wild cells, showing that not only the levels of gene expression but also the levels of enzyme activities increased in the Al-tolerant mutant. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/S0378-1097(03)00886-3

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Increased blood-DNA breakage was observed in diseased pearl oysters. They showed significant formation of 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA), whereas the oysters that had a low mortality rate from the disease had high activity of superoxide dismutase (SOD) and low amounts of 8-OHdG and MDA. These results suggest that radical damage had occurred only in the diseased pearl oysters with the cytolysis of their haemocytes, which was related to the mass mortality of the Japanese pearl oysters.

DOI： 10.1271/bbb.67.2470

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A cDNA encoding barley phospholipid hydroperoxide glutathione peroxidase (PHGPX)-like protein was cloned and sequenced by the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The cDNA comprised 846 bp, and included an open reading frame which encodes a polypeptide of 169 amino acid residues with a molecular mass of 18,532 Da. The deduced amino acid sequence showed significant identity to plant putative PHGPXs and mammalian PHGPXs. The cloned gene was expressed in Escherichia coli cells to produce an extra protein, which showed a molecular mass similar to the deduced one, and the clone cells were much more tolerant to NaCl stress than the host cells. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S1381-1177(03000104-8

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Language：English   Publisher：ELSEVIER SCIENCE BV  

An earthworm, Lumbricus rubellus, produces alkaline serine proteases that are greater than trypsins in their activity and stability. The proteases which were purified from the earthworm were composed of six isozyme proteins. Each isozyme consisted of a single polypeptide chain which was derived from the different genes. The enzymes had activity and were stable at below 60 degreesC over a wide range of pH 2-11 and were strongly resistant to organic solvents and detergents. Moreover, they retain full activity for long years at room temperature. They acted on various proteins, such as elastin as well as fibrin, and some peptides, such as beta-amyloid 1-40 and solubilized actual fibrin clots of whole blood in a rat's vena cava. They also catalyzed the hydrolysis of various esters. The cDNAs encoding the proteases were cloned and sequenced. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of an isozyme protein was expressed in Pichia pastoris to produce the active protease in the culture medium. The proteases contributed to the production of the "earthworm autolysate". The extracts of the autolysate could be used as a "peptone substitute" in media for the efficient growth of microorganisms. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S1381-1177(03)00082-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The amino acid sequence of the earthworm-serine protease, isozyme C, which shows not only elastase-like activity but also trypsin-like activity, was determined. The catalytic triad of the trypsin family, His, Asp, Ser, was conserved in isozyme C, but the primary substrate determinant of trypsin, Asp, was missing in isozyme C, the same as in elastase. One of the two Gly at the entrance of the substrate-binding pocket of trypsin was replaced by Val as in elastase, however, the other was replaced by Ser whereas Thr is present in elastase. Furthermore, isozyme C also showed esterase-like activity, which was applicable for the synthesis of useful substances. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S1381-1177(03)00105-X

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Language：English   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

The acylated plant pigments synthesized by lipasecatalyzed transesterification with aromatic acids were compared in respect of their light-resistance and radicalscavenging ability. With both the flavonols and anthocyanins, their acylated derivatives were more stable against illumination with fluorescent light than their non-acylated glucosides. Their radical-scavenging ability partially decreased or was retained by acylation to the glucoside molecules.

DOI： 10.1271/bbb.67.1828

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A cDNA encoding an O-methyltransferase (OMT) was isolated from salt-tolerant barley roots by subtraction hybridization with cDNAs of salt-tolerant barley roots as a tester cDNA and cDNAs of the salt-sensitive barley roots as a driver cDNA. The deduced amino acid sequence showed significant identity with plant caffeic acid/5-hydroxyferulic acid OMTs. Southern blot analysis showed that the OMT gene was a single copy in both salt-tolerant and -sensitive barley. The cloned gene was expressed in a wheat germ cell-free system to produce the OMT, which had methylating activity for caffeic acid. Northern blot analysis showed that the OMT gene was expressed constitutively in the salt-tolerant barley roots and the expression level was increased 1.5 times by salt stress, but the salt-sensitive barley showed no expression of the gene in roots and leaves.

DOI： 10.1271/bbb.67.966

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Transglucosylation activities of spinach alpha-glucosidase I and IV, which have different substrate specificity for hydrolyzing activity, were investigated. In a maltose mixture, alpha-glucosidase I, which has high activity toward not only maltooligosaccharides but also soluble starch and can hydrolyze isomaltose, produced maltotriose, isomaltose, and panose, and alpha-glucosidase IV, which has high activity toward maltooligosaccharides but faint activity toward soluble starch and isomaltose, produced maltotriose, kojibiose, and 2,4-di-alpha-D-glucosyl-glucose. Transglucosylation to sucrose by alpha-glucosidase I and IV resulted in the production of theanderose and erlose, respectively, showing that spinach alpha-glucosidase I and IV are useful to synthesize the alpha-1,6-glucosylated and alpha-1,2- and 1,4-glucosylated products, respectively.

DOI： 10.1271/bbb.67.1160

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The chimeric alpha-glucosidases of Mucor javanicus and Aspergillus oryzae, which has high activity toward not only maltooligosaccharides but also soluble starch and has high activity toward maltooligosaccharides but faint activity toward soluble starch, respectively, were constructed by shuffling the C-terminal regions where low homology is observed between the two enzymes. The chimera genes were expressed in Pichia pastoris to produce and secrete the enzymes that have predicted molecular masses in the culture medium. The two chimeric M javanicus alpha-glucosidases, of which the N- and C-terminal regions are substituted for those of A. oryzae, respectively, decreased in soluble starch-hydrolyzing activity, however, increased in maltose-hydrolyzing activity by 2.1 and 4.9 times higher than that of the native form of M javanicus alpha-glucosidase, respectively. The chimeric enzymes changed on the V-max values for maltose significantly, whereas the K-m values were similar to that of the native enzyme. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S1570-9639(02)00525-3

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Earthworm serine protease is more stable and is less affected by organic solvents and detergent than other proteases. However, it is inactivated, probably by autolysis, at 60degreesC or above under alkaline conditions. Further stabilization was managed by chemical modification of the enzyme with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and phenylglyoxal to protect the activity from the autolytic inactivation. Stabilization was possible also under acidic conditions, in which the stability of the enzyme was rather low, by immobilization with folded sheet mesoporous material. Thus, further stabilization of the enzyme has been achieved by chemical modification or immobilization.

DOI： 10.1271/bbb.66.2739

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Language：Japanese  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zu. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%).

DOI： 10.1128/JB.183.22.6694-6698.2001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：URBAN & FISCHER VERLAG  

Farnesyl pyrophosphate (FPP) synthase catalyzes the biosynthesis of geranyl pyrophosphate (GPP) and FPP, which are the precursors of terpenoids. such as essential oils, from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). gDNA and cDNA encoding hop (Humulus lupulus L.) FPP synthase was cloned by the Inverse PCR method and the Cassette-mediated PCR method and sequenced. The deduced amino acid sequence showed a high identity to plant FPP synthases. The cloned gene was expressed in Escherichia coli cells to produce the FPP synthase. Northern blot analysis revealed that the FPP synthase gene was expressed in the lupulin gland, bract, leaf, and stem of hop and expressed more strongly In the lupulin gland than in other tissues.

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An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents. cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship. The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively. The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by posttranslational processing. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.

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Various soil samples were collected to screen the presence of microorganisms which have ability to degrade TOE. One strain (AKU-883) with good TOE degrading activity was isolated and identified as Burkholderia cepacia and the extracellular enzyme was purified to homogeneity. The purification was achieved by ultrafiltration. Super Q anion-exchange chromatography and Superdex 200HR gel-filtration in the presence of Triton X. The enzyme was purified to 85-fold. and specific activity of 4.910 kU mg protein(-1). The peak preparation on gel filtration showed a single band of 34 kDa on SDS-PAGE and native PAGE which indicate the monomeric nature of the enzyme. The pr of the enzyme was 6.3. The enzyme showed the maximum activity at pH 9 and 65 degreesC. and was stable in the range of pH 5-10 and up to 60 degreesC. Almost all the activity (92%) was kept after incubation for more than 1 week at 50 degreesC (pH 7.3). High activities remained even in water-miscible solvents such as ethanol, dimethyl formamide, diisopropyl ether, and dioxane, The N-terminal 16 amino acid residues were determined as A-N-G-Y-A-A-T-R-Y-P-I-I-L-V-G-G. which showed a consensus sequence for lipases from Burkholderia species. Thus the enzyme was concluded to be a kind of lipase. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1097(01)00016-7

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Language：English   Publisher：FEDERATION AMER SOC EXP BIOL  

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A cDNA encoding spinach α-glucosidase was cloned and sequenced by the reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA comprised 2867 bp, and included an open reading frame which encodes a polypeptide of 903 amino acid residues. The calculated molecular mass of 101 kDa was larger than those of native α-glucosidases in spinach seeds, which are 78. 78, 82, and 82 kDa by SDS-PAGE for α-glucosidase I, II, III, and IV, respectively. The deduced amino acid sequence included those of tryptic peptides from native enzymes. Southern blot analysis suggested that the α-glucosidase gene was a single copy gene. These results indicate the possibility that the multiplicity of α-glucosidase in spinach occurs via post translational modification.

DOI： 10.1023/A:1005766003923

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Language：Japanese   Publisher：社団法人日本農芸化学会  

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Language：English   Publishing type：Research paper (scientific journal)  

The strongest fibrinolytic protease (F-III-2) in the six enzyme proteins purified from earthworm, Lumhricus rubellus [N. Nakajima et al., Biosci. Biotech. Biochem., 57, 1726–1730 (1993)] has been modified chemically with fragmented human serum albumin (mol. wt., 10,000–30,000). The modified enzyme lost the antigenicity of the native enzyme and reacted with the antisera against human serum albumin, the human serum albumin fragments, and the conjugate with the native enzyme to form precipitation lines, which fused with each other. The conjugate was significantly more resistant to inactivation by protease inhibitors in rat plasma. The enzyme was a non-hemorrhagic protein and did not induce platelet aggregation. The enzyme kept potent proteolytic activity for fibrin and fibrinogen than that of human plasmin. The enzyme easily solubilized actual fibrin clots (thrombi) of whole blood induced by thrombin in a rat’s vena cava. The continuous fibrinolysis for fibrin suspension in an enzyme reactor system using the modified enzyme immobilized to oxirane-activated acrylic beads has been achieved without any inactivation of the activity at least for more than 1 month. The N-terminal amino acid sequence of the protein was also investigated and the sequence showed local similarity to those of the serine proteases such as plasmin and chymotrvpsin. © 1996, Taylor &amp
Francis Group, LLC. All rights reserved.

DOI： 10.1271/bbb.60.293

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

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Four molecular forms of α-glucosidase were isolated from spinach seeds by several kinds of chromatography. The molecular masses of α-glucosidases I, II, III, and IV were 78, 78, 82, and 82kDa by SDS-PAGE, and 62, 62, 190, and 70kDa by gel filtration, respectively. α-Glucosidases I and II showed similar enzymatic properties, in which the Km for soluble starch was about 10 times lower than that for maltose, and they had higher activity not only toward malto-oliosaccharides but also toward α-glucans. The optimum pH was 4.5-5.5 and about 50% of the activity remained after incubation at 70°C for 20min. On the other hand, α-glucosidases III and IV showed similar enzymatic properties, in which the Km for maltose was 3-4 times lower than that for soluble starch, and they had high activity toward malto-oligosaccharides but faint activity toward α-glucans. The optimum pH was 4.5-5.0 and no activity was found after incubation at 70°C for 20min. However, anti-α-glucosidase III serum formed precipitation specifically with α-glucosidase III. © 1995, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.

DOI： 10.1271/bbb.59.673

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

The active site of α-glucosidase from Mucor javanicus IFO 4570 was investigated by kinetic studies. Competition between maltose and soluble starch, and linearity of Lineweaver-Burk plots for the mixed substrates were observed. The dependence of the apparent maximum velocities agreed with those predicted for a single active site mechanism. These results suggest that the enzyme hydrolyzes maltose and soluble starch at a single active site. © 1994 Taylor &amp
Francis Group LLC.

DOI： 10.1080/bbb.58.1535

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Language：English   Publishing type：Rapid communication, short report, research note, etc. (scientific journal)  

The rate parameters for maltooligosaccharides of α-glucosidase from Mucor javanicus were studied. The Km and k0 values (mM, s-1) for maltose, maltotriose, -tetraose, -pentaose, -hexaose, and -heptaose were estimated to be (0.60, 334)
(0.29, 271)
(0.27, 312)
(0.23, 285)
(0.14, 230)
(0.11, 184) in this order. The subsite affinities in the active site were 1.78, 5.31, 0.317, 0.126, 0.039, 0.168, and 0.002 kcal/mol for subsites 1, 2, 3, 4, 5, 6, and 7, respectively. From these results, it is considered that extended subsites exist in the enzyme, where maltooligosaccharides could be bound. © 1993, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.

DOI： 10.1271/bbb.57.1378

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We have developed a simple and efficient method for oligonucleotide-directed mutagenesis with double-stranded (plasmid) DNA as a template. The template was simply and rapidly prepared by cell lysis and the following DNA denaturation with alkali. The chain elongation was performed with phosphorothioate-modified nucleotide at 37°C. After the selective digestion of original DNA with NciI and exonuclease III, the desired mutated gene was obtained at a high frequency (about 70%). © 1989.

DOI： 10.1016/0003-2697(89)90134-6

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Language：English  

We have developed a simple and efficient method for oligonucleotide-directed mutagenesis with double-stranded (plasmid) DNA as a template. The template was simply and rapidly prepared by cell lysis and the following DNA denaturation with alkali. The chain elongation was performed with phosphorothioate-modified nucleotide at 37°C. After the selective digestion of original DNA with NciI and exonuclease III, the desired mutated gene was obtained at a high frequency (about 70%). © 1989.

DOI： 10.1016/0003-2697(89)90134-6

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Language：English   Publisher：ELSEVIER SCI IRELAND LTD  

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