DOCSLIB.ORG

(12) United States Patent (10) Patent No.: US 8,129,590 B2 Maranta Et Al

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(12) United States Patent (10) Patent No.: US 8,129,590 B2 Maranta Et Al US008129590B2 (12) United States Patent (10) Patent No.: US 8,129,590 B2 Maranta et al. (45) Date of Patent: Mar. 6, 2012 (54) POLYPEPTIDES HAVING ACETYLXYLAN (56) References Cited ESTERASE ACTIVITY AND POLYNUCLEOTDESENCODING SAME U.S. PATENT DOCUMENTS 5,681,732 A 10, 1997 De Graaffet al. (75) Inventors: Michelle Maranta, Davis, CA (US); 5,763,260 A * 6/1998 De Graaffet al. ............ 435,274 Kimberly Brown, Elk Grove, CA (US) FOREIGN PATENT DOCUMENTS EP O 507 369 3, 1992 (73) Assignee: Novozymes A/S, Bagsvaerd (DK) JP 2001054383. A 2, 2001 WO WO 2005/OO 1036 1, 2005 (*) Notice: Subject to any disclaimer, the term of this WO 2009073709 A1 6, 2009 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 196 days. Margolles-Clark et al., Accetyl Xylan esterase from Trichoderma reesei contains an active-site serine residue and a cellulose-binding (21) Appl. No.: 12/327,347 domain, 1996, Eur: J. Biochem. 237: 553-560. Henrissat B. A classification of glycosylhydrolases based on amino Filed: Dec. 3, 2008 acid sequence similarities, 1991, Biochem. J. 280: 309-316. (22) Henrissat et al., Updating the sequence-based classification of glycosyl hydrolases, 1996, Biochem. J. 316: 695-696. (65) Prior Publication Data Sundberg et al., 1991, Biotechnol. Appl. Biochem. 13: 1-11. US 201O/OO43105 A1 Feb. 18, 2010 Sundberg et al. Purification and Properties of Two Acetylxylan Esterases of Trichoderma reesei, 1991, Biotechnol. Appl. Biochem. 13: 1-11. Related U.S. Application Data Written Opinion of the International Searching Authority for PCT/ (60) Provisional application No. 60/992,995, filed on Dec. US2008/085.380. 6, 2007. * cited by examiner Primary Examiner — Brent T Page (51) Int. C. (74) Attorney, Agent, or Firm — Robert L. Starnes; Eric J. CI2N 15/31 (2006.01) Fechter CI2N 15/82 (2006.01) CI2N IS/10 (2006.01) (57) ABSTRACT CI2N 15/52 (2006.01) The present invention relates to isolated polypeptides having AOIH 5/00 (2006.01) acetylxylan esterase activity and isolated polynucleotides (52) U.S. C. ........ 800/288: 800/284; 800/295: 800/278: encoding the polypeptides. The invention also relates to 435/320.1; 435/468; 536/23.1:536/23.2: nucleic acid constructs, vectors, and host cells comprising the 536/23.7 polynucleotides as well as methods of producing and using (58) Field of Classification Search ........................ None the polypeptides. See application file for complete search history. 27 Claims, 4 Drawing Sheets U.S. Patent Mar. 6, 2012 Sheet 1 of 4 US 8,129,590 B2 JASÐVÌMÔXIIJ,EN3 LIZ G09 L0.9 IZL 998 U.S. Patent Mar. 6, 2012 Sheet 2 of 4 US 8,129,590 B2 g|-61-I 600I T80I GZZI U.S. Patent Mar. 6, 2012 Sheet 3 of 4 US 8,129,590 B2 Pst (7918) Na2Tpipromoter Bcil (7802) Bcli (7509) EcoRI (7494) Pst (7404) Bcf. (7288) s Aval (430) Pna2 minus TATA box ins AXE2 Bcli (6995 Aval (760) Sali (6980) NXmal (1125) Pmei (6975) Aval (1125) Apa Li (6297) Bell (1356) AMG Terminator WEcoRV (1440) coRV (1573) Clai (1956) Bcli (2014) Saci (2187) -Lactamase EcoRV (2266) Apa Li (2737) Apa L (5051) EcoRV (2856) ANDS Apa LI (4554) Pst (3498) Pnei (4328) Bam H. (3536) Pst (4072) mal (3780) Aval (3780) Smal (3782) Pst (3886) Fig. 2 U.S. Patent Mar. 6, 2012 Sheet 4 of 4 US 8,129,590 B2 HindIII (235) Kpni (245) Saci (251) Apa LI (4790 am Hl (253) CoE1 or EcoRI (284) st (690) Aval (736) Aval (1066) pHinsAXE2 5228 bp Xmal (1431) bla Aval (1431) Small (1433) SSEcoRI (1599) Apa L (3544) VPst (1608) EcoRV (1611) Not (1626) Ava (1632) Nicol (3177) Xbal (1644) kan f1 Ori Psti (2798) BCII (2586) Fig. 3 US 8,129,590 B2 1. 2 POLYPEPTDES HAVING ACETYLXYLAN Margolles-Clark et al., 1996, Eur: J. Biochem. 237: 553 ESTERASE ACTIVITY AND 560, disclose an acetylxylan esterase from Trichoderma POLYNUCLEOTDESENCODING SAME reesei. Sundberg and Poutanen, 1991, Biotechnol. Appl. Bio chem. 13: 1-11, disclose the purification and properties of two CROSS-REFERENCE TO RELATED acetylxylan esterases of Trichoderma reesei. WO 2005/ APPLICATION 001036 discloses an acetylxylan esterase gene from Tricho derma reesei. U.S. Pat. No. 5,681,732 discloses an acetylxy This application claims the benefit of U.S. Provisional lan esterase gene from Aspergillus niger. U.S. Pat. No. 5,763, Application No. 60/992,995, filed Dec. 6, 2007, which appli 260 discloses methods for altering the properties of cation is incorporated herein by reference. 10 acetylated Xylan. The present invention relates to polypeptides having REFERENCE TO ASEQUENCE LISTING acetylxylan esterase activity and polynucleotides encoding the polypeptides. This application contains a Sequence Listing in computer 15 readable form. The computer readable form is incorporated SUMMARY OF THE INVENTION herein by reference. The present invention relates to isolated polypeptides hav REFERENCE TO A DEPOSIT OF BIOLOGICAL ing acetylxylan esterase activity selected from the group con MATERIAL sisting of: (a) a polypeptide comprising an amino acid sequence hav This application contains a reference to a deposit of bio ing at least 75% identity to the mature polypeptide of SEQID logical material, which deposit is incorporated herein by ref NO: 2; CCC. (b) a polypeptide encoded by a polynucleotide that hybrid 25 izes under at least medium-high stringency conditions with BACKGROUND OF THE INVENTION (i) the mature polypeptide coding sequence of SEQID NO: 1. (ii) the cDNA sequence contained in the mature polypeptide 1. Field of the Invention coding sequence of SEQ ID NO: 1, or (iii) a full-length The present invention relates to isolated polypeptides hav complementary strand of (i) or (ii); ing acetylxylan esterase activity and isolated polynucleotides 30 (c) a polypeptide encoded by a polynucleotide comprising encoding the polypeptides. The invention also relates to a nucleotide sequence having at least 75% identity to the nucleic acid constructs, vectors, and host cells comprising the mature polypeptide coding sequence of SEQID NO: 1; and polynucleotides as well as methods of producing and using (d) a variant comprising a Substitution, deletion, and/or the polypeptides. insertion of one or more (several) amino acids of the mature 2. Description of the Related Art 35 Plant cell wall polysaccharides constitute generally 90% of polypeptide of SEQID NO: 2. the plant cell wall and can be divided into three groups: The present invention also relates to isolated polynucle cellulose, hemicellulose, and pectin. Cellulose represents the otides encoding polypeptides having acetylxylan esterase major constituent of cell wall polysaccharides. Hemicellulo activity, selected from the group consisting of ses are the second most abundant constituent of plant cell 40 (a) a polynucleotide encoding a polypeptide comprising an walls. The major hemicellulose polymer is xylan. The struc amino acid sequence having at least 75% identity to the ture of xylans found in cell walls of plants can differ signifi mature polypeptide of SEQID NO: 2: cantly depending on their origin, but they always contain a (b) a polynucleotide that hybridizes under at least medium beta-1,4-linked D-xylose backbone. The beta-1,4-linked high stringency conditions with (i) the mature polypeptide D-Xylose backbone can be substituted by various side groups, 45 coding sequence of SEQID NO: 1, (ii) the cDNA sequence Such as L-aribinose, D-galactose, acetyl, feruloyl, p-couma contained in the mature polypeptide coding sequence of SEQ royl, and glucuronic acid residues. ID NO: 1, or (iii) a full-length complementary strand of (i) or The biodegradation of the Xylan backbone depends on two (ii); classes of enzymes: endoxylanases and beta-xylosidases. (c) a polynucleotide comprising a nucleotide sequence Endoxylanases (EC 3.2.1.8) cleave the Xylan backbone into 50 having at least 75% identity to the mature polypeptide coding Smaller oligosaccharides, which can be further degraded to sequence of SEQID NO: 1; and xylose by beta-xylosidases (EC 3.2.1.37). Other enzymes (d) a polynucleotide encoding a variant comprising a Sub involved in the degradation of Xylan include, for example, stitution, deletion, and/or insertion of one or more (several) acetylxylan esterase, arabinase, alpha-glucuronidase, feru amino acids of the mature polypeptide of SEQID NO: 2. loyl esterase, and p-coumaric acid esterase. 55 The present invention also relates to nucleic acid con Acetylxylan esterase (EC 3.1.1.6) removes the O-acetyl structs, recombinant expression vectors, recombinant host groups from positions 2 and/or 3 on the beta-D-Xylopyrano cells comprising the polynucleotides, and methods of produc Syl residues of acetylxylan. Acetylxylan plays an important ing a polypeptide having acetylxylan esterase activity. role in the hydrolysis of Xylan because the acetyl side groups The present invention also relates to methods of inhibiting can interfere sterically with the approach of enzymes that 60 the expression of a polypeptide having acetylxylan esterase cleave the backbone. Removal of the acetyl side groups facili activity in a cell, comprising administering to the cell or tates the action of endoxylanases. A classification system for expressing in the cell a double-stranded RNA (dsRNA) mol carbohydrate esterases, based on sequence similarity, has led ecule, wherein the dsRNA comprises a subsequence of a to the definition of 13 families, seven of which contain polynucleotide of the present invention. The present also acetylxylan esterases (Henrissat B., 1991, Biochem. J. 280: 65 relates to a double-stranded inhibitory RNA (dsRNA) mol 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: ecule, wherein optionally the dsRNA is a siRNA or a miRNA 695-696). molecule. US 8,129,590 B2 3 4 The present invention also relates to methods for degrading pure, preferably at least 5% pure, more preferably at least a Xylan-containing material with a polypeptide having 10% pure, more preferably at least 20% pure, more preferably acetylxylan esterase activity.
Load more

Recommended publications
  • Advances in Chitin/Chitosan Characterization and Applications
    Advances in Chitin/Chitosan Characterization and Applications Edited by Marguerite Rinaudo and Francisco M. Goycoolea Printed Edition of the Special Issue Published in Polymers www.mdpi.com/journal/polymers Advances in Chitin/Chitosan Characterization and Applications Advances in Chitin/Chitosan Characterization and Applications Special Issue Editors Marguerite Rinaudo Francisco M. Goycoolea MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade Special Issue Editors Marguerite Rinaudo Francisco M. Goycoolea University of Grenoble Alpes University of Leeds France UK Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Polymers (ISSN 2073-4360) from 2017 to 2018 (available at: https://www.mdpi.com/journal/polymers/ special issues/chitin chitosan) For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Article Number, Page Range. ISBN 978-3-03897-802-2 (Pbk) ISBN 978-3-03897-803-9 (PDF) c 2019 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Special Issue Editors ..................................... ix Preface to ”Advances in Chitin/Chitosan Characterization and Applications” .........
    [Show full text]
  • 1471-2164-15-164.Pdf
    Meinhardt et al. BMC Genomics 2014, 15:164 http://www.biomedcentral.com/1471-2164/15/164 RESEARCH ARTICLE Open Access Genome and secretome analysis of the hemibiotrophic fungal pathogen, Moniliophthora roreri, which causes frosty pod rot disease of cacao: mechanisms of the biotrophic and necrotrophic phases Lyndel W Meinhardt1*, Gustavo Gilson Lacerda Costa2, Daniela PT Thomazella3, Paulo José PL Teixeira3, Marcelo Falsarella Carazzolle2, Stephan C Schuster4, John E Carlson5, Mark J Guiltinan6, Piotr Mieczkowski7, Andrew Farmer8, Thiruvarangan Ramaraj8, Jayne Crozier9, Robert E Davis10, Jonathan Shao10, Rachel L Melnick1, Gonçalo AG Pereira3 and Bryan A Bailey1 Abstract Background: The basidiomycete Moniliophthora roreri is the causal agent of Frosty pod rot (FPR) disease of cacao (Theobroma cacao), the source of chocolate, and FPR is one of the most destructive diseases of this important perennial crop in the Americas. This hemibiotroph infects only cacao pods and has an extended biotrophic phase lasting up to sixty days, culminating in plant necrosis and sporulation of the fungus without the formation of a basidiocarp. Results: We sequenced and assembled 52.3 Mb into 3,298 contigs that represent the M. roreri genome. Of the 17,920 predicted open reading frames (OFRs), 13,760 were validated by RNA-Seq. Using read count data from RNA sequencing of cacao pods at 30 and 60 days post infection, differential gene expression was estimated for the biotrophic and necrotrophic phases of this plant-pathogen interaction. The sequencing data were used to develop a genome based secretome for the infected pods. Of the 1,535 genes encoding putative secreted proteins, 1,355 were expressed in the biotrophic and necrotrophic phases.
    [Show full text]
  • Changes in the Sclerotinia Sclerotiorum Transcriptome During Infection of Brassica Napus
    Seifbarghi et al. BMC Genomics (2017) 18:266 DOI 10.1186/s12864-017-3642-5 RESEARCHARTICLE Open Access Changes in the Sclerotinia sclerotiorum transcriptome during infection of Brassica napus Shirin Seifbarghi1,2, M. Hossein Borhan1, Yangdou Wei2, Cathy Coutu1, Stephen J. Robinson1 and Dwayne D. Hegedus1,3* Abstract Background: Sclerotinia sclerotiorum causes stem rot in Brassica napus, which leads to lodging and severe yield losses. Although recent studies have explored significant progress in the characterization of individual S. sclerotiorum pathogenicity factors, a gap exists in profiling gene expression throughout the course of S. sclerotiorum infection on a host plant. In this study, RNA-Seq analysis was performed with focus on the events occurring through the early (1 h) to the middle (48 h) stages of infection. Results: Transcript analysis revealed the temporal pattern and amplitude of the deployment of genes associated with aspects of pathogenicity or virulence during the course of S. sclerotiorum infection on Brassica napus. These genes were categorized into eight functional groups: hydrolytic enzymes, secondary metabolites, detoxification, signaling, development, secreted effectors, oxalic acid and reactive oxygen species production. The induction patterns of nearly all of these genes agreed with their predicted functions. Principal component analysis delineated gene expression patterns that signified transitions between pathogenic phases, namely host penetration, ramification and necrotic stages, and provided evidence for the occurrence of a brief biotrophic phase soon after host penetration. Conclusions: The current observations support the notion that S. sclerotiorum deploys an array of factors and complex strategies to facilitate host colonization and mitigate host defenses. This investigation provides a broad overview of the sequential expression of virulence/pathogenicity-associated genes during infection of B.
    [Show full text]
  • Supplementary Table S1 List of Proteins Identified with LC-MS/MS in the Exudates of Ustilaginoidea Virens Mol
    Supplementary Table S1 List of proteins identified with LC-MS/MS in the exudates of Ustilaginoidea virens Mol. weight NO a Protein IDs b Protein names c Score d Cov f MS/MS Peptide sequence g [kDa] e Succinate dehydrogenase [ubiquinone] 1 KDB17818.1 6.282 30.486 4.1 TGPMILDALVR iron-sulfur subunit, mitochondrial 2 KDB18023.1 3-ketoacyl-CoA thiolase, peroxisomal 6.2998 43.626 2.1 ALDLAGISR 3 KDB12646.1 ATP phosphoribosyltransferase 25.709 34.047 17.6 AIDTVVQSTAVLVQSR EIALVMDELSR SSTNTDMVDLIASR VGASDILVLDIHNTR 4 KDB11684.1 Bifunctional purine biosynthetic protein ADE1 22.54 86.534 4.5 GLAHITGGGLIENVPR SLLPVLGEIK TVGESLLTPTR 5 KDB16707.1 Proteasomal ubiquitin receptor ADRM1 12.204 42.367 4.3 GSGSGGAGPDATGGDVR 6 KDB15928.1 Cytochrome b2, mitochondrial 34.9 58.379 9.4 EFDPVHPSDTLR GVQTVEDVLR MLTGADVAQHSDAK SGIEVLAETMPVLR 7 KDB12275.1 Aspartate 1-decarboxylase 11.724 112.62 3.6 GLILTLSEIPEASK TAAIAGLGSGNIIGIPVDNAAR 8 KDB15972.1 Glucosidase 2 subunit beta 7.3902 64.984 3.2 IDPLSPQQLLPASGLAPGR AAGLALGALDDRPLDGR AIPIEVLPLAAPDVLAR AVDDHLLPSYR GGGACLLQEK 9 KDB15004.1 Ribose-5-phosphate isomerase 70.089 32.491 32.6 GPAFHAR KLIAVADSR LIAVADSR MTFFPTGSQSK YVGIGSGSTVVHVVDAIASK 10 KDB18474.1 D-arabinitol dehydrogenase 1 19.425 25.025 19.2 ENPEAQFDQLKK ILEDAIHYVR NLNWVDATLLEPASCACHGLEK 11 KDB18473.1 D-arabinitol dehydrogenase 1 11.481 10.294 36.6 FPLIPGHETVGVIAAVGK VAADNSELCNECFYCR 12 KDB15780.1 Cyanovirin-N homolog 85.42 11.188 31.7 QVINLDER TASNVQLQGSQLTAELATLSGEPR GAATAAHEAYK IELELEK KEEGDSTEKPAEETK LGGELTVDER NATDVAQTDLTPTHPIR 13 KDB14501.1 14-3-3
    [Show full text]
  • Characterization of Acetylxylan Esterase from White-Rot Fungus Irpex Lacteus
    J. Appl. Glycosci., 66, 131–137 (2019) doi: 10.5458/jag.jag.JAG-2019_0007 ©2019 The Japanese Society of Applied Glycoscience Regular Paper Characterization of Acetylxylan Esterase from White-Rot Fungus Irpex lacteus Acetylxylan Esterase from Irpex lacteus (Received May 8, 2019; Accepted September 5, 2019) (J-STAGE Advance Published Date: October 24, 2019) Sangho Koh,1 Seika Imamura,2 Naoto Fujino,3 Masahiro Mizuno,1,2,4, † Nobuaki Sato,5 Satoshi Makishima,4,5 Peter Biely,6 and Yoshihiko Amano1,2,4 1Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University (4–17–1 Wakasato, Nagano 380–8553, Japan) 2Department of Biomedical Engineering, Graduate School of Science and Technology, Shinshu University (4–17–1 Wakasato, Nagano 380–8553, Japan) 3Biomaterial in Tokyo Co., Ltd. (5–23–32 Seki Bidg. 4F, Higashioi, Shinagawa-ku, Tokyo 140–0011, Japan) 4Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu University (4–17–1 Wakasato, Nagano 380–8553, Japan) 5B Food Science Co., Ltd. (24–12, Kitamachi, Chita, Aichi 478–0046, Japan) 6Institute of Chemistry, Slovak Academy of Sciences (Dúbravská cesta 9, SK 845 38 Bratislava, Slovak Republic) Abstract: The carbohydrate esterase family 1 (CE1) in CAZy contains acetylxylan esterases (AXEs) and feruloyl esterases (FAEs). Here we cloned a gene coding for an AXE belonging to CE1 from Irpex lacteus (IlAXE1). IlAXE1 was heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified and characterized. IlAXE1 hydrolyzed p-nitrophenyl acetate, α-naphthyl acetate and 4- methylumbelliferyl acetate, however, it did not show any activity on ethyl ferulate and methyl p- coumarate.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 8,561,811 B2 Bluchel Et Al
    USOO8561811 B2 (12) United States Patent (10) Patent No.: US 8,561,811 B2 Bluchel et al. (45) Date of Patent: Oct. 22, 2013 (54) SUBSTRATE FOR IMMOBILIZING (56) References Cited FUNCTIONAL SUBSTANCES AND METHOD FOR PREPARING THE SAME U.S. PATENT DOCUMENTS 3,952,053 A 4, 1976 Brown, Jr. et al. (71) Applicants: Christian Gert Bluchel, Singapore 4.415,663 A 1 1/1983 Symon et al. (SG); Yanmei Wang, Singapore (SG) 4,576,928 A 3, 1986 Tani et al. 4.915,839 A 4, 1990 Marinaccio et al. (72) Inventors: Christian Gert Bluchel, Singapore 6,946,527 B2 9, 2005 Lemke et al. (SG); Yanmei Wang, Singapore (SG) FOREIGN PATENT DOCUMENTS (73) Assignee: Temasek Polytechnic, Singapore (SG) CN 101596422 A 12/2009 JP 2253813 A 10, 1990 (*) Notice: Subject to any disclaimer, the term of this JP 2258006 A 10, 1990 patent is extended or adjusted under 35 WO O2O2585 A2 1, 2002 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 13/837,254 Inaternational Search Report for PCT/SG2011/000069 mailing date (22) Filed: Mar 15, 2013 of Apr. 12, 2011. Suen, Shing-Yi, et al. “Comparison of Ligand Density and Protein (65) Prior Publication Data Adsorption on Dye Affinity Membranes Using Difference Spacer Arms'. Separation Science and Technology, 35:1 (2000), pp. 69-87. US 2013/0210111A1 Aug. 15, 2013 Related U.S. Application Data Primary Examiner — Chester Barry (62) Division of application No. 13/580,055, filed as (74) Attorney, Agent, or Firm — Cantor Colburn LLP application No.
    [Show full text]
  • ABSTRACT SHU, XIAOMEI. Pathogenesis and Host Response
    ABSTRACT SHU, XIAOMEI. Pathogenesis and Host Response During Infection of Maize Kernels by Aspergillus flavus and Fusarium verticillioides. (Under the direction of Dr. Gary A. Payne.) Developing maize kernels are vulnerable to colonization by microbes. When colonization allows proliferation of the microbe at the expense of the host, disease occurs. The ascomycete fungal pathogens Aspergillus flavus and Fusarium verticillioides are capable of colonizing maize kernels, causing ear rots and contamination of the kernel with mycotoxins. These diseases lead to significant losses of crop yield and quality, and constitute a threat to food safety and human health. Thus, the significance of these diseases has prompted extensive research efforts to understand these plant-parasite interactions. However, pathogenesis and resistance mechanisms remain poorly characterized, hampering the development of effective control strategies. No commercial maize lines are completely resistant to these fungi. We applied an integrated approach consisting of histology, in situ gene expression and transcriptional profiling to better understand the nature of the interactions that occur between maize kernels and these fungi. Maize inbred line B73 was hand pollinated and inoculated with either A. flavus or F. verticillioides by wounding the kernel with a needle bearing conidia. Histological staining of the kernel sections revealed fungal mycelium in kernels adjacent to the inoculation site by 48 hours post inoculation (hpi), and in all tissues at 96 hpi. Compared with F. verticillioides, A. flavus more aggressively colonized kernel tissue and formed a unique biofilm-like structure around the scutellum. Transcriptome profiling using RNA-sequencing (RNA-seq) coupled with pathway analysis showed that these fungi were recognized by the kernel tissues prior to visible colonization.
    [Show full text]
  • Analysis of Functional Genes in Carbohydrate Metabolic Pathway of Anaerobic Rumen Fungus Neocallimastix Frontalis PMA02
    1555 Asian-Aust. J. Anim. Sci. Vol. 22, No. 11 : 1555 - 1565 November 2009 www.ajas.info Analysis of Functional Genes in Carbohydrate Metabolic Pathway of Anaerobic Rumen Fungus Neocallimastix frontalis PMA02 Mi Kown* 2, Jaeyong Song1, 3, Jong K. Ha3, Hong-Seog Park4 and Jongsoo Chang1, * 1 Department of Agricultural Science, Korea National Open University, Korea ABSTRACT : Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastixfrontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five「-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated.
    [Show full text]
  • Biohydrogen Production by the Thermophilic Bacterium Caldicellulosiruptor Saccharolyticus: Current Status and Perspectives
    Life 2013, 3, 52-85; doi:10.3390/life3010052 OPEN ACCESS life ISSN 2075-1729 www.mdpi.com/journal/life Review Biohydrogen Production by the Thermophilic Bacterium Caldicellulosiruptor saccharolyticus: Current Status and Perspectives Abraham A. M. Bielen *, Marcel R. A. Verhaart, John van der Oost and Servé W. M. Kengen Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands; E-Mails: [email protected] (M.R.A.V.); [email protected] (J.O.); [email protected] (S.W.M.K.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +31-317-482-105; Fax: +31-317-483-829. Received: 5 December 2012; in revised form: 6 January 2013 / Accepted: 7 January 2013 / Published: 17 January 2013 Abstract: Caldicellulosiruptor saccharolyticus is one of the most thermophilic cellulolytic organisms known to date. This Gram-positive anaerobic bacterium ferments a broad spectrum of mono-, di- and polysaccharides to mainly acetate, CO2 and hydrogen. With hydrogen yields approaching the theoretical limit for dark fermentation of 4 mol hydrogen per mol hexose, this organism has proven itself to be an excellent candidate for biological hydrogen production. This review provides an overview of the research on C. saccharolyticus with respect to the hydrolytic capability, sugar metabolism, hydrogen formation, mechanisms involved in hydrogen inhibition, and the regulation of the redox and carbon metabolism. Analysis of currently available fermentation data reveal decreased hydrogen yields under non-ideal cultivation conditions, which are mainly associated with the accumulation of hydrogen in the liquid phase. Thermodynamic considerations concerning the reactions involved in hydrogen formation are discussed with respect to the dissolved hydrogen concentration.
    [Show full text]
  • Carboxylic Ester Hydrolases in Bacteria: Active Site, Structure, Function and Application
    crystals Review Carboxylic Ester Hydrolases in Bacteria: Active Site, Structure, Function and Application Changsuk Oh 1 , T. Doohun Kim 2,* and Kyeong Kyu Kim 1,* 1 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Korea; [email protected] 2 Department of Chemistry, College of Natural Science, Sookmyung Women’s University, Seoul 04310, Korea * Correspondence: [email protected] (T.D.K.); [email protected] (K.K.K.) Received: 4 October 2019; Accepted: 7 November 2019; Published: 14 November 2019 Abstract: Carboxylic ester hydrolases (CEHs), which catalyze the hydrolysis of carboxylic esters to produce alcohol and acid, are identified in three domains of life. In the Protein Data Bank (PDB), 136 crystal structures of bacterial CEHs (424 PDB codes) from 52 genera and metagenome have been reported. In this review, we categorize these structures based on catalytic machinery, structure and substrate specificity to provide a comprehensive understanding of the bacterial CEHs. CEHs use Ser, Asp or water as a nucleophile to drive diverse catalytic machinery. The α/β/α sandwich architecture is most frequently found in CEHs, but 3-solenoid, β-barrel, up-down bundle, α/β/β/α 4-layer sandwich, 6 or 7 propeller and α/β barrel architectures are also found in these CEHs. Most are substrate-specific to various esters with types of head group and lengths of the acyl chain, but some CEHs exhibit peptidase or lactamase activities. CEHs are widely used in industrial applications, and are the objects of research in structure- or mutation-based protein engineering. Structural studies of CEHs are still necessary for understanding their biological roles, identifying their structure-based functions and structure-based engineering and their potential industrial applications.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2012/0266329 A1 Mathur Et Al
    US 2012026.6329A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0266329 A1 Mathur et al. (43) Pub. Date: Oct. 18, 2012 (54) NUCLEICACIDS AND PROTEINS AND CI2N 9/10 (2006.01) METHODS FOR MAKING AND USING THEMI CI2N 9/24 (2006.01) CI2N 9/02 (2006.01) (75) Inventors: Eric J. Mathur, Carlsbad, CA CI2N 9/06 (2006.01) (US); Cathy Chang, San Marcos, CI2P 2L/02 (2006.01) CA (US) CI2O I/04 (2006.01) CI2N 9/96 (2006.01) (73) Assignee: BP Corporation North America CI2N 5/82 (2006.01) Inc., Houston, TX (US) CI2N 15/53 (2006.01) CI2N IS/54 (2006.01) CI2N 15/57 2006.O1 (22) Filed: Feb. 20, 2012 CI2N IS/60 308: Related U.S. Application Data EN f :08: (62) Division of application No. 1 1/817,403, filed on May AOIH 5/00 (2006.01) 7, 2008, now Pat. No. 8,119,385, filed as application AOIH 5/10 (2006.01) No. PCT/US2006/007642 on Mar. 3, 2006. C07K I4/00 (2006.01) CI2N IS/II (2006.01) (60) Provisional application No. 60/658,984, filed on Mar. AOIH I/06 (2006.01) 4, 2005. CI2N 15/63 (2006.01) Publication Classification (52) U.S. Cl. ................... 800/293; 435/320.1; 435/252.3: 435/325; 435/254.11: 435/254.2:435/348; (51) Int. Cl. 435/419; 435/195; 435/196; 435/198: 435/233; CI2N 15/52 (2006.01) 435/201:435/232; 435/208; 435/227; 435/193; CI2N 15/85 (2006.01) 435/200; 435/189: 435/191: 435/69.1; 435/34; CI2N 5/86 (2006.01) 435/188:536/23.2; 435/468; 800/298; 800/320; CI2N 15/867 (2006.01) 800/317.2: 800/317.4: 800/320.3: 800/306; CI2N 5/864 (2006.01) 800/312 800/320.2: 800/317.3; 800/322; CI2N 5/8 (2006.01) 800/320.1; 530/350, 536/23.1: 800/278; 800/294 CI2N I/2 (2006.01) CI2N 5/10 (2006.01) (57) ABSTRACT CI2N L/15 (2006.01) CI2N I/19 (2006.01) The invention provides polypeptides, including enzymes, CI2N 9/14 (2006.01) structural proteins and binding proteins, polynucleotides CI2N 9/16 (2006.01) encoding these polypeptides, and methods of making and CI2N 9/20 (2006.01) using these polynucleotides and polypeptides.
    [Show full text]
  • Mode of Action of Acetylxylan Esterase from Streptomyces Lividans: a Study with Deoxy and Deoxy-Fluoro Analogues of Acetylated Methyl H-D-Xylopyranoside
    Biochimica et Biophysica Acta 1622 (2003) 82–88 www.bba-direct.com Mode of action of acetylxylan esterase from Streptomyces lividans: a study with deoxy and deoxy-fluoro analogues of acetylated methyl h-D-xylopyranoside Peter Bielya,*,Ma´ria Mastihubova´ a, Gregory L. Coˆte´ b, Richard V. Greeneb a Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 84238 Bratislava, Slovak Republic b Fermentation Biotechnology Research Unit, National Center for Agricultural Research, Agricultural Research Service, United States Department of Agriculture, Peoria, IL, USA Received 6 March 2003; received in revised form 26 May 2003; accepted 12 June 2003 Abstract Streptomyces lividans acetylxylan esterase removes the 2- or 3-O-acetyl groups from methyl 2,4-di-O-acetyl- and 3,4-di-O-acetyl h-D- xylopyranoside. When the free hydroxyl group was replaced with a hydrogen or fluorine, the rate of deacetylation was markedly reduced, but regioselectivity was not affected. The regioselectivity of deacetylation was found to be independent of the prevailing conformation of the substrates in solution as determined by 1H-NMR spectroscopy. These observations confirm the importance of the vicinal hydroxyl group and are consistent with our earlier hypothesis that the deacetylation of positions 2 and 3 may involve a common ortho-ester intermediate. Another possible role of the free vicinal hydroxyl group could be the activation of the acyl leaving group in the deacetylation mechanism. Involvement of the free hydroxyl group in the enzyme–substrate binding is not supported by the results of inhibition experiments in which methyl 2,4-di-O-acetyl h-D-xylopyranoside was used as substrate and its analogues or methyl h-D-xylopyranoside as inhibitors.
    [Show full text]