DOCSLIB.ORG

Combining Functional Metagenomics and Glycoanalytics to Identify Enzymes That Facilitate Structural Characterization of Sulfated N‑Glycans Léa Chuzel1,2 , Samantha L

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Combining Functional Metagenomics and Glycoanalytics to Identify Enzymes That Facilitate Structural Characterization of Sulfated N‑Glycans Léa Chuzel1,2 , Samantha L Chuzel et al. Microb Cell Fact (2021) 20:162 https://doi.org/10.1186/s12934-021-01652-w Microbial Cell Factories RESEARCH Open Access Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans Léa Chuzel1,2 , Samantha L. Fossa2, Madison L. Boisvert2, Samanta Cajic1 , René Hennig3 , Mehul B. Ganatra2, Udo Reichl1,4 , Erdmann Rapp1,3 and Christopher H. Taron2* Abstract Background: Sulfate modifcation of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modifcation and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifcations is hampered in part by a lack of enzymes that enable site-specifc detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fuorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO4 on N-glycans. Results: Our screen identifed a sugar-specifc sulfatase that specifcally removes sulfate from GlcNAc-6-SO4 when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated gly- can but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO4 (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated. Conclusion: The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifcations of glycans. The discovered enzymes represent new specifcities that can help resolve the presence of GlcNAc-6-SO4 in N-glycan structural analyses. Keywords: Sulfatase, Functional metagenomics, Glycoanalytics, Glycan analysis, Human microbiome, Glycan sulfation, N-acetylglucosamine-6-sulfate Background glycans (O-glycans)) residues. It is estimated that more Most eukaryotic proteins are covalently modifed with than half of all mammalian proteins bear some form carbohydrates (“glycans”) at certain asparagine (N-linked of glycosylation [1]. Additionally, the surface of mam- glycans (N-glycans)) or serine/threonine (O-linked malian cells may contain upwards of 10 million N- and O-linked glycans attached to extracellular proteins [1]. Glycans play many important roles in secretory protein *Correspondence: [email protected] structure and function including assisting in protein fold- 2 New England Biolabs, Ipswich, MA 01938, USA ing, trafcking, tissue distribution, and persistence in the Full list of author information is available at the end of the article mammalian bloodstream [2]. As such, glycosylation is © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chuzel et al. Microb Cell Fact (2021) 20:162 Page 2 of 17 considered an intrinsic part of a secretory protein’s over- analysis. Structural characterization of sulfated glycans all structure. Glycan structure itself has wide-ranging has been accomplished using mass spectrometry (e.g., importance in both basic biology and pharmaceutical sci- LC–MS/MS or MALDI-TOF) of released glycans or ence [2–5]. Aberrant glycosylation has been implicated glycopeptides [14, 22–25]. Tese methods can be com- in numerous diseases [6, 7] and changes in N-glycans of promised by poor ionization of sulfated species and the serum glycoproteins has yielded promising disease bio- natural low abundance of sulfated N- and O-glycans. markers [7–9]. Additionally, N-glycan composition can Methods to enrich samples for sulfated glycans could afect the function and efcacy of many glycoprotein aid their analysis. Glycan-specifc binding proteins have drugs like certain monoclonal antibodies, growth fac- recently been used to isolate glycans possessing certain tors and fusion proteins [10, 11], and is a critical quality structural features from complex samples [26–30]. How- attribute that is monitored during drug manufacturing. ever, no sugar sulfate-specifc binding proteins have yet Protein glycosylation is inherently complex due to the been applied to enrich sulfated glycans. Additional ana- manner in which glycans are assembled and processed lytical methods involving multiplexed capillary gel elec- by cells. Glycan synthesis does not occur through copy- trophoresis with laser-induced fuorescence detection ing or interpreting a biological template (like DNA, RNA, (xCGE-LIF) [31–33] and high/ultra-performance liquid or protein synthesis). Instead glycans are synthesized chromatography coupled to fuorescence detector (H/ via complex biosynthetic pathways involving numer- UPLC-FLR) methods [34–37] are also routinely used to ous glycosyltransferases that assemble diferent mono- separate glycans and match their migration- and reten- saccharides into specifc structures. Tere are over 200 tion-times, respectively, to those of known glycans in ref- glycosyltransferases encoded in the human genome and erence databases. Tese methods of glycan analysis often expression of many varies by cell type, tissue type or in use highly specifc exoglycosidases to enzymatically con- response to environmental factors. Tis results in sub- frm the presence or absence of certain sugars [38–40]. stantial glycan structural heterogeneity. For example, However, enzymes that remove site-specifc sugar sulfa- over 140 diferent N-glycan structures have been identi- tion or sulfated sugars are currently missing from the gly- fed on glycoproteins present in human serum [12]. Add- coanalysis toolbox. ing to this complexity, glycans can also be modifed with In this study, we sought to improve the range of ana- sulfate, phosphate, methyl or acetyl groups on certain lytical enzymes available for analysis of sulfated glycans. sugars. Tese groups are collectively termed ‘post-glyco- We successfully applied high-throughput functional sylation modifcations’ (PGMs) and each imparts a vari- metagenomic screening to identify enzymes that manip- ety of biological functions (See [13] for a review). ulate GlcNAc-6-SO4, a sulfated sugar found in some One major type of PGM involves enzymatic modifca- O-glycans and certain mammalian N-glycan outer arms. tion of glycans with sulfate. Sulfation has been observed We devised a novel coupled screening assay using a fuo- on all classes of glycans, including N- and O-glycans. Sul- rescent GlcNAc-6-SO4 analog and an exogenous hexosa- fation occurs in the Golgi where a sulfate group is trans- minidase. In this assay the coupling enzyme can act upon ferred from 3′-phosphoadenosine-5′-phosphosulfate the substrate and release the fuorophore only if the sul- (PAPS) to specifc positions on sugars by sulfotrans- fate group was frst hydrolyzed by an enzyme expressed ferases. Sulfation of N-acetylglucosamine (GlcNAc- from a metagenomic clone. Our screen identifed a sul- 6-SO4), N-acetylgalactosamine (GalNAc-6-SO4 and fatase and hexosaminidase that each act exclusively upon GalNAc-4-SO4), galactose (Gal-3-SO4 and Gal-6-SO4) GlcNAc-6-SO4. We further employed xCGE-LIF-based and mannose (Man-6-SO4) has been observed in N- and glycoanalysis to evaluate these enzymes activity on N-gly- O-glycans [14–20] (Fig. 1). Sulfation of N- and O-glycans can substrates and showed their suitability for structural has been implicated in many biological processes. Sul- characterization and enrichment of N-glycans bearing fated N-glycans are prevalent on both infuenza virus A terminal GlcNAc-6-SO4. neuraminidase and hemagglutinin surface proteins and likely play a role in virus replication and virulence [14, Results Functional screening for sulfatases 21]. Additionally, a GlcNAc-6-SO4 α2-6-sialyl LacNAc N-glycan epitope on B lymphocytes is a preferred ligand A fosmid library containing large DNA inserts (~ 40 kb) for the CD22 receptor, indicating a role in immunoregu- from human gut microbiota was created in E. coli. lation [13]. Recently, sulfated N-glycans present on serum Te library diversity was assessed by analyzing cloned immunoglobulin G were proposed as potential biomark- fragments from 24 randomly picked transformants. ers for rheumatoid arthritis [22]. Sequencing
Load more

Recommended publications
  • Restriction Endonucleases
    Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic) 9 Restriction Endonucleases Derek Robinson, Paul R. Walsh, and Joseph A. Bonventre Background Information . 226 Which Restriction Enzymes Are Commercially Available? . 226 Why Are Some Enzymes More Expensive Than Others? . 227 What Can You Do to Reduce the Cost of Working with Restriction Enzymes? . 228 If You Could Select among Several Restriction Enzymes for Your Application, What Criteria Should You Consider to Make the Most Appropriate Choice? . 229 What Are the General Properties of Restriction Endonucleases? . 232 What Insight Is Provided by a Restriction Enzyme’s Quality Control Data? . 233 How Stable Are Restriction Enzymes? . 236 How Stable Are Diluted Restriction Enzymes? . 236 Simple Digests . 236 How Should You Set up a Simple Restriction Digest? . 236 Is It Wise to Modify the Suggested Reaction Conditions? . 237 Complex Restriction Digestions . 239 How Can a Substrate Affect the Restriction Digest? . 239 Should You Alter the Reaction Volume and DNA Concentration? . 241 Double Digests: Simultaneous or Sequential? . 242 225 Genomic Digests . 244 When Preparing Genomic DNA for Southern Blotting, How Can You Determine If Complete Digestion Has Been Obtained? . 244 What Are Your Options If You Must Create Additional Rare or Unique Restriction Sites? . 247 Troubleshooting . 255 What Can Cause a Simple Restriction Digest to Fail? . 255 The Volume of Enzyme in the Vial Appears Very Low. Did Leakage Occur during Shipment? . 259 The Enzyme Shipment Sat on the Shipping Dock for Two Days.
    [Show full text]
  • Alpha-Agarases Define a New Family of Glycoside Hydrolases, Distinct from Beta-Agarase Families
    Alpha-agarases define a new family of glycoside hydrolases, distinct from beta-agarase families Didier Flament, Tristan Barbeyron, Murielle Jam, Philippe Potin, Mirjam Czjzek, Bernard Kloareg, Gurvan Michel To cite this version: Didier Flament, Tristan Barbeyron, Murielle Jam, Philippe Potin, Mirjam Czjzek, et al.. Alpha- agarases define a new family of glycoside hydrolases, distinct from beta-agarase families. Applied and Environmental Microbiology, American Society for Microbiology, 2007, 73 (14), pp.4691-4694. 10.1128/AEM.00496-07. hal-00616725 HAL Id: hal-00616725 https://hal.univ-brest.fr/hal-00616725 Submitted on 5 Sep 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 Short communication 2 3 Alpha-agarases define a new family of glycoside hydrolases, distinct from 4 beta-agarase families 5 6 Didier FLAMENT §, Tristan BARBEYRON, Murielle JAM, Philippe POTIN, Mirjam 7 CZJZEK, Bernard KLOAREG and Gurvan MICHEL* 8 9 Centre National de la Recherche Scientifique; Université Pierre et Marie Curie-Paris6, Unité 10 Mixte de Recherche 7139 "Marine
    [Show full text]
  • Isolation and Characterization of an Antarctic Flavobacterium Strain with Agarase and Alginate Lyase Activities
    vol. 37, no. 3, pp. 403–419, 2016 doi: 10.1515/popore-2016-0021 Isolation and characterization of an Antarctic Flavobacterium strain with agarase and alginate lyase activities Paris LAVÍN1*, Cristian ATALA2, Jorge GALLARDO-CERDA1, Marcelo GONZALEZ-ARAVENA1, Rodrigo DE LA IGLESIA3, Rómulo OSES4, Cristian TORRES-DÍAZ5, Nicole TREFAULT6, Marco A. MOLINA-MONTENEGRO4,7 and H. Dail LAUGHINGHOUSE IV8 1 Departamento Científico, Instituto Antártico Chileno (INACH), Punta Arenas, Chile 2 Laboratorio de Anatomía y Ecología Funcional de Plantas, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Campus Curauma, Valparaíso, Chile <[email protected]> 3 Departamento de Genética Molecular y Microbiología. Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile 4 Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Facultad de Ciencias del Mar, Universidad Católica del Norte, Coquimbo, Chile 5 Departamento de Ciencias Básicas, Facultad de Ciencias, Universidad del Bío-Bío, Chillán, Chile 6 Centro de Genómica y Bioinformática, Facultad de Ciencias, Universidad Mayor, Santiago, Chile 7 Instituto de Ciencias Biológicas, Universidad de Talca, Avda. Lircay s/n, Talca, Chile 8 Institute for Bioscience and Biotechnology Research (IBBR), Rockville, MD, USA * Corresponding author Abstract: Several bacteria that are associated with macroalgae can use phycocolloids as a carbon source. Strain INACH002, isolated from decomposing Porphyra (Rhodo- phyta), in King George Island, Antarctica, was screened and characterized for the ability to produce agarase and alginate-lyase enzymatic activities. Our strain INACH002 was identified as a member of the genus Flavobacterium, closely related to Flavobacterium faecale, using 16S rRNA gene analysis. The INACH002 strain was characterized as psy- chrotrophic due to its optimal temperature (17 ºC) and maximum temperature (20°C) of growth.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 8,124,103 B2 Yusibov Et Al
    USOO81241 03B2 (12) United States Patent (10) Patent No.: US 8,124,103 B2 Yusibov et al. (45) Date of Patent: *Feb. 28, 2012 (54) INFLUENZA ANTIGENS, VACCINE 5,383,851 A 1/1995 McKinnon, Jr. et al. 5,403.484 A 4/1995 Ladner et al. COMPOSITIONS, AND RELATED METHODS 5,417,662 A 5/1995 Hjertman et al. 5,427,908 A 6/1995 Dower et al. (75) Inventors: Vidadi Yusibov, Havertown, PA (US); 5,466,220 A 11/1995 Brenneman Vadim Mett, Newark, DE (US); 5,480,381 A 1/1996 Weston 5,502,167 A 3, 1996 Waldmann et al. Konstantin Musiychuck, Swarthmore, 5,503,627 A 4/1996 McKinnon et al. PA (US) 5,520,639 A 5/1996 Peterson et al. 5,527,288 A 6/1996 Gross et al. (73) Assignee: Fraunhofer USA, Inc, Plymouth, MI 5,530,101 A 6/1996 Queen et al. 5,545,806 A 8/1996 Lonberg et al. (US) 5,545,807 A 8, 1996 Surani et al. 5,558,864 A 9/1996 Bendig et al. (*) Notice: Subject to any disclaimer, the term of this 5,565,332 A 10/1996 Hoogenboom et al. patent is extended or adjusted under 35 5,569,189 A 10, 1996 Parsons U.S.C. 154(b) by 0 days. 5,569,825 A 10/1996 Lonberg et al. 5,580,717 A 12/1996 Dower et al. This patent is Subject to a terminal dis 5,585,089 A 12/1996 Queen et al. claimer. 5,591,828 A 1/1997 Bosslet et al.
    [Show full text]
  • Degradation of Marine Algae-Derived Carbohydrates by Bacteroidetes
    Article Degradation of Marine Algae‐Derived Carbohydrates by Bacteroidetes Isolated from Human Gut Microbiota Miaomiao Li 1,2, Qingsen Shang 1, Guangsheng Li 3, Xin Wang 4,* and Guangli Yu 1,2,* 1 Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering, School of Medicine and pharmacy, Ocean University of China, Qingdao 266003, China; [email protected] (M.L.); [email protected] (Q.S.) 2 Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China 3 DiSha Pharmaceutical Group, Weihai 264205, China; gsli‐[email protected] 4 State Key Laboratory of Breeding Base for Zhejiang Sustainable Pest and Disease Control and Zhejiang Key Laboratory of Food Microbiology, Academy of Agricultural Sciences, Hangzhou 310021, China * Correspondence: [email protected] (X.W.); [email protected] (G.Y.); Tel.: +86‐571‐8641‐5216 (X.W.); +86‐532‐8203‐1609 (G.Y.) Academic Editor: Paola Laurienzo Received: 2 January 2017; Accepted: 20 March 2017; Published: 24 March 2017 Abstract: Carrageenan, agarose, and alginate are algae‐derived undigested polysaccharides that have been used as food additives for hundreds of years. Fermentation of dietary carbohydrates of our food in the lower gut of humans is a critical process for the function and integrity of both the bacterial community and host cells. However, little is known about the fermentation of these three kinds of seaweed carbohydrates by human gut microbiota. Here, the degradation characteristics of carrageenan, agarose, alginate, and their oligosaccharides, by Bacteroides xylanisolvens, Bacteroides ovatus, and Bacteroides uniforms, isolated from human gut microbiota, are studied. Keywords: carrageenan; agarose; alginate; oligosaccharides; Bacteroides xylanisolvens; Bacteroides ovatus; Bacteroides uniforms 1.
    [Show full text]
  • Saccharification Enzyme Composition with Bacillus Subtilis Alpha-Amylase
    (19) TZZ _ _T (11) EP 2 291 526 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 9/28 (2006.01) C12N 9/34 (2006.01) 13.08.2014 Bulletin 2014/33 C12P 7/00 (2006.01) (21) Application number: 09759442.8 (86) International application number: PCT/US2009/046296 (22) Date of filing: 04.06.2009 (87) International publication number: WO 2009/149283 (10.12.2009 Gazette 2009/50) (54) Saccharification enzyme composition with Bacillus subtilis alpha-amylase Zusammensetzung von Verzuckerungsenzymen enthaltend Bacillus subtilis alpha-Amylase Composition d’enzymes de saccharification comprenant une alpha-amylase de Bacillus subtilis (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR • KONSOULA ET AL: "Alpha-amylases and HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL glucoamylases free or immobilized in calcium PT RO SE SI SK TR alginate gel capsules for synergistic hydrolysis of crude starches" AMINO ACIDS, vol. 33, 2007, (30) Priority: 06.06.2008 US 59535 P page XIII, XP002552827 01.04.2009 US 165856 P • YEESANG ET AL: "Sago starch as a low-cost carbon source for exopolysaccharide production (43) Date of publication of application: by Lactobacillus kefiranofaciens" WORLD 09.03.2011 Bulletin 2011/10 JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 24, 15 November 2007 (73) Proprietor: Danisco US Inc. (2007-11-15), pages 1195-1201, XP019617015 Palo Alto, CA 94304 (US) • DE MORAES ET AL: "Development of yeast strains for the efficient utilisation of starch: (72) Inventors: evaluation of constructs that express alpha- • BRENEMAN, Suzanne amylase and glucoamylase separately or as Orfordville bifunctional fusion proteins" APPLIED WI 53576 (US) MICROBIOLOGY AND BIOTECHNOLOGY, vol.
    [Show full text]
  • 12) United States Patent (10
    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
    [Show full text]
  • Asp271 Is Critical for Substrate Interaction with the Surface Binding Site in Β-Agarase a from Zobellia Galactanivorans
    Downloaded from orbit.dtu.dk on: Sep 28, 2021 Asp271 is critical for substrate interaction with the surface binding site in -agarase A from Zobellia galactanivorans Wilkens, Casper; Tiwari, Manish K.; Webb, Helen; Jam, Murielle; Czjzek, Mirjam; Svensson, Birte Published in: Proteins: Structure, Function, and Bioinformatics Link to article, DOI: 10.1002/prot.25614 Publication date: 2019 Document Version Peer reviewed version Link back to DTU Orbit Citation (APA): Wilkens, C., Tiwari, M. K., Webb, H., Jam, M., Czjzek, M., & Svensson, B. (2019). Asp271 is critical for substrate interaction with the surface binding site in -agarase A from Zobellia galactanivorans. Proteins: Structure, Function, and Bioinformatics, 87(1), 34-40. https://doi.org/10.1002/prot.25614 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Research Article Proteins: Structure, Function and Bioinformatics DOI 10.1002/prot.25614 Title: Asp271 is critical for substrate interaction with the surface binding site in β-agarase A from Zobellia galactanivorans Short title: Asp271 in AgaA SBS Authors: Casper Wilkens1,*,#, Manish K.
    [Show full text]
  • Cazymes Catalogue 2021
    cazymes 2021 Visit the Online Store www.nzytech.com Newsletter NZYWallet Instantaneous Quotes Subscribe our newsletter to receive NZYWallet is a prepaid account Do you need an urgent quote? Just awesome news and promotions. that oers the flexibility you need add your products to Cart, proceed to focus on your research. With to Checkout, select quote and it’s NZYWallet you can buy any done! product from our Online Store, check your up-to-date balance and track your latest orders. Contact our customer service at [email protected] or your local sales representative for more information. Follow us: 2021 NZYTech NZYTech 2 Visit the Online Store www.nzytech.com Newsletter NZYWallet Instantaneous Quotes Subscribe our newsletter to receive NZYWallet is a prepaid account Do you need an urgent quote? Just awesome news and promotions. that oers the flexibility you need add your products to Cart, proceed to focus on your research. With to Checkout, select quote and it’s NZYWallet you can buy any done! product from our Online Store, check your up-to-date balance and track your latest orders. Contact our customer service at [email protected] or your local sales representative for more information. Follow us: cazymes 3 4 2021 NZYTech NZYTech OVERVIEW 8 GLYCOSIDE HYDROLASES 10 Acetylgalactosaminidases Acetylglucosaminidases Agarases Amylases @ a glance Amylomaltases Arabinanases Arabinofuranosidases Arabinopyranosidases Arabinoxylanases Carrageenases Cellobiohydrolases Cellodextrinases Cellulases Chitinases Chitosanases Dextranases Fructanases Fructofuranosidases
    [Show full text]
  • Transfer of Carbohydrate-Active Enzymes from Marine Bacteria to Japanese Gut Microbiota
    Vol 464 | 8 April 2010 | doi:10.1038/nature08937 LETTERS Transfer of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota Jan-Hendrik Hehemann1,2{, Gae¨lle Correc1,2, Tristan Barbeyron1,2, William Helbert1,2, Mirjam Czjzek1,2 & Gurvan Michel1,2 Gut microbes supply the human body with energy from dietary not possess the critical residues needed for recognition of agarose or polysaccharides through carbohydrate active enzymes, or k-carrageenan12. CAZymes1, which are absent in the human genome. These enzymes To identify their substrate specificity we cloned and expressed these target polysaccharides from terrestrial plants that dominated diet five GH16 genes in Escherichia coli. However, only Zg1017 and the throughout human evolution2. The array of CAZymes in gut catalytic module of Zg2600 were expressed as soluble proteins and microbes is highly diverse, exemplified by the human gut symbiont could be further analysed (Supplementary Fig. 2). As predicted, these Bacteroides thetaiotaomicron3, which contains 261 glycoside proteins had no activity on commercial agarose (Supplementary hydrolases and polysaccharide lyases, as well as 208 homologues Fig. 3) or k-carrageenan. Consequently, we screened their hydrolytic of susC and susD-genes coding for two outer membrane proteins activity against natural polysaccharides extracted from various marine involved in starch utilization1,4. A fundamental question that, to macrophytes (data not shown). Zg2600 and Zg1017 were found to be our knowledge, has yet to be addressed is how this diversity evolved active only on extracts from the agarophytic red algae Gelidium, by acquiring new genes from microbes living outside the gut. Here Gracilaria and Porphyra, as shown by the release of reducing ends we characterize the first porphyranases from a member of the (Fig.
    [Show full text]
  • Molecular Cloning, Expression, and Functional Characterization of the Β-Agarase Agab-4 from Paenibacillus Agarexedens
    Chen et al. AMB Expr (2018) 8:49 https://doi.org/10.1186/s13568-018-0581-8 ORIGINAL ARTICLE Open Access Molecular cloning, expression, and functional characterization of the β‑agarase AgaB‑4 from Paenibacillus agarexedens Zeng‑Weng Chen1, Hui‑Jie Lin1, Wen‑Cheng Huang1, Shih‑Ling Hsuan2, Jiunn‑Horng Lin1 and Jyh‑Perng Wang1* Abstract In this study, a β-agarase gene, agaB-4, was isolated for the frst time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883- amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purifed from the soluble frac‑ tion of E. coli cell lysate through immobilized metal ion afnity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specifcity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confrmed to be neoagarotetraose. Purifed rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degrada‑ tion for the production of neoagarotetraose.
    [Show full text]
  • Carbohydrate Analysis: Enzymes, Kits and Reagents
    2007 Volume 2 Number 3 FOR LIFE SCIENCE RESEARCH ENZYMES, KITS AND REAGENTS FOR ANALYSIS OF: AGAROSE ALGINIC ACID CELLULOSE, LICHENEN AND GLUCANS HEMICELLULOSE AND XYLAN CHITIN AND CHITOSAN CHONDROITINS DEXTRAN HEPARANS HYALURONIC ACID INULIN PEPTIDOGLYCAN PECTIN Cellulose, one of the most abundant biopolymers on earth, is a linear polymer of β-(1-4)-D-glucopyranosyl units. Inter- and PULLULAN intra-chain hydrogen bonding is shown in red. STARCH AND GLYCOGEN Complex Carbohydrate Analysis: Enzymes, Kits and Reagents sigma-aldrich.com The Online Resource for Nutrition Research Products Only from Sigma-Aldrich esigned to help you locate the chemicals and kits The Bioactive Nutrient Explorer now includes a • New! Search for Plants Dneeded to support your work, the Bioactive searchable database of plants listed by physiological Associated with Nutrient Explorer is an Internet-based tool created activity in key areas of research, such as cancer, dia- Physiological Activity to aid medical researchers, pharmacologists, nutrition betes, metabolism and other disease or normal states. • Locate Chemicals found and animal scientists, and analytical chemists study- Plant Detail pages include common and Latin syn- in Specific Plants ing dietary plants and supplements. onyms and display associated physiological activities, • Identify Structurally The Bioactive Nutrient Explorer identifies the while Product Detail pages show the structure family Related Compounds compounds found in a specific plant and arranges and plants that contain the compound, along with them by chemical family and class. You can also comparative product information for easy selection. search for compounds having a similar chemical When you have found the product you need, a structure or for plants containing a specific simple mouse click connects you to our easy online compound.
    [Show full text]