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2019-20 NEB Catalog Technical Reference

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Reference Appendix Technical Support – Featured Tools & for scientists, by scientists Resources As a partner to the scientific community, New England Biolabs is committed to Optimizing Restriction Enzyme Reactions providing top quality tools and scientific expertise. This philosophy still stands, 290 and has led to long-standing relationships with many of our fellow scientists. Performance Chart for NEB's commitment to scientists is the same regardless of whether or not they 293 Restriction Enzymes purchase product from NEB: their ongoing research is supported by our catalog, Troubleshooting Guide website and technical staff. 349 for Cloning NEB's technical support model is unique as it utilizes most of the scientists at NEB. Several of our product lines have designated technical support scientists 334 Methylation Sensitivity assigned to servicing customers in those application areas. Any questions regarding a product could be dealt with by one of the technical support Guidelines for scientists, the product manager who manufactures it, the product development 337 PCR Optimization scientist who optimizes it, or a researcher who uses the product in their daily Cleavage Close to the research. As such, customers are supported by scientists and often experts in 343 End of DNA Fragments the product or its application. To access technical support: 288 Online Interactive Tools Call 1-800-632-7799 (Monday – Friday: 9:00 am - 6:00 pm EST) Submit an online form at www.neb.com/techsupport View NEB TV Episode #22 Email [email protected] to learn more about our Technical Support program. International customers can contact a local NEB subsidiary or distributor. For more information see inside back cover. Visit the Tools & Resources tab at www.neb.com to find additional online tools, video tech tips and tutorials to help Learn more you in your research. about NEB's tech support program. 286 Online Interactive Tools 288 PCR, qPCR & RT-qPCR Databases, Mobile Apps & General Guidelines for PCR Optimization 337 Freezer Program Locator 289 PCR Troubleshooting Guide 338 Restriction Endonucleases Optimization Tips for Luna qPCR 339 Optimization Tips for Luna One-Step RT-qPCR 340 Optimizing Restriction Enzyme Reactions 290 Luna qPCR Troubleshooting Guide 341 Double Digestion 291 Luna One-Step RT-qPCR Troubleshooting Guide 342 Types of Restriction Enzymes 291 Cleavage Close to the End of DNA Fragments 343 Troubleshooting Guide 292 Activity of Restriction Enzymes in PCR Buffers 344–345 Performance Cloning Performance Chart for Restriction Enzymes 293–298 Getting Started with Molecular Cloning 346 Activity of Enzymes at 37°C 299 Traditional Cloning Quick Guide 347–348 Activity of DNA Modifying Enzymes in CutSmart Buffer 299 Troubleshooting Guide for Cloning 349–351 Tips for Avoiding Star Activity 300 Optimization Tips for Your Cloning Reaction 352–353 High-Fidelity (HF) Restriction Enzymes 301 Nucleic Acid Purification Reduced Star Activities of HF Enzymes 301 Troubleshooting Guide for DNA Cleanup Time-Saver Qualified Restriction Enzymes 302–303 & Plasmid Purification Using Monarch Kits 354 Isoschizomers/Recognition Sequences Choosing Sample Input Amounts When Using the Monarch Genomic DNA Purification Kit 355 Cross Index of Recognition Sequences 304–306 Troubleshooting Guide for Genomic DNA Palindromic Penta-nucleotide Purification Using Monarch Kits 356 Recognition Sequences 305 Choosing Sample Input Amounts When Using the Specificities Greater Than 6 Bases 305 Monarch Total RNA Miniprep Kit 357 Interrupted Palindromes 305–306 Troubleshooting Guide for Total RNA Extraction Multiple & Nonpalindromic & Purification using Monarch Kits 358 Recognition Sequences 306–309 Alphabetized List of NEB Recognition Sequences 309–310 Transformation Isoschizomers 311–327 Genetic Markers 359–360 Properties of Restriction Enzymes Enhancing Transformation Efficiency 361 Protein Expression with T7 Express Strains 362 Survival in a Reaction 328–329 Cleavage of Supercoiled DNA 329 NGS Library Preparation Generating New Cleavage Sites DNA/RNA Input Guidelines for NGS Library Prep 363 Recleavable Filled-in 5´ Overhangs 330 Protein Expression Recleavable Blunt Ends 331 Labeling with SNAP-tag Technology Compatible Cohesive Ends 332–333 Troubleshooting Guide 364 Frequencies of Restriction Sites in Methylation Sensitivity Sequenced DNAs 365–368 Dam, Dcm and CpG Methylation 334–336 DNA Maps 369–381 Odds and Ends General Molecular Biology Information 382–385 Index 386 One or more of these products are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please email us at [email protected]. The use of these products may require you to obtain additional third party intellectual property rights for certain applications. Your purchase, acceptance, and/or payment of and for NEB’s products is pursuant to NEB’s Terms of Sale at https://www.neb.com/support/terms-of-sale. NEB does not agree to and is not bound by any other terms or conditions, unless those terms and conditions have been expressly agreed to in writing by a duly authorized officer of NEB. 287 Online Interactive Tools Use the Tools & Resources tab at www.neb.com to access our growing selection of interactive technical tools. These tools can also be accessed directly in the footer of every web page. NEB scientists are often involved in the development of online tools that will aid in their research. We are now making these tools and in some circumstances, the source code, available for you to evaluate. To learn more, visit www.neb.com/NEBetaTools. Online Tools Competitor Cross-Reference Tool NEBcutter® V2.1 Use this tool to select another company's product and find out Identify the restriction sites within your DNA sequence using which NEB product is compatible. Choose either the product NEBcutter. Choose between Type II and commercially available name or catalog number from the available selections, and this Type III restriction enzymes to digest your DNA. NEBcutter will tool will identify the recommended NEB product. indicates cut frequency and methylation-state sensitivity. ® DNA Sequences and Maps Tool NEBioCalculator Use this tool for your scientific calculations and conversions With the DNA Sequences and Maps Tool, find the nucleotide for DNA and RNA. Options include conversion of mass to sequence files for commonly used molecular biology tools, moles, ligation amounts, conversion of OD to concentration, including plasmid, viral and bacteriophage vectors. dilution and molarity. Additional features include sgRNA template oligo design and qPCR library quantification. ® Double Digest Finder NEBcloner Use this tool to find the right products and protocols for each Use this tool to guide your reaction buffer selection step (digestion, end modification, ligation, transformation when setting up double digests, a common timesaving and mutagenesis) of your next traditional cloning experiment. procedure. Choosing the right buffers will help you to Also, find other relevant tools and resources to enable protocol avoid star activity and loss of product. optimization. ® Enzyme Finder NEBuilder Assembly Tool Use this tool to design primers for your DNA assembly Use this tool to select restriction enzymes by name, reaction, based on the entered fragment sequences and the sequence, overhang or type. Enter your sequence polymerase being used for amplification. using single letter code nomenclature, and Enzyme Finder will identify the right enzyme for the job. Glycan Analyzer PCR Fidelity Estimator Use this tool to interpret ultra or high pressure liquid Estimate the percentage of correct DNA copies (those chromatography (UPLC/HPLC) N-glycan profiles without base substitution errors) per cycle of PCR for following exoglycosidase digestions. selected DNA polymerases. NEB Golden Gate Assembly Tool PCR Selector Use this tool to assist with in silico DNA construct design for Use this tool to help select the right DNA polymerase Golden Gate DNA assembly. It enables the accurate design of for your PCR setup. Whether your amplicon is long, primers with appropriate Type IIS restriction sites and overlaps, complex, GC-rich or present in a single copy, the PCR quick import of sequences in many formats and export of the selection tool will identify the perfect DNA polymerase final assembly, primers and settings. for your reaction. NEBaseChanger® Tm Calculator NEBaseChanger can be used to design primers specific to Determine the optimal annealing temperature for your amplicon the mutagenesis experiment you are performing using the with our Tm Calculator. Simply input your DNA polymerase, Q5® Site-Directed Mutagenesis Kit. This tool will also calculate primer concentration and your primer sequence, and the Tm APPENDIX a recommended custom annealing temperature based on the Calculator will guide you to successful reaction conditions. sequence of the primers by taking into account any mismatches. NEBNext® Selector Thermostable Ligase Reaction Temperature Calculator Use this tool to guide you through the selection of NEBNext This tool will help you estimate an optimal reaction temperature reagents for next generation sequencing sample preparation. to minimize mismatch for thermostable ligation of two adjacent ssDNA probes annealed to a template. 288 www.neb.com · [email protected] TECHNICAL SUPPORT Online Resources Online Tools (continued) Read Coverage Calculator This tool allows for easy calculation of values associated with read coverage in NGS protocols. Additional Databases
Recommended publications
  • Restriction Endonucleases

    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.
  • 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

    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
  • Isolation and Characterization of an Antarctic Flavobacterium Strain with Agarase and Alginate Lyase Activities

    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.
  • (12) United States Patent (10) Patent No.: US 8,124,103 B2 Yusibov Et Al

    (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.
  • 1. Restriction Endonucleases (® Fermentas 2006)

    1. Restriction Endonucleases (® Fermentas 2006)

    We guarantee that these products are Introduction ..........................................................................................................2 free of contaminating activities. Restriction Endonucleases Guide Our stringent quality control with the most FastDigest™ Restriction Endonucleases .........................................................2 Fermentas Restriction Endonucleases............................................................3 advanced tests guarantees you pure 1 Alphabetic List of Commercially Available Restriction Endonucleases.........6 products for your experiments. ISO9001 Recognition Specifi cities...............................................................................16 and ISO14001 is your assurance of con- Commercial Restriction Enzymes Sorted by the Type of DNA Ends Generated sistency and lot-to-lot reproducibility. Enzymes Generating 5’-protruding Ends.......................................................18 Enzymes Generating 3’-protruding Ends.......................................................18 ™ PureExtreme Quality will provide the Enzymes Generating Blunt Ends...................................................................19 performance you need for your most Enzymes Cleaving DNA on the Both Sides of Their Recognition Sequences.....19 demanding experiments. PureExtreme™ Quality PureExtreme™ Quality Guarantee..................................................................20 Activity Assay.................................................................................................20
  • Biology of DNA Restriction THOMAS A

    Biology of DNA Restriction THOMAS A

    MICROBIOLOGICAL REVIEWS, June 1993, p. 434-450 Vol. 57, No. 2 0146-0749/93/020434-17$02.00/0 Copyright © 1993, American Society for Microbiology Biology of DNA Restriction THOMAS A. BICKLE'* AND DETLEV H. KRUGER2 Department ofMicrobiology, Biozentrum, Basel University, Klingelbergstrasse 70, CH-4056 Basel, Switzerland, 1 and Institute of Virology, Charite School ofMedicine, Humboldt University, D-0-1040 Berlin, Gernany2 INTRODUCTION ........................................................................ 434 TYPE I R-M SYSTEMS ........................................................................ 434 Type I Systems Form Families of Related Enzymes ...................................................................435 Structure of hsd Genes ........................................................................ 435 Evolution of DNA Sequence Recognition by Recombination between hsdS Genes .........................***436 Mutations Affecting Modification Activity........................................................................ 437 TYPE II R-M SYSTEMS........................................................................ 437 Evolutionary Aspects ........................................................................ 437 Control of Expression of Type II RM Genes .....................................................438 Cytosine Can Be Methylated on Either C-5 Or NA: Consequences for Mutagenesis...............438 Type II Restriction Endonucleases That Require Two Recognition Sites for Cleavage.439 What Is the Function of Type IIS Enzymes.440
  • Degradation of Marine Algae-Derived Carbohydrates by Bacteroidetes

    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.
  • Anza Restriction Enzyme Cloning System Complete, One-Buffer System— for Beautifully Simple Cloning Cloning Has Never Been Simpler

    Anza Restriction Enzyme Cloning System Complete, One-Buffer System— for Beautifully Simple Cloning Cloning Has Never Been Simpler

    Anza Restriction Enzyme Cloning System Complete, one-buffer system— for beautifully simple cloning Cloning has never been simpler Finally, forget the frustrations of finding compatible buffers and sorting through protocols for your restriction enzyme digests—start getting reliable results for your downstream experiments. The Invitrogen™ Anza™ Restriction Enzyme Cloning System is a complete system, comprised of: DNA modifying enzymes 128 restriction enzymes + 5 DNA modifying enzymes Anza T4 DNA Ligase Master Mix Anza Thermosensitive All Anza™ restriction enzymes work together cohesively and are fully functional Alkaline Phosphatase with the single Anza™ buffer. Anza T4 Polynucleotide Kinase The system offers: Anza DNA Blunt End Kit • One buffer for all restriction enzymes Anza DNA End Repair Kit • One digestion protocol for all DNA types • Complete digestion in 15 minutes • Overnight digestion without star activity Convenient buffer formats All Anza restriction enzymes come with an Anza 10X Buffer and an Anza 10X Red Buffer to give you the flexibility you require. The red buffer includes a density reagent containing red and yellow tracking dyes that migrate with 800 bp DNA fragments and faster than the 10 bp DNA fragments, respectively, in a 1% agarose gel. This eliminates tedious dye addition steps prior to gel loading and is compatible with downstream applications.* * For applications that require analysis by fluorescence excitation, Anza 10X Buffer is recommended, as the Anza 10X Red Buffer may interfere with some fluorescence measurements. 2 A one-buffer system Eliminate the frustration of trying to find compatible buffers for all your enzymes. All Anza restriction enzymes allow for complete digestion using a single Anza buffer.
  • 2019-20 NEB Catalog Technical Reference

    2019-20 NEB Catalog Technical Reference

    Reference Appendix Technical Support – Featured Tools & for scientists, by scientists Resources As a partner to the scientific community, New England Biolabs is committed to Optimizing Restriction Enzyme Reactions providing top quality tools and scientific expertise. This philosophy still stands, 290 and has led to long-standing relationships with many of our fellow scientists. Performance Chart for NEB's commitment to scientists is the same regardless of whether or not they 293 Restriction Enzymes purchase product from NEB: their ongoing research is supported by our catalog, Troubleshooting Guide website and technical staff. 349 for Cloning NEB's technical support model is unique as it utilizes most of the scientists at NEB. Several of our product lines have designated technical support scientists 334 Methylation Sensitivity assigned to servicing customers in those application areas. Any questions regarding a product could be dealt with by one of the technical support Guidelines for scientists, the product manager who manufactures it, the product development 337 PCR Optimization scientist who optimizes it, or a researcher who uses the product in their daily Cleavage Close to the research. As such, customers are supported by scientists and often experts in 343 End of DNA Fragments the product or its application. To access technical support: 288 Online Interactive Tools Call 1-800-632-7799 (Monday – Friday: 9:00 am - 6:00 pm EST) Submit an online form at www.neb.com/techsupport View NEB TV Episode #22 Email [email protected] to learn more about our Technical Support program. International customers can contact a local NEB subsidiary or distributor. For more information see inside back cover.
  • Saccharification Enzyme Composition with Bacillus Subtilis Alpha-Amylase

    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.
  • 12) United States Patent (10

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

    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.