Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
    • JB Special Collection
    • JB Classic Spotlights
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JB
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Bacteriology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
    • JB Special Collection
    • JB Classic Spotlights
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JB
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
ENZYMES AND PROTEINS

Molecular and Biochemical Characterization of α-Glucosidase and α-Mannosidase and Their Clustered Genes from the Thermoacidophilic Archaeon Picrophilus torridus

Angel Angelov, Mateusz Putyrski, Wolfgang Liebl
Angel Angelov
1Institute of Microbiology and Genetics, University of Goettingen, Grisebachstrasse 8, D-37077 Goettingen, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mateusz Putyrski
1Institute of Microbiology and Genetics, University of Goettingen, Grisebachstrasse 8, D-37077 Goettingen, Germany
2Institute of Microbiology, Department of Bacterial Genetics, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wolfgang Liebl
1Institute of Microbiology and Genetics, University of Goettingen, Grisebachstrasse 8, D-37077 Goettingen, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: wliebl@gwdg.de
DOI: 10.1128/JB.00757-06
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Additional Files
  • FIG. 1.
    • Open in new tab
    • Download powerpoint
    FIG. 1.

    Phylogenetic tree of selected members of glycoside hydrolase family 31. The EC numbers for protein sequences with known substrate specificities are given in parentheses; the numbers at the nodes are bootstrap confidence values. The following abbreviations are used (accession numbers are in parentheses): PTO-AglA, P. torridus AglA (AAT42677); SSO-MalA, S. solfataricus MalA (AAK43151); SSO-XylS, S. solfataricus XylS (AAK43123); TAC, T. acidophilum (CAC11443); TVO, T. volcanium (BAB60467); BTH, B. thermoamyloliquefaciens (BAA76396); TTH, T. thermophilus (AAS82549); ATH-Aglu1, Arabidopsis thaliana Aglu1 (AAB82656); ATH-Xyl1, A. thaliana Xyl1 (AAD05539); HSA-GAA, Homo sapiens GAA (CAA68763); HSA-SI, H. sapiens SI (CAA45140); BTA-Agl, Bos taurus Agl (AAF81637); RNO-SI, Rattus norvegicus SI (AAA65097); GLE-Glq1, Gracilariopsis lemaneiformis Glq1 (CAB51910); ECO-YihQ, E. coli YihQ (AAB03011); ECO-YicI, E. coli YicI (AAA62009).

  • FIG. 2.
    • Open in new tab
    • Download powerpoint
    FIG. 2.

    SDS-PAGE analysis of the steps in the purification of recombinant P. torridus α-glucosidase (A) and α-mannosidase (B). (A) Lane 1, molecular weight marker; lane 2, E. coli BL21RS::p24-aglA cellular extract (8 μg); lane 3, heat-treated extract (5 μg); lane 4, AglA pooled fractions after anion-exchange chromatography (2 μg); lane 5, AglA pooled fractions after gel filtration chromatography (2 μg). (B) Lane 1, molecular weight marker; lane 2, E. coli BL21RS::p24-manA cellular extract (8 μg); lane 3, ManA pooled fractions after hydrophobic interaction chromatography (5 μg); lane 4, ManA pooled fractions after anion-exchange chromatography (3 μg); lane 5, ManA pooled fractions after gel filtration chromatography (0.7 μg).

  • FIG. 3.
    • Open in new tab
    • Download powerpoint
    FIG. 3.

    Thin-layer chromatography analysis of the reaction products of P. torridus α-glucosidase (A) and α-mannosidase (B). (C) Schematic diagram of the structure of α3,α6-mannopentaose. The common core mannotriose structure found in N-glycans is indicated by boldface type. The reactions were carried out in 20 μl of 50 mM acetate buffer (pH 5.0) with 0.2% substrate and with (+) or without (−) 3.6 μg of purified AglA enzyme at 85°C (A) or with (+) or without (−) 0.3 μg of purified ManA at 65°C (B) for 4 h. (A) Lane 1, G1 to G7 maltooligosaccharide standards; lanes 2 and 3, maltose; lanes 4 and 5, maltotriose; lanes 6 and 7, maltopentaose; lanes 8 and 9, maltohexaose. (B) Lane 1, mannose; lanes 2 and 3, 1,2-α-mannobiose; lanes 4 and 5, 1,3-α-mannobiose; lanes 6 and 7, methyl-O-1,4-α-mannobiose; lanes 8 and 9, 1,6-α-mannobiose; lanes 10 and 11, α3,α6-mannopentaose.

  • FIG. 4.
    • Open in new tab
    • Download powerpoint
    FIG. 4.

    Temperature inactivation kinetics of recombinant P. torridus α-mannosidase at 80°C. The purified enzyme (0.1 mg/ml) was incubated at 80°C in 50 mM acetate buffer (pH 5.0) with different additives, samples were withdrawn, and the residual activities were determined as described in Materials and Methods. Relative activity is expressed as the percentage of the activity without heat treatment.

  • FIG. 5.
    • Open in new tab
    • Download powerpoint
    FIG. 5.

    Inhibition profile of P. torridus α-mannosidase. The enzyme (0.26 μg) was incubated for 15 min at 70°C in 50 mM acetate buffer (pH 5.0) with pNP-Man and different concentrations of the inhibitors deoxymannojirimycin (A) and swainsonine (B). (C) Dixon plot of the inhibition by swainsonine at two different substrate concentrations, resulting in a Ki of 1.22 μM.

  • FIG. 6.
    • Open in new tab
    • Download powerpoint
    FIG. 6.

    (A) Genome region containing the manA and aglA genes (gray arrows). Start and stop codons are underlined, putative TATA box regions are enclosed in boxes, and the transcription start sites identified are indicated by bent arrows. (B) Northern blot analysis of the aglA gene. Lane 1 contained 2.5 μg total P. torridus RNA, and lanes 2 and 3 contained 5 μg total RNA isolated by two different methods (see Materials and Methods). The radioactive probe used for hybridization is indicated by a solid bar in panel A.

Tables

  • Figures
  • Additional Files
  • TABLE 1.

    Primers used in this study

    PrimerSequence (5′-3′)Description
    984.RC-rev1TATAATGAGCCGGTGTCCTG manA 5′-RACE inner
    984.RC-rev2CAGTGTCCAATTGCCAAGATG manA 5′-RACE outer
    985.RC-rev1TTAAGGCCTATGTCGATCTC aglA 5′-RACE inner
    985.RC-rev2TTGATATCTGGTGCCCGAGTG aglA 5′-RACE outer
    984.FGGATTCCTGTAGGAGGTATG manA quantitative RT-PCR forward
    984.RATGAATCCGGTAGCCAAC manA quantitative RT-PCR reverse
    985.FGGAGCGCACTTCACTACAC aglA quantitative RT-PCR forward
    985.RGCCTCGTATGTTGCCTTAGC aglA quantitative RT-PCR reverse
  • TABLE 2.

    Purification of AglA and ManA

    EnzymeStepTotal protein (mg)Total activity (U)Sp act (U · mg−1)Yield (%)Purification (fold)
    AglACell extract171.3151.70.881001
    Heat treatment35.195.12.7162.73.1
    Source Q1.0122.722.5214.925.6
    Superdex 2000.4114.234.569.439.3
    ManACell extract2,166541.50.251001
    Phenyl Sepharose3801900.5035.12
    Source Q1.9238.620.17.280.5
    Superdex 2000.137.759.21.4237.5
  • TABLE 3.

    Characteristics of AglA and ManA

    EnzymeSubstrateOptimum pHOptimum temp (°C)Km Vmaxkcat (s−1)
    AglA p-Nitrophenyl-α-glucoside5.0870.94 ± 0.1451.8 ± 1.5765.1
    Maltose5.0873.7 ± 0.32149.7 ± 3.9188.1
    ManA p-Nitrophenyl-α-mannoside5.2700.49 ± 0.0356.5 ± 3.1105.1

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Fig. S1, Multiple sequence alignment of α-glucosidase enzymes from P. torridus, T. acidophilum, and their homologs with known structures from S. solfataricus and E. coli
      Zipped PDF document, 1.4MB.
    • Supplemental file 2 - Fig. S2, Multiple sequence alignment of α-mannosidase enzymes from B. taurus, H. sapiens, P. torridus, T. volcanium, S. solfataricus, and S. tokodaii Zipped PDF document, 72K.
PreviousNext
Back to top
Download PDF
Citation Tools
Molecular and Biochemical Characterization of α-Glucosidase and α-Mannosidase and Their Clustered Genes from the Thermoacidophilic Archaeon Picrophilus torridus
Angel Angelov, Mateusz Putyrski, Wolfgang Liebl
Journal of Bacteriology Oct 2006, 188 (20) 7123-7131; DOI: 10.1128/JB.00757-06

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Bacteriology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Molecular and Biochemical Characterization of α-Glucosidase and α-Mannosidase and Their Clustered Genes from the Thermoacidophilic Archaeon Picrophilus torridus
(Your Name) has forwarded a page to you from Journal of Bacteriology
(Your Name) thought you would be interested in this article in Journal of Bacteriology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Molecular and Biochemical Characterization of α-Glucosidase and α-Mannosidase and Their Clustered Genes from the Thermoacidophilic Archaeon Picrophilus torridus
Angel Angelov, Mateusz Putyrski, Wolfgang Liebl
Journal of Bacteriology Oct 2006, 188 (20) 7123-7131; DOI: 10.1128/JB.00757-06
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Thermoplasmales
alpha-Glucosidases
alpha-Mannosidase

Related Articles

Cited By...

About

  • About JB
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jbacteriology

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0021-9193; Online ISSN: 1098-5530