Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

High-activity barley alpha-amylase by directed evolution.

Dominic W S Wong1, Sarah B Batt, Charles C Lee

  • 1Western Regional Research Center, USDA-Agricultural Research Service, Albany, CA 94710, USA. dwsw@pw.usda.gov

The Protein Journal
|January 8, 2005
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Molecular Mechanisms of Autism Spectrum Disorder.

Cells·2026
Same author

Binaural Processing Deficits in Autism Spectrum Disorder.

Audiology research·2026
Same author

Prenatal Intraportal Delivery of Polymeric Nanoparticles to Fetal Rhesus Monkeys (<i>Macaca mulatta</i>).

Human gene therapy·2026
Same author

Effects of Blueberry Supplementation on Depression and Anxiety Symptoms in a Rural Louisiana Population.

Nutrients·2025
Same author

Impact of GLP-1 Agonist on Surgical Wound Complications Following Plastic and Reconstructive Surgery: A Propensity Matched Cohort Large Database Analysis.

Plastic and reconstructive surgery·2025
Same author

Correction: Polyhydroxyalkanoate production in Pseudomonas putida from alkanoic acids of varying lengths.

PloS one·2025

Directed evolution of barley alpha-amylase in yeast significantly enhanced enzyme activity. A mutant (Mu322) exhibited 1000x total and 20x specific activity compared to wild-type, showcasing successful protein engineering.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Barley alpha-amylase isozyme 2 is a key enzyme in starch degradation.
  • Enhancing enzyme properties through protein engineering is crucial for industrial applications.
  • Saccharomyces cerevisiae offers a robust system for recombinant protein expression and modification.

Purpose of the Study:

  • To improve the activity and specific activity of barley alpha-amylase through directed evolution.
  • To identify beneficial mutations conferring enhanced enzymatic properties.
  • To characterize the engineered barley alpha-amylase mutant.

Main Methods:

  • Cloning and constitutive secretion of wild-type barley alpha-amylase in Saccharomyces cerevisiae.
  • Directed evolution employing screening methods like halo formation on starch agar and high-throughput liquid assays with dye-labeled starch.

Related Experiment Videos

  • Quantification of recombinant enzyme concentration via immunodetection for specific activity calculation.

    • Main Results:

    • Three rounds of directed evolution yielded a mutant (Mu322) with significantly enhanced enzymatic performance.
    • Mu322 demonstrated a 1000-fold increase in total activity and a 20-fold increase in specific activity compared to the wild-type enzyme.
    • Five amino acid substitutions (Q44H, R303K, F325Y, T94A, R128Q) were identified in the engineered mutant, with Q44H and R303K involving highly conserved residues.

    Conclusions:

    • Directed evolution is an effective strategy for enhancing barley alpha-amylase activity in yeast.
    • Specific mutations, particularly those in conserved regions and the raw starch-binding fragment, are responsible for the observed improvements.
    • The engineered Mu322 mutant represents a promising candidate for applications requiring high alpha-amylase activity.