Applied Biochemistry and Biotechnology

, Volume 161, Issue 1, pp 301–312

Directed Evolution of a Thermophilic β-glucosidase for Cellulosic Bioethanol Production

  • Elizabeth Hardiman
  • Moreland Gibbs
  • Rosalind Reeves
  • Peter Bergquist
Article

DOI: 10.1007/s12010-009-8794-6

Cite this article as:
Hardiman, E., Gibbs, M., Reeves, R. et al. Appl Biochem Biotechnol (2010) 161: 301. doi:10.1007/s12010-009-8794-6

Abstract

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a β-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant β-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (kcat/KM) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (kcat) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (Ki) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.

Keywords

β-glucosidaseDirected evolutionRandom drift mutagenesisIn vitro compartmentalisationFluorescence-activated cell sorting

Copyright information

© Humana Press 2009

Authors and Affiliations

  • Elizabeth Hardiman
    • 1
  • Moreland Gibbs
    • 1
    • 2
  • Rosalind Reeves
    • 1
    • 2
  • Peter Bergquist
    • 1
    • 2
    • 3
    • 4
  1. 1.Biomolecular Frontiers Research Centre, Department of Chemistry and Biomolecular SciencesMacquarie UniversitySydneyAustralia
  2. 2.Applimex Systems Pty. Ltd.SydneyAustralia
  3. 3.Department of Molecular Medicine & PathologyUniversity of Auckland Medical SchoolAucklandNew Zealand
  4. 4.Department of Chemistry and Biomolecular SciencesMacquarie UniversitySydneyAustralia