Increasing the substrate specificity of Bacillus stearothermophillus lactate dehydrogenase by DNA shuffling

Baris Binay, Debbie K. Shoemark, Richard B. Sessions, Anthony R. Clarke, Nevin Gul Karaguler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

Abstract

Previously it is suggested that a single mutation (Q102R) in the lactate dehydrogenase (LDH) gene from Bacillus stearothermophillus (bs) could switch the substrate specificity by 3 orders of magnitude from lactate to malate and produce a highly efficient malate dehydrogenase (MDH). In order to examine if random mutation and screening could improve this, a DNA-shuffling method would be used to generate a mutant LDH and recombinant LDH genes which will be later used for transforming Escherichia coli. The recombinant colonies are blotted and screened for their ability to catalyse the oxidation of malate. The most active MDH produced by this method is only slightly more efficient than the rationally designed Q102R variant. In addition to this mutation, the shuffled version incorporates further seven residue changes which are chemically conservative. These experiments demonstrate that the blind shuffling can achieve a huge shift in specificity which was a known, highly effective single-site mutation designed using structural knowledge.

Original languageEnglish
Pages (from-to)118-123
Number of pages6
JournalBiochemical Engineering Journal
Volume48
Issue number1
DOIs
Publication statusPublished - 15 Dec 2009

Funding

This work is supported by Turkish State Planning Organisation in Advanced Technologies Program and by TUBITAK (Project No. 102T178 ). The authors would like to thank Mrs K. Moreton for her assistance and technical expertise.

FundersFunder number
TUBITAK102T178
Turkish State Planning Organisation in Advanced Technologies Program

    Keywords

    • Molecular modelling
    • NAD-dependent lactate dehydrogenase enzyme
    • Protein engineering
    • Recombinant protein
    • Steady-state kinetic
    • Substrate specificity

    Fingerprint

    Dive into the research topics of 'Increasing the substrate specificity of Bacillus stearothermophillus lactate dehydrogenase by DNA shuffling'. Together they form a unique fingerprint.

    Cite this