Applied Microbiology and Biotechnology

, Volume 102, Issue 7, pp 3203–3215 | Cite as

Engineering a thermostable highly active glucose 6-phosphate dehydrogenase and its application to hydrogen production in vitro

Biotechnologically relevant enzymes and proteins


Glucose 6-phosphate dehydrogenase (G6PDH) is one of the most important dehydrogenases responsible for generating reduced NADPH for anabolism and is also the rate-limiting enzyme in the Entner-Doudoroff pathway. For in vitro biocatalysis, G6PDH must possess both high activity and good thermostability due to requirements of efficient use and low expense of biocatalyst. Here, we used directed evolution to improve thermostability of the highly active G6PDH from Zymomonas mobilis. Four generations of random mutagenesis and Petri-dish-based double-layer screening evolved the thermolabile wild-type enzyme to the thermostable mutant Mut 4-1, which showed a more than 124-fold increase in half-life time (t1/2) at 60 °C, a 3.4 °C increase in melting temperature (T m ), and a 5 °C increase in optimal temperature (Topt), without compromising the specific activity. In addition, the thermostable mutant was conducted to generate hydrogen from maltodextrin via in vitro synthetic biosystems (ivSB), gaining a more than 8-fold improvement of productivity rate with 76% of theoretical yield at 60 °C. Thus, the engineered G6PDH has been shown to effectively regenerate NADPH at high temperatures and will be applicable for NAD(P)H regeneration in numerous in vitro biocatalysis applications.


Glucose 6-phosphate dehydrogenase Thermostability High activity Hydrogen Directed evolution 



This project could not have been carried out without the support of the Biological Systems Engineering Department, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA and Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences. This study was supported by the Department of Energy, Office of Energy Efficiency and Renewable Energy, Fuel Cell Technologies Office under Award Number DE-EE0006968. The manuscript was edited by Ryan S. Senger.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Biological Systems Engineering DepartmentVirginia TechBlacksburgUSA
  2. 2.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina

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