In vitro conversion of glycerol to lactate with thermophilic enzymes

Chalisa Jaturapaktrarak,Kohsuke Honda,Suchada Chanprateep Napathorn,Hisao Ohtake,Maria Cheng,Kenji Okano

doi:10.1186/s40643-014-0018-4

Chalisa Jaturapaktrarak, Kohsuke Honda + Show 4 more

Open Access

https://doi.org/10.1186/s40643-014-0018-4

Copy DOI

Abstract

Abstract Background In vitro reconstitution of an artificial metabolic pathway has emerged as an alternative approach to conventional in vivo fermentation-based bioproduction. Particularly, employment of thermophilic and hyperthermophilic enzymes enables us a simple preparation of highly stable and selective biocatalytic modules and the construction of in vitro metabolic pathways with an excellent operational stability. In this study, we designed and constructed an artificial in vitro metabolic pathway consisting of nine (hyper)thermophilic enzymes and applied it to the conversion of glycerol to lactate. We also assessed the compatibility of the in vitro bioconversion system with methanol, which is a major impurity in crude glycerol released from biodiesel production processes. Results The in vitro artificial pathway was designed to balance the intrapathway consumption and regeneration of energy and redox cofactors. All enzymes involved in the in vitro pathway exhibited an acceptable level of stability at high temperature (60°C), and their stability was not markedly affected by the co-existing of up to 100 mM methanol. The one-pot conversion of glycerol to lactate through the in vitro pathway could be achieved in an almost stoichiometric manner, and 14.7 mM lactate could be produced in 7 h. Furthermore, the in vitro bioconversion system exerted almost identical performance in the presence of methanol. Conclusions Many thermophilic enzymes exhibit higher stability not only at high temperatures but also in the presence of denaturants such as detergents and organic solvents than their mesophilic counterparts. In this study, compatibilities of thermophilic enzymes with methanol were demonstrated, indicating the potential applicability of in vitro bioconversion systems with thermophilic enzymes in the conversion of crude glycerol to value-added chemicals.

Highlights

In vitro reconstitution of an artificial metabolic pathway has emerged as an alternative approach to conventional in vivo fermentation-based bioproduction
10 kg of crude glycerol, which is the primary byproduct of the biodiesel industry, is released for every 100 kg of biodiesel and the growing production of biodiesel has resulted in a worldwide surplus of crude glycerol [16]
We focused on the high operational stabilities of thermophilic enzymes and employed them as modules to construct an in vitro synthetic pathway for the conversion of glycerol to lactate, which is one of the most important and versatile biomass-derived chemical [20], in the presence of methanol

Summary

Introduction

In vitro reconstitution of an artificial metabolic pathway has emerged as an alternative approach to conventional in vivo fermentation-based bioproduction. Installation of an artificially engineered metabolic pathway in living organisms often leads to a competition with natural metabolic pathways for intermediates organic solvents than their mesophilic counterparts [13,14], and activities of some thermophilic enzymes are even improved with organic solvents [15] These excellent stabilities of thermophilic enzymes allow great flexibility in the operational conditions of in vitro bioconversion systems. We focused on the high operational stabilities of thermophilic enzymes and employed them as modules to construct an in vitro synthetic pathway for the conversion of glycerol to lactate, which is one of the most important and versatile biomass-derived chemical [20], in the presence of methanol

Methods

Results

Conclusion