Counteraction of Trehalose on N, N-Dimethylformamide-Induced Candida rugosa Lipase Denaturation: Spectroscopic Insight and Molecular Dynamic Simulation
March
Counteraction of Trehalose on N, N-Dimethylformamide-Induced Candida rugosa Lipase Denaturation: Spectroscopic Insight and Molecular Dynamic Simulation
Xin Yang 1 2
Ling Jiang 2
Yigang Jia 2
Yi Hu 1 2
Qing Xu 1 2
Xian Xu 1 2
He Huang 0 2
0 College of Pharmaceutical Sciences, Nanjing Tech University , Nanjing 210009 , PR China
1 College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University , Nanjing 210009 , PR China , 2 College of Food Science and Light Industry, Nanjing Tech University , Nanjing 210009 , PR China
2 Editor: Rajagopal Subramanyam, University of Hyderabad , INDIA
Candida rugosa lipase (CRL) has been widely used as a biocatalyst for non-aqueous synthesis in biotechnological applications, which, however, often suffers significant loss of activity in organic solvent. Experimental results show that trehalose could actively counteract the organic-solvent-induced protein denaturation, while the molecular mechanisms still don't unclear. Herein, CRL was used as a model enzyme to explore the effects of trehalose on the retention of enzymatic activity upon incubation in N,N-dimethylformamide (DMF). Results showed that both catalytic activity and conformation changes of CRL influenced by DMF solvent were inhibited by trehalose in a dose-dependent fashion. The simulations further indicated that the CRL protein unfolded in binary DMF solution, but retained the native state in the ternary DMF/trehalose system. Trehalose as the second osmolyte added into binary DMF solution decreased DMF-CRL hydrogen bonds efficiently, whereas increased the intermolecular hydrogen bondings between DMF and trehalose. Thus, the origin of its denaturing effects of DMF on protein is thought to be due to the preferential exclusion of trehalose as well as the intermolecular hydrogen bondings between trehalose and DMF. These findings suggest that trehalose protect the CRL protein from DMF-induced unfolding via both indirect and direct interactions.
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OPEN ACCESS
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The authors are grateful for the support of
the National Science Foundation, National Basic
Research Program of China, the National High
Technology Research and Development Program of
China, the Natural Science Foundation of Jiangsu
Province, and the Six Talent Peaks Project in Jiangsu
Province. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Introduction
In the past decade, biocatalysis has been established as a scalable and green technology
alternative to traditional chemocatalysis in the production of industrial and specialty chemicals, foods
as well as pharmaceuticals, both in the laboratory and on industrial scale [
1
]. Lipases (EC
3.1.1.3) have been proved to be versatile biocatalysts for catalyzing kinds of reactions, e.g.,
transesterification, esterification, asymmetric hydrolysis as well as organic synthetic reaction in
Competing Interests: The authors have declared
that no competing interests exist.
water-restricted environment [
2
]. Natural lipases are often highly selective catalysts, but are
not sufficiently tolerant to organic solvents. Polar solvents (e.g., N,N-dimethylformamide
(DMF), dimethyl sulfoxide (DMSO), formamide) can easily take away water molecular from
the protein surface and compete firmly for hydrogen bondings between proteins and water
molecules, which often denature the protein state to an unfolded one with the loss of specific
binding activity [
3
]. For example, an aqueous medium containing DMF even at levels of <10%
would result in nearly 30% loss of enzyme activity [
4
]. From the industrial view points, how to
improve the stability and the enzyme activity of lipases in typical organic solvent like DMF has
became a prosperous and powerful area of research.
Various technical methods have been developed to enhance the performance of organic
solvent tolerance of lipases from four main aspects, including directed evolution of
lipaseproducing strains, rational design of lipase genes, chemical modification and immobilization
of existing lipase products [
5,6
]. For example, it is reported that Proteus mirabilis lipase has
been successfully reengineered by directed evolution for improved thermostability as well as
tolerance to DMF [7]. In our previous studies, the performance of lipases in organic solvents
(e.g., DMF, methanol) has been improved by modifying with different kinds of functional
ionic liquids as well as immobilizing onto the surface of mesoporous SBA-15 [
8, 9
]. It is also
well-known that chemical chaperones as protein stabilizers can affect the stability and
catalytic activity of different proteins [10]. Furthermore, these low-molecular-weight chemicals
do not covalently modify the proteins. Such advantages would encourage the use of chemical
chaperones to assist in the improvement of lipase a (...truncated)