Rational design of a Yarrowia lipolytica derived lipase for improved thermostability
Graphical abstract
Introduction
Lipases (triacylglycerol hydrolases, E.C. 3.1.1.3) are a class of enzymes that catalyze the hydrolysis or formation of triglycerides. In addition, they are a versatile group of enzymes and some express other catalytic activities, such as phospholipase, lysophospholipase, cholesterol esterase, cutinase, amidase and other esterase type activities. Generally, lipases have a preference for their substrate type and are enantioselective, which makes them increasingly popular in the food, detergent, chemical, and pharmaceutical industries [1]. Lipases have emerged as one of the leading biocatalysts/bio-accelerators with proven potential for contributing to the underexploited multibillion-dollar bio-industry, through both in situ lipid metabolism and multifaceted ex situ industrial applications. Among commercial enzymes, >20 microbial lipases have recently been produced in large-scale industrial processes by companies such as Novozymes, Genencor, Amano, and others [2].
Regardless of process conditions, thermostability is often a key factor in successful industrial bioprocesses or food processing, since reaction rates typically increase exponentially with temperature, until the point of enzyme denaturation [3]. Thus, the engineering of thermostable lipases from biological entities for use in industrial reactors is an important goal. The most commonly used approaches for obtaining thermostable lipases are various immobilization techniques and enzyme mutagenesis [4,5]. In enzyme mutagenesis, non-rational and rational design are two general methods employed to obtain thermostable mutants of a target enzyme [6]. Firstly, numerous studies have shown that directed evolution based on error-prone PCR and DNA shuffling can be used to enhance the performance of diverse enzymes at elevated temperatures [7]. However, screening large numbers of colonies (usually >104) is time-consuming and is not feasible for enzymes expressed in slow-growing hosts. Therefore, the rational design approach, based on a detailed understanding of enzyme structure and function, has become another popular method to improve thermostability. Generally, thermophilic enzymes are more rigid, more stable and show lower root-mean-square deviation (RMSD) values than their mesophilic counterparts. Common stabilization strategies, such as introducing disulfide bonds or salt bridges, will result in lower RMSD values. In addition, enhancing intramolecular interactions, such as ionic interactions and/or hydrophobic interactions, can also lead to a decrease in the overall RMSD value of an enzyme [8]. Therefore, lowering the overall RMSD is an important factor for enhancing an enzyme's thermostability. In recent years, a number of strategies have been developed for identifying the weak-point residues of an enzyme as specific targets for mutation [9,10]. Molecular dynamics (MD) simulations proved to be useful tools for understanding enzyme structure and behavior. Using MD simulations, it is possible to observe different enzyme motions at various temperatures. MD simulations at various temperatures are necessary not only to predict the weak points of an enzyme, but also to verify its rigidity [11].
In the present research, the lipase LIP2 from Yarrowia lipolytica was rationally mutated to improve its thermostability. LIP2 belongs to the same family as Thermomyces lanuginosus lipase, a well-known lipase with many applications in the field of detergents and biotechnological processes [12]. Furthermore, it is an ideal candidate for enzyme replacement therapy because it shows highest activity at low pH and is not repressed by bile salts. In addition, LIP2 is an efficient biocatalyst for hydrolysis in water and for esterification in organic solvents [13]. This catalyst is used in numerous industrial applications due to its high enantioselectivity and wide range of substrates. However, the Y. lipolytica lipase also has a drawback: it is sensitive to thermal and interfacial denaturation and works only under mild conditions. Thus, improving the thermostability of LIP2 is highly desirable.
In this study, a rational design strategy was developed to improve the thermostability of LIP2. MD simulations at different temperatures were first performed to predict the most intensely fluctuating sidechains, which were then considered to be thermally weak-point residues. After analyzing the simulation results, computational design was applied to the target residues to lower the overall RMSD of the enzyme. The strategy developed here was successfully applied to design a LIP2 variant with high thermostability, and therefore holds great promise for improving the heat resistance of many other enzymes.
Section snippets
Strains, plasmids and reagents
The plasmid pPIC9K and Pichia pastoris GS115 (Invitrogen) were used as the expression vector and the host strain, respectively. Escherichia coli strain DH5ɑ (Life Technologies Inc., USA) and plasmid PUCm-T (Sangon, Shanghai, China) were routinely used for molecular cloning. DNA polymerase, restriction enzymes, LA Taq enzyme and T4-DNA ligase were from TaKaRa (Dalian, China). Primers were synthesized by the GENEWIZ Biotechnology Corporation (Suzhou, China). All reagents were of analytical grade
Exploring the molecular mechanism of the thermolability of LIP2 by MD simulations
Despite its many desirable characteristics, the comparatively low thermolability of LIP2 greatly impedes its broader industrial usage. In order to improve its thermostability, the molecular mechanism of the thermolability of LIP2 was probed by MD simulations at different temperatures. The values of RMSD, Rg, SASA and the number of internal hydrogen bonds of LIP2 at 303, 343 and 383 K were calculated over time from 0 to 100 ns. The values of RMSD and Rg are related to the structural stability of
Conclusions
In this study, a computational design scheme based on MD simulations to improve lipase stability at high temperatures was proposed. Firstly, MD simulations were performed to explore the molecular characteristics of LIP2 at 303, 343 and 383 K. Based on the results of RMSF calculation, the unstable regions A111-H126 and G207-K221 were identified. Subsequently, the mutants S115P and V213P were determined according to the residue with the highest RMSF value in that unstable region and the proline
Acknowledgements
This research was supported by the National Key Research and Development Program (2018YFA0901700), the National High-Tech Research and Development Plan of China (Grant number: 2013AA102803; Task number: 2013AA102803C), the National Natural Science Foundation of China (NSFC, Grant number: 81373309) and the Tianjin natural science funding (18JCYBJC43400).
Conflicts of interest
The authors declare no conflicts of interest.
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These three authors contributed equally to this work.