Rational design of a Yarrowia lipolytica derived lipase for improved thermostability

https://doi.org/10.1016/j.ijbiomac.2019.07.070Get rights and content

Abstract

To improve the thermostability of the lipase LIP2 from Yarrowia lipolytica, molecular dynamics (MD) simulations at various temperatures were used to investigate the common fluctuation sites of the protein, which are considered to be thermally weak points. Two of these residues were selected for mutations to improve the enzyme's thermostability, and the variants predicted by MD simulations to have improved thermostability were expressed in Pichia pastoris GS115 for further investigations. According to the proline rule, the high fluctuation site S115 or V213 was replaced with proline residue, the two lipase mutants S115P and V213P were obtained. The mutant V213P exhibited evidently enhanced thermostability with an approximately 70% longer half-life at 50 °C than that of the parent LIP2 expressed in P. pastoris. The temperature optimum of V213P was 42 °C, which was about 5.0 °C higher than that of the parent LIP2, while its specific catalytic activity was comparable to that of the parent and reached 876.5 U/mg. The improved thermostability of V213P together with its high catalytic efficiency indicated that the rational design strategy employed here can be efficiently applied for structure optimization of industrially important enzymes.

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.

References (38)

  • K. Watanabe et al.

    Protein thermostabilization by proline substitutions

    J. Mol. Catal. B Enzym.

    (1998)
  • H. Yu et al.

    The role of proline substitutions within flexible regions on thermostability of luciferase

    BBA-Proteins Proteom

    (2015)
  • L. Li et al.

    Enhancing thermostability of Yarrowia lipolytica lipase 2 through engineering multiple disulfide bonds and mitigating reduced lipase production associated with disulfide bonds

    Enzym. Microb. Technol.

    (2019)
  • A.K. Singh et al.

    Overview of fungal lipase: a review

    Appl. Biochem. Biotechnol.

    (2012)
  • M.E. Bruins et al.

    Thermozymes and their applications: a review of recent literature and patents

    Appl. Biochem. Biotechnol.

    (2001)
  • S. Cesarini et al.

    Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

    BMC Biotechnol.

    (2014)
  • J.L. Porter et al.

    Directed evolution of enzymes for industrial biocatalysis

    ChemBioChem

    (2016)
  • P.A. Tizei et al.

    Selection platforms for directed evolution in synthetic biology

    Biochem. Soc. Trans.

    (2016)
  • T. Tu et al.

    Improvement in Thermostability of an Achaetomium sp. strain Xz8 Endopolygalacturonase via the optimization of charge-charge interactions

    Appl. Environ. Microbiol.

    (2015)
  • Cited by (31)

    • Rational engineering of phospholipase C from Bacillus cereus HSL3 for simultaneous thermostability and activity improvement

      2022, Journal of Biotechnology
      Citation Excerpt :

      It is well known that substitution with proline residues reduces the conformational degree of freedom in the main polypeptide chain, thus increasing protein thermostability. Several attempts have been made to improve protein thermostability by proline substitutions in other enzymes (Zhang et al., 2019; Anbar et al., 2012; Liu et al., 2019). For example, Tian et al (Tian et al., 2010).

    • Employment of polysaccharides in enzyme immobilization

      2021, Reactive and Functional Polymers
      Citation Excerpt :

      To overcome such issues, different solvents have been broadly exploited as reaction media [27]. Other important applications of enzymes constitute (i) recovery of cheese [29,30], (ii) enhancing the flavor and production of enzyme-modified cheese (EMC) [31], (iii) bio-detergents [32–34], (iv) chemical catalysts [35,36]- the unification of biocatalysis and chemocatalysis renders the eco-friendly characteristic of biocatalysts, strong reactivity of chemical catalysts, reduced costs, and less generation of wastes, decreased operational time, easy handling of unstable intermediates and hence better selectivity and yield of the process [36], (v) agriculture products such as pesticides, insects, etc [37], and (vi) enhanced thermotolerance [38]. All the aforementioned desirable qualities of enzymes are frequently obstructed due to (i) less operational stability, (ii) high cost of production, (iii) moderate shelf-life, (iv) substrate and product inhibitions due to steric exclusion of the substrate from an inhibition site with interference with the active site of an enzyme [39] and thus cumbersome recovery and reusability [1,40–42].

    • Improving the activity and stability of Bacillus clausii alkaline protease using directed evolution and molecular dynamics simulation

      2021, Enzyme and Microbial Technology
      Citation Excerpt :

      The results are shown in Fig. 4b. The secondary structure of the Gly95-Gly100 region is a loop, and it therefore has greater flexibility compared with other secondary structures, given that proline is a rare imine amino acid and, according to the proline rule [49], the introduction of proline can improve the regional stability of the loop region and ultimately enhance the overall structural stability of the protein [50,51]. It can be seen from Fig. 4 that the Gly95-Gly100 loop region in the WT protease was close to its active sites Asp32, His62, Ser215, and in the G95P mutant, this loop region was significantly distant those active sites.

    View all citing articles on Scopus
    1

    These three authors contributed equally to this work.

    View full text