Elsevier

Bioorganic Chemistry

Volume 104, November 2020, 104214
Bioorganic Chemistry

Activity and specificity studies of the new thermostable esterase EstDZ2

https://doi.org/10.1016/j.bioorg.2020.104214Get rights and content

Highlights

  • Activity and specificity of the new thermostable carboxyl esterase EstDZ2 was studied extensively for the first time.

  • EstDZ2 is an extremely active enzyme in the hydrolysis of aryl-substituted medium chain carboxylates.

  • EstDZ2 was tested for the first time in the kinetic resolution of secondary alcohols and showed (R) enantioselectivity.

  • Docking experiments revealed activity and stereoselectivity profile of EstDZ2.

Abstract

In this paper, we study the activity and specificity of EstDZ2, a new thermostable carboxyl esterase of unknown function, which was isolated from a metagenome library from a Russian hot spring. The biocatalytic reaction employing EstDZ2 proved to be an efficient method for the hydrolysis of aryl p-, o- or m-substituted esters of butyric acid and esters of secondary alcohols. Docking studies revealed structural features of the enzyme that led to activity differences among the different substrates.

Introduction

Lipolytic enzymes (EC 3.1.1.x) catalyze the hydrolysis of carboxyl ester bonds and they can be further grouped into several subclasses based on their substrate specificity and their capacity to hydrolyze esters in solution and emulsion. EC 3.1.1.1 represents carboxylesterases, enzymes which are active on solutions of short acyl chain esters [1]. The second more widely used subclass includes lipases, which are represented as EC 3.1.1.3 enzymes [2]. It is generally established that lipases have the unique capacity to cleave carboxylic ester bonds of water-insoluble long chain triacylglycerols including natural fats. Moreover, lipases, unlike esterases, are interfacially activated [3].

However, this classical distinction between lipases and esterases should no longer be considered as the main distinction between these hydrolytic enzymes, as kinetic and structural studies have revealed that there are lipases that can hydrolyze water-insoluble-long-chain esters without bearing a lid domain [4]. Therefore, the distinction between esterases and lipases should no longer be correlated with the physical state of the substrate and/or the presence of a lid domain in the enzyme molecule [5].

Lipolytic enzymes constitute one of the most abundant class of enzymes and they have received great attention as they participate in a variety of biological processes due to their functional diversity [6]. Their functions are pivotal for the human body via digestion, as they provide building blocks for biosynthesis, help the detoxification and provide carbon sources for energy [7]. In addition to their fundamental biological importance, lipolytic enzymes have many potential industrial and medicinal applications. Increasing interest revolves around their potential applications in the pulp- and paper-making industries [8]. Besides their ability to catalyze hydrolysis, lipolytic enzymes can also catalyze synthesis of ester bonds or transesterification reactions in non-aqueous media, following the thermodynamic reversion of the hydrolysis reaction route [9]. All these features render them protagonist biocatalysts in the industry [10].

Their biotechnological significance is reflected by the fact that there is an enormous effort to find techniques and methods, which will allow exploration of microbial organisms residing in extreme environments [11]. Metagenomics is one of the approaches, which aims to address this issue and bypass the limitations imposed by current culturing techniques [12]. Such recently developed techniques have provided access to new esterases or lipases bearing novel sequences and important features, such as thermostability, which is a crucial prerequisite for industrial applications.

EstDZ2 is a new lipolytic enzyme, which we have recently isolated from a metagenomic sample from a hot spring located in the Kamchatka Peninsula (Russia) as a part of the international project Hotzyme, which aims at identifying novel thermostable hydrolytic enzymes derived from hot springs with properties suitable for industrial applications [13]. The isolated esterase, was cloned, purified from Escherichia coli and characterized biochemically [14]. According to activity and specificity studies EstDZ2 can efficiently catalyze the hydrolysis of short to medium-length acyl chains esters [15]. The biochemical characterization proved that this new esterase is a thermostable enzyme with a half-life of more than six hours and excellent stability at high concentrations of organic solvents [15]. These properties suggest that EstDZ2 could be utilized in many different industrial applications. Furthermore, EstDZ2 is an interesting enzyme from a phylogenetic point of view, since its amino acid sequence did not cluster with that of any previously characterized bacterial esterolytic enzyme and, thus, it was found to belong to a putative new family of bacterial esterolytic enzymes, for which we have proposed the index XV [15].

In this work, we have examined in more detail the biochemical profile of the new thermostable esterase EstDZ2 by studying its reactivity and specificity towards aryl p-substituted esters of butyric acid and esters of secondary alcohols, and have found that EstDZ2 can successfully hydrolyze a wide range of esters. In silico analysis reveals that the activity of this new enzyme is a function of the geometry of the active site and the structure of the bulky substrate. These are the first reported reactions catalyzed by EstDZ2 and may eventually lead to utilization of this enzyme as a potent catalyst in organic synthesis.

Section snippets

Results and discussion

Recently published detailed biochemical characterization of EstDZ2 revealed that the three-dimensional modelled structure of the new hydrolytic enzyme is characterized by the α/β hydrolase fold, forming a twisted β-sheet [15]. The catalytic triad is constituted by residues of Ser, Asp and His, which are typical for esterolytic enzymes. Due to its novel sequence and functional characteristics, EstDZ2 is classified to a new family of bacterial esterolytic enzymes [15]. To investigate its

Conclusions

A detailed activity and specificity analysis of the new thermostable carboxyl esterase EstDZ2 was performed in this work. EstDZ2 was found to be highly active in the hydrolysis of aryl-substituted esters of butyric acid bearing subtituents in the ortho position of the aromatic ring. While sole steric effects could be explained rationally, the impact of altered electronic properties did not follow a clear trend in all cases. These results indicate that substrate acceptance is mainly determined

General

NMR spectra were recorded on Bruker Avance series 500 and 300 spectrometers at room temperature. Chemical shifts (δ) are reported in ppm relative to the residual solvent peak (CDCl3, δ: 7.26, 13CDCl3, δ: 77.16) and the multiplicity of each signal is designated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Coupling constants (J) are quoted in Hz. Column chromatographic separations were carried out using a flash chromatography system with

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References (21)

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    EKR has proven to be an extraordinary complementary technique to traditional asymmetric catalysis for the production of enantioenriched compounds [42]. Within either the wild-type (WT), or variants bearing specific mutations, an enzyme’s active site can provide an inherently chiral environment, resulting in a highly enantiodiscriminating catalysis, and effective resolutions when presented with racemic substrates [43]. EKR, therefore, continues to discovering transformations that produce interesting molecules in enantioenriched form with synthetically useful applications and high selectivity.

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