Construction of a novel lipolytic fusion biocatalyst GDEst-lip for ...

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J Ind Microbiol Biotechnol (2017) 44:799–815DOI 10.1007/s10295-017-1905-4

BIOCATALYSIS - ORIGINAL PAPER

Construction of a novel lipolytic fusion biocatalyst GDEst‑lip for industrial application

Renata Gudiukaite1 · Mikas Sadauskas2 · Audrius Gegeckas1 · Vilius Malunavicius1 · Donaldas Citavicius1

Received: 10 November 2016 / Accepted: 7 January 2017 / Published online: 19 January 2017 © Society for Industrial Microbiology and Biotechnology 2017

substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physico-chemical characteristics of target enzymes for industrial applications.

Keywords Biotechnology of thermophiles · Geobacillus lipases · Protein engineering · Geobacillus esterases · Fusion enzymes

Introduction

Microbial lipolytic enzymes are classified into two major families: lipases (EC 3.1.1.3) and carboxylesterases (EC 3.1.1.1) [7, 11]. Their activity strongly depends on a highly conserved catalytic triad Ser-Asp-His and α/β hydro-lase fold [7, 50]. Even though they share similar catalytic mechanisms and molecular structures, lipases and ester-ases hydrolyze different substrates [32]. Carboxylesterases show preferential activity toward acylglycerols with short chains (<10 carbon atoms). In contrast, lipases catalyze the hydrolysis of long chain acylglycerols (>10 carbon atoms) [21, 32]. Because of their regio- and stereospecific-ity and substantial activity in organic solvents, lipases and esterases have been recognized as very useful biocatalysts in industrial applications, such as the production of phar-maceuticals, leather, detergents, foods and medical diag-nostics [11, 29, 31, 33, 61, 63]. Lipases and esterases are also important in the cosmetic and perfume industry for the synthesis of flavor esters or mono- and di-acylglycerols that can be used as an emollient in personal care products [28]. The demand of lipolytic enzymes continues to increase for the synthesis of biopolymers, biodiesel and in lipid modifi-cation [4, 12, 18, 52, 71].

Abstract The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The result-ing open reading frame of 1497 nucleotides encoded a pro-tein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93–97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7–12) and tem-perature (5–85 °C) range and displayed maximum activ-ity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6–12, thermoactivity (5–85 °C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability,

During this work, Prof. Dr. D. J. Citavicius passed away. We remember him always with great respect and admiration.

Electronic supplementary material The online version of this article (doi:10.1007/s10295-017-1905-4) contains supplementary material, which is available to authorized users.

* Renata Gudiukaite [email protected]

1 Institute of Biosciences, Vilnius University, Sauletekio Avenue 7, 10257 Vilnius, Lithuania

2 Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University, Sauletekio Avenue 7, 10257 Vilnius, Lithuania

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800 J Ind Microbiol Biotechnol (2017) 44:799–815

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During the last 15 years since Bacillus stearothermophi-lus L1 lipase was described [36] the focus on the lipases and esterases produced by Geobacillus bacteria strongly increased. These microorganisms synthesize lipases and esterases which can be active at extreme conditions (alka-line pH, high temperature, organic solvents or detergents in reaction environment) and have a great potential for application in bioconversion and ecotechnology [16, 17, 26, 59]. Industrial processes often take place under harsh conditions that are hostile to microorganisms and their bio-catalysts. For these reasons thermoactive and thermostable enzymes are necessary [21]. Another aspect—the number of resources for natural enzymes is decreasing. New and versatile biocatalysts are required, which can be used in different industries: chemical synthesis, cleaning industry, leather and pulp processing. These industrial areas require biocatalysts, which are stable and active at not favorable environmental conditions. One of the strategies to improve the thermoactivity, thermostability and other physicochem-ical properties of industrial enzymes is a developing of syn-thetic fused chimeric enzymes.

Fusion proteins are composed of two or more differ-ent protein domains integrated into one molecule and have been developed as a class of novel biomolecules with multi-functional properties [14, 73]. Synthetic fusion proteins can be designed to achieve improved properties, enhanced bio-activities or generate new functionality of target enzymes [73]. The recombinant DNA technology by genetically fus-ing protein coding open reading frames or post-translational modification are the most commonly used strategies to cre-ate fusion proteins [21]. The construction of a recombinant chimeric/fusion protein is a standard and simple method used to increase the expression of target proteins and to facilitate purification steps [9, 69]. Gene fusion techniques were also successfully applied in immunology for devel-opment of immunoassays using chimeras between anti-body fragments and green fluorescent protein variants [5, 6]. Many examples of chimeric proteins (with bifunctional activity) are related to chimeric hemicellulases, endo-1,4-β-xylanases, β-xylosidases [15] or other enzymes, which are involved in degradation of biomass [22]. Examples of fused chimeric microbial enzymes include, but are not limited to, the following [1, 2, 30, 37, 39, 42, 46, 55]. Lipase B from Candida antarctica was successfully fused with cellulose-binding domain of cellulase A [27, 56]; Geobacillus sp. T1 lipase was fused with cellulase binding domain [54] and the secretion of B. stearothermophilus L1 lipase in Saccharo-myces cerevisiae was enhanced by translational fusion with cellulose-binding domain of Trichoderma harzianum endo-glucanase II (THEG) [3]. Notably, no fusion enzymes com-posed of lipase and esterase domains were described to date.

In this study chimeric lipolytic biocatalyst named GDEst-lip was designed, cloned into pET-21c (+) vector,

expressed in Escherichia coli BL21 (DE3) cells and puri-fied using IMAC methodology. Then physicochemical and kinetic analysis was performed. This new synthetic GDEst-lip enzyme was composed of previously described lipase GD-95 from Geobacillus sp. strain 95 [24] and GDEst-95 esterase produced by the same strain. GDEst-95 esterase is a 55-kDa esterase, which was cloned, purified, characterized and is the first such esterase enzyme produced by Geoba-cillus that was applied in protein engineering experiments. Since GD-95 lipase and GDEst-95 esterase displayed very different physicochemical properties it was hypothesized that chimeric GDEst-lip biocatalyst should exhibit char-acteristics of both enzymes or show intermediate values when properties were opposite. In this research two fused enzymes, composed of two GD-95 lipase domains (GDLip-lip) and two GDEst-95 esterase domains (GDEst-est) were also constructed and analyzed. These constructs showed that addition of identical domain can affect the thermoactivity, thermostability and other physicochemical and catalytic properties of monomeric Geobacillus lipases and esterases.

Materials and methods

Bacterial strains, plasmids and culture conditions

Geobacillus sp. strain 95 was previously isolated in Lithu-ania in the Vilnius University Faculty of Natural Sciences, Department of Microbiology and Biotechnology, from oil well [24]. For DNA manipulations, Geobacillus sp. strain 95 was grown in Luria–Bertani (LB) broth [58] at 60 °C with agitation (180 rpm). The E. coli strains used in DNA manipulations were: E. coli DH5α (ϕ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 phoA supE44 gyrA96 λ-thi-1 relA1) (Invitrogen) and E. coli BL21 (DE3) (λcIts857 indl Sam7 nin5 lacUV-T7 gene 1) (Novagen). E. coli transformants were grown in LB medium supple-mented with 100 μg/ml ampicillin at 37 °C with agita-tion (150–180 rpm). Vector pTZ57R/T (Thermo Fisher Scientific) was used as the cloning vector for the esterase and chimeric genes in E. coli DH5α strain and pET-21c (+) (Novagen) for enzymes’ expression in E. coli BL21 (DE3). LB agar containing 100 μg/ml ampicillin, 0.5 mM isopropyl-β-D thiogalactopyranoside (IPTG) (Thermo Fisher Scientific), 20 μg/mL 5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside (X-Gal) (Thermo Fisher Scientific) and 0.5% emulsified tributyrin (TCI Europe) was used to screen the recombinant clones harboring pTZ57R/T plas-mids with inserted esterase and chimeric genes. The same LB agar medium with ampicillin and tributyrin, but with-out IPTG and X-Gal was used for the screening of E. coli BL21 (DE3) transformants harboring pET-21c (+) plas-mids with target inserts.

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DNA manipulation

Genomic DNA of Geobacillus sp. strain 95 was extracted according to the method described by Sambrook and Rusell [58]. Plasmid DNA from E. coli transformants was isolated with the Zyppy™ Plasmid Miniprep Kit (Zymo Research). PCR products were purified for sequencing using Gene-JET™ PCR Purification Kit (Thermo Fisher Scientific). GeneJET™ Gel Extraction Kit (Thermo Fisher Scientific) was used to recover DNA fragments from agarose gels. Electrocompetent E. coli DH5α and E. coli BL21 (DE3) cells were prepared using the protocol from Sambrook and Rusell [58].

Amplification of the GDEst‑95 esterase gene

Geobacillus sp. 95 genomic DNA was used as a template for the PCR amplification of GDEst-95 esterase gene using the following primers: GSLE-F-41 (5′-GAC GTG GGA GGG GTG GTG GTT TAT-3′) and GSLE-R + 64 (5′-TTG GCC GTT CCT TTG TTG GTT TAG-3′) (Metabion). Amplification process was carried out in a 50-μL reaction mixture containing 1.5 mM MgCl2, 1× Taq DNA Poly-merase buffer without MgCl2, 0.2 mM dNTP mix, 2.5 U of Taq DNA Polymerase (Thermo Fisher Scientific), 20 pmol of each forward and reverse primers and genomic DNA (10 ng). The conditions for the PCR amplification were as follows: pre-denaturation at 94 °C for 4 min, denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min, exten-sion at 72 °C for 3 min, final extension at 72 °C for 7 min and preservation at 4 °C. Stages 2–4 stages were repeated 29 times. The amplificated product was detected by electro-phoresis through 0.8% agarose gel with ethidium bromide (1 μg/μL) and visualized with UV. The PCR product was purified and sequenced at the Institute of Biotechnology (Lithuania). Homology searches were performed against the sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) using the BLAST program.

Design of recombinant plasmid for gdest‑95 gene expression

After gdest-95 gene amplification and sequencing primers GESTp-31F (5′-GGG ATA AAG CAT ATG GAA CAA ACC GAT GTT G-3′) and GESTp-23R (5′-TAG TCG ACG CGT CCT TGC CAT GC-3′) were designed with intro-duced NdeI and SalI restriction sites for GDEst-95 clon-ing into pET-21c (+) expression vector. The resulting new PCR product was purified and ligated into pTZ57R/T vec-tor according to the manufacturer’s instructions (Thermo Fisher Scientific). The ligation product was transformed into E. coli DH5α electrocompetent cells using a standard

electroporation protocol [58]. After electroporation, the transformation suspension was plated onto LB-TB plates (100 µg/mL ampicillin) and incubated at 37 °C overnight. Recombinant clones were detected as described in Gudiu-kaite et al. [24]. For expression, the esterase gene frag-ment was cleaved from pTZ57R/T-est plasmid with the NdeI and SalI restriction enzymes and ligated into pET-21c (+) expression vector previously digested with the same restriction enzymes and dephosphorylated with calf intes-tinal alkaline phosphatase (Thermo Fisher Scientific). The resulting recombinant plasmid was transformed into E. coli DH5α and then retransformed into E. coli BL21 (DE3) competent cells for expression. Positive clones were identi-fied by lipolytic activity on plates supplemented with tribu-tyrin and double restriction digestion with NdeI and SalI.

Construction of fused gdest‑lip, gdest‑est and gdlip‑lip gene variants

In this work plasmids with inserted gdest-95 esterase gene and previously analyzed GD-95 lipase [24] were used as templates for construction of fused GDEst-lip, GDLip-lip and GDEst-est biocatalysts. The parental GD-95 lipase and GDEst-95 esterase genes were amplificated with primers listed in Table 1. The resulting PCR products were cloned into pTZ57R/T vector and transformed in E. coli DH5α cells. Plasmids were isolated from positive transformants and desired genes were digested with restriction endonu-cleases, listed in Table 1. After restriction the target gene fragments were purified from agarose gel and ligated with each other using T4 ligase (Thermo Fisher Scientific) as specified by manufacturer. The chimeric gene variants were cloned into pET-21c (+) vector for protein expression as previously described.

Expression and purification of GDEst‑95 esterase and synthetic GDEst‑lip, GDEst‑est and GDLip‑lip enzymes

The optimal conditions for expression of GDEst-95, GDEst-est esterase, GDLip-lip lipase and fused GDEst-lip protein were established according to Gudiukaite et al. [24]. Before purification the localization of desired lipases and/or esterases was detected by analyzing solu-ble and insoluble fractions of proteins in SDS-PAGE [38] as described in Gudiukaite et al. [25]. His-tagged recom-binant GDEst-95 esterase and fused protein variants, com-posed of two GD-95 lipase domains, two GDEst-95 ester-ase domains or chimeric GDEst-lip variant were purified to homogeneity by one-step purification protocol using immo-bilized nickel ion affinity chromatography (Profinity™ IMAC Resins, BIO-RAD) following the manufacturer‘s

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purification protocols for native and denaturing conditions with minimal modification. The detailed purification pro-tocol under native conditions is presented in Gudiukaite et al. [24] and for denaturing conditions in Gudiukaite et al. [25]. Protein concentration was determined using biopho-tometer (Eppendorf) and SDS-PAGE analysis was carried out as described in Gudiukaite et al. [24]. A PageRuler™ Unstained Protein Ladder (Thermo Fisher Scientific) and Pierce Unstained Protein MW Marker (Thermo Fisher Sci-entific) were used as a molecular mass marker.

Enzyme assay

Enzymatic activity was monitored spectrophotometrically at 420 nm with p-nitrophenyl (p-NP) dodecanoate (Sigma-Aldrich) as substrate [24]. One unit of enzyme activity was defined as the amount of enzyme that releases 1 µmol of p-NP from substrate per minute. The amount of released p-NP was calculated using a standard curve. The standard curve was prepared using the known amount of p-NP [13]. Due to instability differences of p-NP substrates at different temperatures and various pHs, the reaction mixture used as a negative control was incubated parallely at the given tem-perature or buffers without enzyme solution.

The lipolytic activity in zymogram was detected using tributyrin as substrate as described in Gudiukaite et al. [24]. Gel renaturation procedure was carried out following Levisson et al. [43].

Characterization of GDEst‑95, GDLip‑lip, GDEst‑est and GDEst‑lip lipolytic enzymes

The optimal temperature for esterase and/or lipase activity was determined by carrying out the enzyme assay at tem-peratures ranging from 5 to 90 °C. The effect of tempera-ture on the stability of target enzymes was investigated by

measuring the residual activity at 55 °C after incubation for 30 min at 30–90 °C.

The pH effect was evaluated by testing pH range of 5–12 under assay conditions. Various buffer systems, includ-ing 50 mM acetate buffer (pH 4–6), potassium phosphate buffer (pH 7), Tris–HCl buffer (pH 8), glycine–NaOH buffer (pH 9–11) and Na2HPO3–NaOH buffer (pH 11–12) were used.

The activity of purified GDEst-95 esterase, GDEst-est, GDLip-lip and GDEst-lip lipolytic enzymes was also stud-ied by incubation at room temperature for 30 min with 25% (v/v) organic solvents (dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), acetone, ethanol, methanol, n-butanol, isopropanol) or 0.1% surfactants (Tween-20, Tween-40, Tween-60, Tween-80, Triton X-100, urea) as described in Gudiukaite et al. [24]. The influence of 1 and 10 mM of metal chlorides (K+, Mg2+, Ca2+, Fe2+, Mn2+, Zn2+ and Cu2+) [34] and inhibitors (phenylmethylsulfo-nylfluoride (PMSF), dithiothreitol (DTT), ethylenediamine tetraacetic acid (EDTA), sodium dodecyl sulfate (SDS)) on lipolytic activity of GDEst-95 esterase and GDEst-lip biocatalyst was also investigated. The residual activity was determined under lipase assay conditions as described above. 100% lipolytic activity was recorded without the treatment with metal ions, inhibitors, surfactants or organic solvents.

Acyl chain length preference was determined by hydrol-ysis of different p-NP esters (Sigma-Aldrich) ranging from C2 to C18: p-NP acetate (C2); p-NP butyrate (C4); p-NP hexanoate (C6); p-NP octanoate (C8); p-NP decanoate (C10); p-NP dodecanoate (C12); p-NP myristate (C14); p-NP palmitate (C16) and p-NP stearate (C18). Glycine–NaOH buffer (50 mM, pH 9, 55 °C) was replaced by potas-sium phosphate buffer (50 mM, pH 7, 55 °C) in substrate specificity assays to reduce the instability of short acyl chain p-NP substrates.

Table 1 Summary of the primers used for the design of GDEst-lip, GDEst-est and GDLip-lip synthetic biocatalysts

Fused gene variant

Primer Sequence Inserted restriction site

Amplicon size (bp)

gdest-est GESTp-31FEst95-Rev-SacIEst95-Forv + SacIGESTp-23R

5′-GGG ATA AAG CAT ATG GAA CAA ACC GAT GTT G-3′5′-TAC GAG CTC GCG TCC TTG CCA TGC-3′5′-TAG CGA GCT CAT GGA ACA AAC CGA TGT TG-3′5′-TAG TCG ACG CGT CCT TGC CAT GC-3′

NdeISacISacISalI

~3000

gdlip-lip Gelip95-43-FLip95-Rev + SacILip95-Forv-SacIGelip95R

5′-TGA AGC GCA TAT GGC AGT TTC ACG CGC CAA-3′5′-TAG AGC TCA GGC CGC AAA CTC GC-3′5′-TAG GAG CTC ATG GCA GTT TCA CGC GC-3′5′-TAG CGG CCG CAG GCC GCA AAC TCG C-3′

NdeISacISacINotI

~2400

gdest-lip GESTp-31FEst95-Rev-SacILip95-Forv-SacIGelip95R

5′-GGG ATA AAG CAT ATG GAA CAA ACC GAT GTT G-3′5′-TAC GAG CTC GCG TCC TTG CCA TGC-3′5′-TAG GAG CTC ATG GCA GTT TCA CGC GC-3′5′-TAG CGG CCG CAG GCC GCA AAC TCG C-3′

NdeISacISacINotI

~2700

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Kinetic characterization of GDEst‑95, GDEst‑est, GDLip‑lip and GDEst‑lip lipolytic enzymes

The Michaelis–Menten constant (Km) and Vmax as well as Kcat were calculated using p-NP dodecanoate (C12) as substrate at concentrations ranging from 0 to 50 mM. The temperature, pH, and quantity of the enzyme were kept the same as for the enzyme activity assay described above. Data fitting of the Michaelis–Menten equation was used to determine Vmax and Km parameters, assuming that the reactions followed a simple Michaelis–Menten kinetics. The energy of activation (Ea) for GDEst-95 esterase and synthetic GDEst-est, GDLip-lip and GDEst-lip lipolytic enzymes was calculated with the help of the Arrhenius plot as was presented in Gudiukaite et al. [24].

Statistical analysis

All experiments were performed in triplicate, and the aver-age means derived. Standard deviation from the mean is shown in Tables 2, 4 and 5; Fig. 2. Significant differences between target enzymes were calculated using two-sample t test (http://insilico.net/tools/statistics/ttest; α = 0.05). Only the results with two-tailed p-value less or equal to 0.02 are presented in this paper.

Bioinformatic analysis

Three-dimensional (3D) structure of GDEst-95 was pre-dicted using I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/ [74] and SWISS-MODEL (http://swiss-model.expasy.org [10]). Image of the resulting 3D model was generated using RasMol [57]. Model quality was assessed using ProSA-web server (https://prosa.services.came.sbg.ac.at/prosa.php [70]). Sequence alignment was performed with BLAST (NCBI) and Mega 4.0.2. Ligand-binding sites were analyzed using RaptorX Binding server (http://raptorx.uchicago.edu/BindingSite/ [35]). The pos-sible interactions of catalytic amino acids with other amino acids in protein structure model were analyzed using CAD-score (http://bioinformatics.ibt.lt/cad-score/ [51]) server. Docking experiments were performed using AutoDockTools version 4 [49]. Ligand structures were acquired from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Kollmand charges for all the macromolecule atoms and Gasteiger atomic charges as well as rotatable bonds for the ligands were coordinated by AutoDockTools. A grid bo × 50 × 50 × 50 points with grid spacing of 0.5 Å was created and centered over the active site of GDEst-95 esterase. Docking grid parameters were generated by AutoGrid. Docking parameters were set as default values of the AutoDock hybrid Lamarckian Genetic Algorithm (LGA) and the free energy scoring function of LGA was

used to evaluate the conformations [48]. Docking was performed with a population size of 400 for 50 independ-ent LGA runs. Parameters of the minimum energy con-servation state between macromolecule and the ligand were noted down and top hits from each clusterings were analyzed for possible hydrogen bonds formation and/or hydrophobic interactions between the enzyme and the sub-strate. All images of the docked complexes were generated using PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.

Gene sequence submission

The sequences of Geobacillus sp. 95 16S rRNA and gdest-95 esterase gene were submitted to GenBank (NCBI) with accession numbers KX013769 and KX013768, respec-tively. The complete sequence of the GD-95 lipase gene was submitted to GenBank in previous report with acces-sion number KC609753 [24].

Results and discussion

Cloning and sequence analysis of the esterase from Geobacillus sp. 95

In previous work GD-95 lipase produced by Geobacillus sp. 95 was described [24]. Esterase gene from Geobacil-lus bacteria was amplificated for the purpose of design of GDEst-est and GDLip-lip fusion proteins and GDEst-lip chimeric biocatalyst. A 1497-bp fragment was obtained using the genomic DNA of Geobacillus sp. 95 and prim-ers based on the conserved regions among various esterase sequences. This fragment contained an open reading frame (ORF) encoding a protein of 498 amino acids (GenBank Accession No. KX013768). The sequence alignment in GenBank using BLAST showed high homology (93–98%) with other known Geobacillus carboxylesterases (Fig. S1). This novel esterase was named GDEst-95. It was analyzed with SignalP V3.0 web server (www.cbs.dtu.dk/services/SignalP/) [53], but the potential signal peptide was not pre-dicted. Therefore, molecular weight of native GDEst-95 esterase corresponded to 54.8 kDa. In addition, sequence alignment revealed that GDEst-95 esterase contains the typical catalytic triad composed of Ser194-Glu310-His409 and the consensus motif (Gly-X-Ser-X-Gly) around the active site serine (Fig. S1). Based on amino acid sequence, high homology with other Geobacillus carboxylesterases and biological properties which are discussed in subsequent sections, GDEst-95 esterase can be classified as a member of the family VII of bacterial lipolytic enzymes.

Geobacillus bacteria secrete two types of carboxylester-ases, which differ in molecular weight (30 and 55 kDa).

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http://zhanglab.ccmb.med.umich.edu/I-TASSER/

http://zhanglab.ccmb.med.umich.edu/I-TASSER/

http://swissmodel.expasy.org

http://swissmodel.expasy.org

https://prosa.services.came.sbg.ac.at/prosa.php

https://prosa.services.came.sbg.ac.at/prosa.php

http://raptorx.uchicago.edu/BindingSite/

http://bioinformatics.ibt.lt/cad-score/

https://pubchem.ncbi.nlm.nih.gov/

https://pubchem.ncbi.nlm.nih.gov/

http://www.cbs.dtu.dk/services/SignalP/

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The majority of findings reported to date described 30-kDa Geobacillus esterases [47, 52, 65, 67, 72, 75]. Liu et al. [44] demonstrated that Est30 and Est55 are distantly related with only 14.3% sequence identity. Est55 carboxylesterase was the first described 55-kDa carboxylesterase produced by Geobacillus bacteria [23, 45]. GDEst-95 esterase is therefore the second broadly characterized 55-kDa Geoba-cillus esterase.

Expression analysis and purification of the recombinant GDEst‑95 esterase

pET-21c (+) vector with inserted gdest-95 esterase gene (Fig. 1a) was transformed into E. coli BL21 (DE3) cells. Protein expression was induced with 1 mM IPTG and ana-lyzed by SDS-PAGE (Fig. 1b).

Expression analysis showed the appearance of a large amount of new protein in E. coli BL21 (DE3) cell sample after induction with IPTG (Fig. 1b). The size of this new pro-tein agreed well with the predicted size of GDEst-95 esterase (approximately 55 kDa). The highest yield of recombinant

GDEst-95 esterase was detected at 1–3 h after induction with 1.0 mM IPTG. The same amount of IPTG was used for the expression of GD-95 lipase [24] and other Geobacillus lipases [34, 40, 60, 62]. For the purification, cells of E. coli BL21 (DE3) transformants were harvested 2 h after induc-tion and both soluble and insoluble fractions were analyzed by SDS-PAGE (Fig. 1c). Results demonstrated that GDEst-95 esterase was partially soluble (Fig. 1c) and was success-fully purified using single step Ni2+ affinity chromatography method (Fig. 1c). Figure 1d shows a zymogram of GDEst-95 esterase together with GD-95 lipase as both of these enzymes were important for further experiments.

GDEst-95 esterase was purified using Tris–HCl (pH 8, 20 °C) buffer with 10 mM imidazole under native condi-tions (Fig. 1c). These conditions are more attractive for industrial application compared to the GD-95 lipase, which was purified using the same binding buffer with 250 mM imidazole [24]. The yield of recombinant GDEst-95 ester-ase was 24.18 mg from 200 ml of E. coli BL21 (DE3) cell culture. This amount was twofold lower than the yield of GD-95 lipase (Table 5).

Fig. 1 Cloning (a), expression (b) and purification (c-d) analysis of GDEst-95 esterase. gdest-95 gene fragment after PCR (a) lane M MassRuler DNA ladder mix. SDS-PAGE (12%) expression analysis of recombinant GDEst-95 esterase (b) lane M PageRuler Unstained Protein Ladder, 0–the moment when E. coli BL21 (DE3) cells har-boring pET-21c (+) with gdest-95 gene were treated with IPTG, 1–3 h–time after induction, (+) sample of cells treated with IPTG, (−) sample of cells without IPTG. Purification of GDEst-95 esterase

using IMAC under native conditions (c) lane M PageRuler Unstained Protein Ladder, lane D sample after sonication, lane S soluble pro-teins, lane IB inclusion bodies, lane FT flow through, lanes GDEst-95 esterase–GDEst-95 esterase purified using Tris–HCl (pH 8, 20 °C) buffer with 10 mM imidazole under native conditions. Analysis of GDEst-95 esterase and GD-95 lipase in SDS-PAGE (12%) and zymo-gram (d) lane M PageRuler Unstained Protein Ladder. The circle (a) and boxes (b–e) indicate the location of the target esterase

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Biochemical characterization of GDEst‑95 esterase

GDEst-95 esterase demonstrated high thermoactiv-ity (Fig. 2a). The optimal temperature for the activity of recombinant GDEst-95 esterase was 60 °C. This enzyme retained about 80% of lipolytic activity at 70–75 °C, while the GD-95 lipase possessed less than 20% of its activity at these temperatures (Fig. 2a). The difference was significant with p-value 0.002. The temperature range of GDEst-95 activity was from 5 to 85 °C which was broader compared to the range of GD-95 lipase, whose activity decreased sig-nificantly at 70 °C (Fig. 2a).

Charbonneau et al. [13] found that optimal pH and tem-perature values for EstGtA2 esterase were pH 8 and 50 °C, respectively. Geobacillus kaustophilus HTA426 esterase displayed optimal activity at pH 8 and 60 °C [47]; Est1, Est2, Est3 esterases from Geobacillus sp. at pH 9.5–10 and 65 °C [68]; G. stearothermophilus Est55 esterase at pH 8–9 and 60 °C [23] and EstB esterase from G. thermoleovorans YN at pH 8–9 and 65 °C [66]. To conclude, GDEst-95 esterase showed optimal temperature range similar to know Geobacillus carboxylesterases.

The temperature effect on the stability of GDEst-95 esterase was examined by measuring the remaining activ-ity after incubation at various temperatures at pH 9.0 for 30 min (Fig. 2b). GDEst-95 esterase retained more than 50% of lipolytic activity after incubation at 30–70 °C and completely lost its activity at 90 °C (Fig. 2b). These results suggested that GDEst-95 esterase is not only ther-moactive, but also thermostable and has a great biotech-nological potential. The thermostability as well as ther-moactivity of GDEst-95 was higher than that of GD-95 lipase. Significant differences were detected at 65 °C with

p-value 0.006 and at 70 °C with p-value 0.009 (Fig. 2b). This provided the basis for the fusion of both lipolytic enzymes produced by Geobacillus sp. 95 and raised the hypothesis that fusion of GDEst-95 esterase and GD-95 lipase might increase the thermostability and thermoactiv-ity of GD-95 lipase.

When the effect of pH on the GDEst-95 esterase activ-ity was analyzed it came out that the enzyme exhibited the highest activity at pH 9–10. Approximately 50% of the enzyme activity was observed at pH 8 compared to 100% at pH 9–10 (Fig. 2c). The pH range of GDEst-95 was nar-rower compared to GD-95 lipase (more than 50% of lipol-ytic activity at pH values from 7 to 11) (Fig. 2c). The dif-ferences were significant at pH values 7, 10 and 11.

The substrate preference of GDEst-95 was also exam-ined in this research. According to literature data esterases catalyze the cleavage and formation of short acyl chain esters [20]. The substrate specificity of GDEst-95 esterase was determined at 55 °C, pH 7, using various p-NP esters (Table 2). GDEst-95 esterase displayed hydrolase activity toward p-NP esters with acyl group chain lengths between C2 and C16, with an optimal activity on C4 (p-NP butyrate) and C6 (p-NP hexanoate) (Table 2). The activity drastically decreased with p-NP decanoate (27%) (Table 2). In spite of that, p-NP dodecanoate (C12) was chosen for the analy-sis of other physicochemical and catalytic properties as it was more stable at higher temperatures and pH values than other substrates and it was hydrolyzed by all in this study investigated enzymes. The differences toward various p-NP esters between GDEst-95 esterase and GD-95 lipase were significant for p-NP dodecanoate (p-value 0.002), p-NP myristate (p-value 0.02), p-NP palmitate (p-value 0.008) and p-NP stearate (p-value 0.003).

Table 2 Activity of GD-95 and new GDEst-95 and chimeric GDEst-est, GDLip-lip, GDEst-lip lipases/esterases toward various acyl length p-NP esters

The analysis was carried out at 55 °C, pH 7. Specific activities of 440 U/mg (GD-95), 212 U/mg (GDEst-95) and 45 U/mg (GDEst-est) were recorded as 100% on p-NP butyrate; 424 U/mg (GDEst-lip) on p-NP hexanoate and 88.9 U/mg (GDLip-lip) on p-NP octanoate

ND not detectable

Substrate Relative activity (%) of target enzymes

GD-95 GDEst-95 GDEst-lip GDLip-lip GDEst-est

p-NP acetate (C2) 87 ± 1.41 83 ± 1.41 100 53 ± 4.24 49 ± 7.07

p-NP butyrate (C4) 100 100 97 ± 2.24 75 ± 1.41 100

p-NP hexanoate (C6) 95 ± 4.07 96 ± 3.66 100 80 ± 12.02 52 ± 0.71

p-NP octanoate (C8) 88 ± 4.95 82 ± 3.54 62 ± 4.24 100 52 ± 4.95

p-NP decanoate (C10) 49 ± 9.19 27 ± 2.83 31 ± 2.12 26 ± 1.04 44 ± 3.54

p-NP dodecanoate (C12) 80 ± 1.41 18 ± 0.71 52 ± 1.41 40 ± 4.95 34 ± 3.54

p-NP myristate (C14) 43 ± 7.78 7 ± 1.41 24 ± 0.71 16 ± 2.12 17 ± 1.41

p-NP palmitate (C16) 35 ± 2.83 3 ± 2.12 22 ± 1.41 14 ± 2.12 11 ± 2.12

p-NP stearate (C18) 26 ± 2.12 ND 25 ± 1.41 7 ± 1.41 ND

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Docking experiments confirmed experimental results, which suggested that GDEst-95 esterase prefers short acyl chain length substrates (Table 3). Complex of GDEst-95 and p-NP hexanoate as substrate showed the lowest kI (dissociation constant) and binding energy (ΔG). The smaller the dissociation constant, the more tightly bound the ligand is, or the higher the affinity between the sub-strate and the protein is. The relative lipolytic activity of

GDEst-95 esterase toward p-NP hexanoate varied between 93 and 100% (Table 2). GDEst-95 esterase showed the highest kI and binding energy values for p-NP palmi-tate and p-NP stearate (Table 3), which corresponded to experimental results (weak p-NP palmitate hydrolysis and undetected hydrolysis of p-NP stearate, Table 2). Although GDEst-95 carboxylesterase showed high kI and binding energy for p-NP acetate (1.41 × 103 µM and −3.89 kcal/

Fig. 2 Effect of temperature on the activity (a) and stability (b) as well as pH influence (c) on the activity of purified GD-95 [24] and GDLip-lip lipases, GDEst-95 and GDEst-est esterases and fused GDEst-lip esterase/lipase. Straight line with a circle–GD-95 lipase, straight line with a triangu-lar–GDLip-lip lipase, dotted line with a square–GDEst-95 esterase, dotted line with rhom-bus GDEst-est esterase, dashed line with triangular GDEst-lip fused enzyme. Assays were performed at various tempera-tures (5–95 °C) (a) or various pH (c) under enzyme assay conditions. The remaining activ-ity was assayed under enzyme assay conditions after the purified recombinant enzymes were incubated at the indicated temperature (30–90 °C) for 30 min (b). Specific activi-ties of 400 U/mg (GD-95) and 545 U/mg (GDEst-lip) were recorded as 100% at 55 °C, 89 U/mg (GDEst-95) at 60 °C, 66 U/mg (GDEst-est) at 70 °C and 1120 U/mg (GDLip-lip) at 65 °C (a); specific activities of 203 U/mg (GD-95), 62 U/mg (GDEst-95), 48 U/mg (GDEst-est), 92 U/mg (GDLip-lip) and 573 U/mg (GDEst-lip) were recorded as 100% (b); 400 U/mg (GD-95), 100 U/mg (GDEst-95), 50 U/mg (GDEst-est), 100 U/mg (GDLip-lip) and 588 U/mg (GDEst-lip) were recorded as 100% at pH 9 (c)

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mol, respectively) it displayed about 83% of its lipolytic activity toward this substrate. The GDEst-95 esterase also possessed similar values of kI and binding energies for p-NP butyrate, p-NP octanoate and p-NP decanoate (Table 3).

In this report the influence of various organic solvents (Table 4), detergents (Table 4), inhibitors (Table 5) and metal ions (Table 5) on the enzyme activity of GDEst-95 was also determined. High specific activity after treatment with various organic solvents is a hallmark of lipases and esterases and is important for application of these enzymes in industrial ester synthesis and biofuel production [8, 64].

Surfactants are used in lipase-catalyzed reactions because they can stabilize the open conformation of the enzyme and prevent it from aggregation [26]. GDEst-95 esterase displayed increased activity after 30 min treatment with isopropanol (activation by 29%), n-butanol (activation by 14%), Triton X-100 (activation by 13%), Tween 40 (acti-vation by 26%), Tween 60 (activation by 36%), Tween 80 (activation by 11%) and 10 mM DTT (activation by 12%) (Tables 4, 5). It is important to note that various Tweens completely inhibited the lipolytic activity of GD-95 lipase (Table 4). The lipolytic activity of GDEst-95 esterase strongly decreased after treatment with DMSO, PMSF and SDS (Tables 4, 5). Significant differences between GDEst-95 esterase and GD-95 lipase were estimated in cases of ethanol, Triton X-100 and Tween 20–80, PMSF (1 and 10 mM), SDS (10 mM) and DTT (1 mM). As indicated above, the effect of the same organic solvent, inhibitor or surfactant on lipases/esterases may be different. One such example is isopropanol. Isopropanol merely affected the activity of G. stearothermophilus JC [34] and G. thermo-leovorans YN lipases [65], but strongly inhibited Geobacil-lus sp. ARM lipase [19]. Tweens 20–80 increased lipolytic activity of G. zalihae T1 lipase [41], but Tween-80 strongly inhibited G. stearothermophilus lipase JC [34]. It is impor-tant to note that GDEst-95 esterase was strongly inhibited by PMSF. The strong inhibitory effect of PMSF (more than 70% inhibition) indicates that the GDEst-95 is a ser-ine esterase. The L2 lipase [60], G. kaustophilus HTA426 esterase [62], T1 lipase [41] were also strongly inhibited by

Table 3 Calculated binding energies (kcal/mol) and dissociation constants (kI) for various substrates of GDEst-95 esterase

Docking analysis was performed with AutoDock

Substrate GDEst-95 esterase

KI (µM) ΔG (kcal/mol)

p-NP acetate (C2) 1.41 × 103 −3.89

p-NP butyrate (C4) 287.04 −4.83

p-NP hexanoate (C6) 65.65 −5.71

p-NP octanoate (C8) 206.74 −5.03

p-NP decanoate (C10) 295.96 −4.81

p-NP dodecanoate (C12) 588.01 −4.41

p-NP myristate (C14) 431.38 −4.59

p-NP palmitate (C16) 1.61 × 103 −3.81

p-NP stearate (C18) 2.75 × 103 −2.47

Table 4 Effect of various organic solvents and surfactants on the enzyme activity of purified GD-95 lipase, GDEst-95 esterase and fused GDLip-lip, GDEst-est and GDEst-lip lipolytic enzymes

Lipase activity without addition of organic solvents and detergents was set as 100%. Specific activities of 207.5 U/mg (GD-95), 93 U/mg (GDEst-95) and 588 U/mg (GDEst-lip) were recorded as 100%

ND not detectable

Effect Relative activity (%) of target enzymes

GD-95 [24] GDEst-95 GDEst-lip GDLip-lip GDEst-est

Organic solvents

DMSO 60 ± 4.4 39 ± 2.12 92 ± 4.24 71 ± 1.32 31 ± 7.78

DMF 113 ± 6.8 81 ± 8.49 63 ± 1.41 82 ± 9.90 86 ± 0.71

Ethanol 122 ± 6.5 67 ± 3.54 70 ± 7.78 78 69 ± 2.12

Methanol 80 ± 2.6 82 ± 12.02 81 ± 0.71 90 ± 9.90 11 ± 4.24

Acetone 90 ± 1.8 87 ± 9.90 70 59 ± 12.02 99 ± 0.71

Isopropanol 104 ± 3.5 129 ± 12.02 105 ± 0.71 74 ± 2.12 85 ± 2.12

n-Butanol 104 ± 4.3 114 ± 2.12 113 ± 4.24 111 128 ± 2.12

Surfactants

Triton X-100 ND 113 ± 2.12 63 ± 6.36 12 ± 0.71 123 ± 2.83

Tween-20 ND 90 ± 4.24 26 ± 7.78 ND 111 ± 5.66

Tween-40 ND 126 ± 7.07 46 ± 2.12 ND 164 ± 7.07

Tween-60 ND 136 ± 6.36 59 ± 1.41 ND 100

Tween-80 ND 111 ± 5.66 38 ± 8.49 ND 100

Urea 110 ± 3.3 89 ± 1.41 100 74 ± 2.12 124 ± 4.24

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PMSF and their catalytic triad is composed of Ser-His-Asp (or Glu in carboxylesterases).

The effect of tested metal ions is presented in Table 5. Our results showed that GDEst-95 esterase retained more than 50% of lipolytic activity after incubation with K+ (1 and 10 mM), Mg2+ (1 and 10 mM), Ca2+ (1 and 10 mM),

Mn2+ (1and 10 mM) and Cu2+ (1 mM), whereas Fe2+ (10 mM) decreased esterase activity by more than 70%. Differences between GDEst-95 esterase and GD-95 lipase after treatment with several metal ions were significant for 1 and 10 mM Fe2+, 1 and 10 mM Mn2+ and 1 mM Zn2+.

Kinetic and catalytic analysis of GDEst‑95 esterase

The GDEst-95 esterase exhibited a Km value of 5.88 mM and Vmax of 5.88 μmol min−1mg−1 when p-NP dodecanoate was used as a substrate. Compared to GD-95 lipase, GDEst-95 esterase displayed lower Kcat (3.27 × 105 min−1), lower catalytic efficiency (5.60 × 104 min−1 mM−1), lower spe-cific activity (100 U/mg) and higher activation energy (50.30 kJ/mol) (Table 6). This was not surprising, as p-NP dodecanoate was not an optimal substrate for GDEst-95, but it allowed a comparison between the two enzymes. The hypothesis that the fusion of GDEst-95 with GD-95 lipase might improve these and other catalytic parameters of both parental enzymes was raised.

Bioinformatic analysis of GDEst‑95 esterase

In this work, three-dimensional (3D) structure of GDEst-95 esterase was predicted using I-TASSER and SWISS-MODEL servers. The 3D structure of GDEst-95 car-boxylesterase with highlighted catalytic amino acids and domains is presented in Fig. 3a. C-score of the model obtained from the I-TASSER server was 1.67. C-score is a confidence score for estimating the quality of predicted models by I-TASSER. C-score is typically in the range of (−5, 2), where a C-score of higher value signifies a model with a high confidence and vice versa [74]. The 3D struc-ture of GDEst-95 esterase was predicted using crystal structure of the G. stearothermophilus carboxylesterase Est55 (pdb: 2OGS) [45] as template. The values of Z-Score in ProSA-web server were −11.81 and −11.8, assessing models predicted with I-TASSER and SWISS-MODEL, respectively. It was chosen to use 3D model predicted by

Table 5 Effect of various metal ions and inhibitors on the enzyme activity of purified GD-95 lipase, GDEst-95 esterase and its fused variant GDEst-lip

Lipolytic activity without the addition of metal ions and inhibitors was set as 100%. Specific activities of 202.9 U/mg (GD-95), 588 U/mg (GDEst-lip) and 86 U/mg (GDEst-95) were recorded as 100%

Effect and concentra-tions (mM)

Relative activity (%) of target enzymes

GD-95 [24] GDEst-95 GDEst-lip

K+ 1 100 92 ± 3.5 89 ± 0.7

10 69 ± 2.7 60 ± 2.8 73 ± 2.1

Mg2+ 1 89 ± 5.9 57 ± 6.1 96 ± 6.3

10 84 ± 3.5 61 ± 5.7 85 ± 7.5

Ca2+ 1 95 ± 4.7 95 ± 3.1 92 ± 5.7

10 96 ± 3.7 89 ± 3.5 83 ± 1.4

Fe2+ 1 3 ± 3.0 51 ± 3.5 64 ± 4.0

10 2 ± 2.0 29 ± 2.8 54 ± 4.9

Mn2+ 1 96 ± 4.0 64 ± 4.9 59 ± 3.5

10 31 ± 4.5 66 ± 2.8 82 ± 5.1

Zn2+ 1 95 ± 5.0 53 ± 0.7 95 ± 2.1

10 78 ± 6.4 51 ± 1.4 76 ± 0.7

Cu2+ 1 79 ± 2.0 96 ± 5.3 100 ± 5.5

10 25 ± 6.7 53 ± 1.41 70 ± 2.1

PMSF 1 92 ± 2.4 63 ± 6.4 88

10 41 ± 3.0 22 ± 1.4 47 ± 3.4

SDS 1 29 ± 4.5 41 ± 4.9 51 ± 5.8

10 29 ± 4.5 2 ± 0.4 4 ± 2.7

EDTA 1 90 ± 5.5 85 ± 2.1 90 ± 7.1

10 78 ± 0.5 72 ± 5.1 71 ± 4.9

DTT 1 100 ± 3.8 43 81 ± 7.2

10 93 ± 6.5 112 ± 8.1 76 ± 2.8

Table 6 Comparative kinetic properties of purified GD-95, GDEst-95, GDLip-lip, GDEst-est and GDEst-lip lipolytic enzymes

The values were determined at 55 °C, pH 9 using p-NP dodecanoate as substrate

Protein Vmax (µmol/min/mg)

Km (mM) Kcat (min−1) Catalytic effi-ciency Kcat/Km (min−1 mM−1)

Activation energy (Ea) (kJ/mol)

Specific activity (U/mg)

Yield (mg) of expressed protein from 200 ml cul-ture (soluble fraction)

GD-95 [24] 40.82 4.35 1.78 × 106 4.10 × 105 24.00 400 44.16

GDEst-95 5.88 5.88 3.27 × 105 5.60 × 104 50.30 100 24.18

GDEst-lip 20.00 14.29 2.00 × 106 1.40 × 105 68.34 600 38.56

GDEst-est 6.32 5.88 7.02 × 106 1.19 × 106 46.05 74.29 10.35

GDLip-lip 10.00 16.67 8.33 × 106 5.00 × 105 32.16 200 13.26

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I-TASSER, as the structure of GD-95 lipase [24] in previ-ous work was also predicted with I-TASSER server.

Crystal structure analysis of the carboxylesterase Est55 from G. stearothermophilus suggested that catalytic triad of Est55 was composed of Ser194, Glu310 and His409. It was also detected that Est55 folds into three domains: a cata-lytic domain, an α/β domain and a regulatory domain as the substrate-binding pocket of Est55 was formed of Phe112, Trp119, Leu225, Leu226, Met274, Leu313, Phe314, Leu316, Val370, Phe371 and Leu411 [45]. GDEst-95 ester-ase displayed 97% sequence similarity to Est55 and had Ser194, Glu310 and His409 at the same positions in amino acid sequence and structure as Est55 esterase. GDEst-95 esterase also had the typical lipase motif G-X-Ser-X-G (G-E-S-A-G) with located catalytic Ser.

Analysis of GDEst-95 esterase in RaptorX Binding server suggested four potential ligands (PG4–tetraethylene glycol; ETM–2-(trimethylammonium) ethyl thiol; NAG-N-acetyl-d-glucosamine; P6G–hexaethylene glycol), but the multiplic-ity only of two ligands (PG4 and ETM) was higher than 40. Pocket multiplicity is used to judge the quality of a predicted pocket. When the value of multiplicity is above 40, there is a high probability that the predicted pocket is real [57]. The multiplicity values of PG4 and ETM were 62 and 56, respec-tively. The analysis of binding residues suggested that in the binding of PG4 the following amino acids are involved: Pro64, Gly108, Leu111, Leu267, Gly270, Ser271, Leu313, Phe314, Thr317, Lys406, Ala407, Cys408 and His409. The ETM ligand bound to Gly107, Gly108, Ala109, Glu193, Ser194, Ala195, Val273, Leu313, Phe314, Lys406, Ala407, Cys408, His409 and Ala410 residues. Liu with co-workers [45] showed that Gly108, Lys406 and Cys408 coordinate iodine atoms, which were used to produce large well-dif-fracting crystals. These authors also suggested that catalytic His409 participates in hydrophobic interactions with Phe314

and Leu313. Our results showed that Gly108, Leu313, Phe314, Lys406, Cys408 and His409 were involved in the binding of both PG4 and ETM ligands. Analysis in CAD-score confirmed that catalytic His409 made significant con-tacts with Ser194 (catalytic amino acid), Leu313, Phe314, Cys408 and Ala410. The second catalytic amino acid Ser194 contacted with Gly107, Gly108, Glu193, Ala195, His409 and Ala410. The third catalytic amino acid Glu310 contacted only with Leu313, Ala407, Cys408 and His409. As most of these amino acids are hydrophobic, it could also be impor-tant for positioning of hydrophobic substrates (for example, various p-NP esters). The analysis of GDEst-95 esterase structure with incorporated p-NP dodecanoate (Fig. 3b, c) suggested that Ala109, Leu111, Leu313, Phe314, Thr317 and Ala410 are localized close to the substrate. Therefore, Gly107, Gly108, Ala109, Leu111, Thr317, Lys406, Ala407 and Ala410 are new candidate amino acids, which could make significant influence on the substrate specificity and activity of GDEst-95 esterase.

Design of GDEst‑est, GDLip‑lip and GDEst‑lip fusion protein

Significant differences between GDEst-95 esterase and GD-95 lipase in terms of thermoactivity, thermostability, resistance to organic solvents, detergents, substrate speci-ficity and catalytic properties generated the idea of fusion of both these lipolytic enzymes. In this work fusion pro-teins composed of two GD-95 lipase and GDEst-95 ester-ase domains were constructed to investigate the influence of additional domain on lipase or esterase activity and functionality. The primers used for design of GDEst-est and GDLip-lip variants and GDEst-lip chimeric protein are presented in Table 1. Schematic presentation of the created new lipolytic biocatalysts is shown in Fig. 4.

Fig. 3 3D structure of GDEst-95 esterase with highlighted cata-lytic triad and amino acids important for the binding of p-NP dode-canoate. The structure was predicted with I-TASSER server (a) and images were generated using RasMol (a–c). The catalytic amino acids are shown as black sticks (a–c). The domains of GDEst-95 (a)

are located according to Lu et al. [45]. Black arrows indicate the N– and C– ends of GDEst-95 esterase (a). The light and dark gray ball sticks (b and c) denote candidate amino acids involved in the binding of substrates. GDEst-95 esterase is represented as wireframe structure (b and c)

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Two amino acids (Glu-Lys) formed after restriction with SacI restriction endonuclease were used as linker. The longer linker was not used due to the instability of fused proteins (data not shown). The order of protein fusion (lipo-lytic domain at N-terminus followed by esterase domain at C-terminus) was chosen according to the observations that GD-95 esterase is being secreted by E. coli although no signal sequence for this protein was detected. In order to prevent the possible proteolytic cleavage between ester-ase and lipase, esterase domain was moved into N-terminal position of GDEst-Lip fusion protein.

Expression and purification of GDEst‑est, GDLip‑lip and GDEst‑lip lipolytic enzymes

The fused gdest-est, gdlip-lip and gdest-lip gene variants were successfully amplificated via PCR and cloned into pTZ57R/T and pET-21c(+) vectors as described above. Next, GDEst-est, GDLip-lip and GDEst-lip proteins were expressed in E. coli BL21 (DE3) cells and purified using Ni2+ affinity chromatography. Fig. S2 shows cloning (Fig. S2a), expression (Fig. S2b) and purification (Fig. S2c) of GDEst-lip chimeric biocatalyst.

SDS-PAGE analysis indicated that the largest amount of GDEst-lip (~100 kDa) was detected after 2–4 h with 1 mM IPTG induction (Fig. S2b). Therefore, recombinant pro-teins were prepared for purification after cells of E. coli BL21 (DE3) transformants producing GDEst-lip esterase/lipase were grown for 3 h after induction. Analysis of sol-uble and insoluble protein fractions showed that GDEst-lip esterase/lipase was soluble and could be purified using IMAC strategy under native conditions (Fig. S2c).

Similar expression results were obtained with GDEst-est and GDLip-lip, except that GDLip-lip lipase formed inclu-sion bodies and the largest amount of protein was detected in insoluble fraction (Fig. S3a). Since GDLip-lip lipase was nonhomogeneous after purification under denatur-ating conditions (Fig. S3a) and affected by urea, soluble fraction (Fig. S3b) was used for further experiments. It is worth noticing that GDEst-lip esterase/lipase was success-fully purified using 10 mM imidazole as was in the case of GDEst-95 esterase. GDEst-est and GDLip-lip lipolytic enzymes were purified using 10 and 250 mM imidazole, respectively (Fig. S3b, c). The yields of purified recombi-nant GDLip-lip, GDEst-est and GDEst-lip fusion biocata-lysts were 13.26, 10.35 and 38.56 mg, respectively, from 200 ml cell culture (Table 5). In this stage GDEst-lip dem-onstrated attractive features for further experiments and production on a large scale (it was easy to purify with a high yield of recombinant protein).

Effect of additional twin domain on physicochemical and catalytic properties of GD‑95 lipase and Est‑95 esterase

pET-21c (+) vector with inserted gdest-95 esterase gene (Fig. 2a). GDLip-lip displayed better abilities to work at higher temperatures (70–85 °C) than monomeric GD-95 lipase, but significant differences were detected only at 70 (p-value 0.012) and 85 °C (p-value 0.00001). At 85 °C GDLip-lip retained 13% of its lipolytic activity, while GD-95 lipase completely lost its activity at the same con-ditions. GDEst-95 and GDEst-est did not show significant differences.

Fig. 4 Schematic representa-tion of generated new biocata-lysts. The same restriction sites are highlighted and Glu-Lys (EL) amino acids represent a linker formed after restriction with SacI

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Thermostability analysis of GDLip-lip and GDEst-est fusion proteins suggested that thermostability of GDEst-est decreased significantly. At 70 °C GDEst-est lost more than 50% of its lipolytic activity, while GDEst-95 esterase pos-sessed nearly 100% activity after incubation at this temper-ature for 30 min. GDLip-lip lipase showed higher stability at mesophilic temperatures, but at 60–75 °C the remaining lipolytic activity was similar to the GD-95 lipase (Fig. 2b).

It is worth noting that lipolytic activity of GDEst-est esterase increased at pH 11 by 40%. GDLip-lip showed significant difference from monomeric GD-95 lipase at pH 10–11 (Fig. 2c).

The substrate specificity analysis suggested that GDLip-lip lipase differed from monomeric GD-95 lipase in its sub-strate specificity, when reaction was carried out at pH 7. Dimeric lipase displayed a threefold lower activity toward long acyl chain (C12–C18) p-NP esters with an optimal substrate p-NP octanoate (Table 2). Fusion of two ester-ase domains reduced the ability of GDEst-95 to hydro-lyze short acyl chain substrates, but improved the activity toward p-NP decanoate, p-NP dodecanoate, p-NP myristate and p-NP palmitate (Table 2).

As shown in Table 4 GDEst-est esterase possessed a similar lipolytic activity after treatment with various organic solvents as GDEst-95 esterase. The only difference was observed in the case of methanol. GDEst-est retained only 11% of its lipolytic activity after 30 min treatment with methanol while GDEst-95 demonstrated 82% of its activity. Also the GDEst-est esterase was not activated by isopropanol (85% relative activity after treatment with isopropanol) (Table 4). Significant differences between GDLip-lip and GD-95 lipase were detected after incuba-tion with DMF, ethanol and isopropanol (Table 4). It was important that GDLip-lip retained 12% of lipolytic activity after treatment with Triton X-100 (Table 4). Triton X-100, Tween-20, Tween-40 and urea activated GDEst-est esterase by 23, 11, 64 and 24%, respectively.

Kinetic parameters were measured using a spectropho-tometric activity assay with p-NP dodecanoate since this substrate was hydrolysed by all in this research investigated enzymes. The Km value for the conversion of p-NP dode-canoate by the GDEst-est esterase was 5.88 mM. GDEst-95 showed the same results (Table 6). The positive effect of fusion of two identical domains was detected in Vmax, Kcat, cat-alytic efficiency and activation energy, but the specific activity (74.29 U/mg) and yield (10.35 mg) of recombinant proteins were twofold lower than that of GDEst-95 esterase (Table 6). Interestingly, the GDEst-est esterase showed one of the best Km, Kcat and catalytic efficiency constants with p-NP dode-canoate as a substrate in this report. GDLip-lip did not dem-onstrate significant positive changes (Table 6). These results confirmed the hypothesis that fusion of identical domains can improve the physicochemical or catalytic properties of target

industrial enzymes, but it appears to depend on each protein individually. The different response of GD-95 lipase and GDEst-95 esterase to fusion may depend on the differences in amino acid sequences and structures. Crystallographic studies are required for further structural experiments.

Physicochemical properties of the new synthetic lipolytic biocatalyst GDEst‑lip and potential for industrial application

Detailed analysis of physicochemical and catalytic proper-ties of GD-95 lipase and GDEst-95 esterase as well as litera-ture data suggested the idea of a design of the new chimeric fusion biocatalyst composed of GD-95 lipase and GDEst-95 esterase domains. It was hypothesized that such novel syn-thetic enzyme named GDEst-lip could have improved fea-tures compared to both parental proteins. We assumed that GDEst-lip esterase/lipase could possess physicochemical and kinetic properties of both parental enzymes or demon-strate new unique characteristics if the features of GD-95 lipase and GDEst-95 esterase were very different, such as the effect of detergents on lipolytic activity.

Our experimental data suggested that at 5–40 °C GDEst-lip displayed thermoactivity profile similar to GDEst-95 esterase. Then, at 50–55 °C it was similar to thermoactiv-ity of GD-95 lipase and temperature optimum of GDEst-lip was at 55–60 °C (Fig. 2a). In the temperature range of 65–75 °C GDEst-lip biocatalyst showed significant differ-ences from both parental enzymes. At 80–85 °C tempera-ture range this enzyme displayed high similarity to GDEst-95 again and at 90 °C it retained 2% of its lipolytic activity. At this temperature both GD-95 lipase and GDEst-95 ester-ase lost their lipolytic activity completely (Fig. 2a). These results confirmed the hypothesis that GDEst-lip biocata-lyst could demonstrate improved characteristics, which are more attractive for industrial application.

One of the reasons for construction of GDEst-lip was the improvement of thermostability of GD-95 lipase. GDEst-lip completely lost its lipolytic activity after incubation at 85 °C for 30 min. This temperature was intermediate between tem-peratures, at wich GD-95 lipase and GDEst-95 esterase were inactivated (Fig. 2b). GDEst-lip demonstrated higher stability than both GD-95 lipase and GDEst-95 esterase at 50 °C. At 65–70 °C GDEst-lip showed unique intermediate thermosta-bility characteristics and after 30 min of incubation at 75 °C GDEst-lip retained 5% of its lipolytic activity. This value was similar to remaining activity of GD-95 lipase. The thermosta-bility of GDEst-lip esterase/lipase at 40 °C and 55–60 °C was similar to GDEst-95 (Fig. 2b).

The purified GDEst-lip esterase/lipase displayed a broad pH activity range of pH 6–12, with an optimum pH of 9–10 in 50 mM glycine-NaOH buffer. At pH 6 and pH 12, GDEst-lip retained 5% of its lipolytic activity (Fig. 2c).

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Significant differences between GDEst-lip and both paren-tal enzymes in terms of pH resistance were not detected.

GDEst-lip possessed 100% activity toward p-NP acetate, p-NP butyrate and p-NP hexanoate (Table 2), but significant differences from parental enzymes were not detected with these substrates. The ability of GDEst-lip to hydrolyze p-NP decanoate, p-NP dodecanoate, p-NP myristate and p-NP palmitate was intermediate between GDEst-95 esterase and GD-95 lipase. In case of p-NP stearate GDEst-lip showed 25% of its lipolytic activity, which was similar to GD-95 lipase (Table 2). Thus GDEst-95 esterase was improved in terms of substrate specificity after chimeric fusion.

Experiments showed that various Tweens and Triton X-100 inhibited GD-95 lipase completely [42]. In con-trast, these detergents activated GDEst-95 esterase. Ideally, GDEst-lip should have possessed about 50% of its lipol-ytic activity after treatment with various Tweens and Triton X-100. Indeed, GDEst-lip demonstrated more than 50% of lipolytic activity after incubation with Triton X-100 and Tween-60 for 30 min (63 and 59%, respectively) (Table 4). Tween-20, Tween-40 and Tween-80 inhibited GDEst-lip enzyme by 74, 54 and 62%, respectively. GDEst-lip chi-meric lipolytic biocatalyst confirmed the expectations and showed higher resistance to detergents than GD-95 lipase.

After incubation with various organic solvents GDEst-lip possessed 60–90% relative lipolytic activity (Table 4). It was activated only by isopropanol and n-butanol (activ-ity increased by 5 and 13%, respectively). It is important to mention that GDEst-lip retained 64 and 54% of its relative activity after treatment with 1 and 10 mM Fe2+, respec-tively (Table 5). This enzyme also demonstrated higher activity after incubation with 1 mM Mg2+, 10 mM Mn2+, 10 mM Cu2+ and 1 mM SDS (Table 5).

Biochemical analysis of GDEst-lip esterase/lipase sug-gested that this novel fused synthetic lipolytic enzyme was a wonderful new biocatalyst for industrial applications and possible to replace chemical catalysts. Additional experi-ments showed that GDEst-lip retained more than 50% of its lipolytic activity after incubation with 25 and 50% hexane and 25% isopropanol at room temperature for 7 days (data not shown). These results confirmed that GDEst-lip esterase/lipase was suitable for synthesis of biofuel, industrial esters or other valuable materials, as these reactions often involve organic solvents and detergents. Based on high lipolytic activity, broad range of pH, temperature and substrate speci-ficity, GDEst-lip was an excellent potential biocatalyst for improvement of modern household and industrial detergents.

Analysis of kinetic characteristics of the chimeric GDEst‑lip esterase/lipase

The new GDEst-lip lipolytic enzyme showed one of the best catalytic characteristics among all lipolytic enzymes

analyzed in this research. GDEst-lip possessed 600 U/mg specific activity when p-NP dodecanoate was used as a substrate. This specific activity was 1.5-fold higher than activity of parental GD-95 lipase and sixfold higher than that of GDEst-95 esterase (Table 6). The new fused enzyme also showed threefold higher Vmax compared to GDEst-95 esterase, though this value was twofold lower than Vmax of GD-95 lipase. Nevertheless, the Vmax of GDEst-lip pos-sessed the second highest Vmax value compared to all five proteins. These results confirmed the hypothesis that chi-meric GDEst-lip enzyme could demonstrate intermediate characteristics. GDEst-lip esterase/lipase also displayed the Kcat similar to GD-95 (2 × 106 min−1), high catalytic efficiency (1.40 × 105 min−1 mM−1), though its activation energy was higher than that of GD-95 lipase, GDEst-95 esterase and fused GDEst-est variant (Table 6). The reduc-tion of activation energy is an object of future research.

Conclusions

The carboxylesterase (GDEst-95) produced by Geobacil-lus sp. 95 strain with molecular size of 55 kDa was iden-tified in this work. This esterase was grouped into family VII of bacterial lipolytic enzymes. GDEst-95 esterase was the first carboxylesterase produced by Geobacillus bacteria with 55 kDa molecular size analyzed in-depth and used in protein engineering experiments. This esterase was cata-lytically active at broad pH (7–12) and temperature ranges (5–85 °C) with an optimum at pH 9 and 60 °C. It was also stable at high temperature (55–70 °C) and showed optimal activity toward short chain acyl derivatives. Because of its physicochemical and kinetic properties GDEst-95 ester-ase has high potential for application in various industrial areas.

In further experiments GDEst-95 esterase together with GD-95 lipase were used for construction of fused lipol-ytic chimeric biocatalyst GDEst-lip. Although both GD-95 lipase and GDEst-95 esterase are α/β hydrolases, they did not show significant similarity in amino acid sequences and possessed different physicochemical properties. It was hypothesized that the new chimeric GDEst-lip protein could have physicochemical characteristics of both parental enzymes. Indeed, GDEst-lip fusion enzyme showed high thermoactivity (5–90 °C) with an optimum at 55–60 °C, thermostability, a broad pH resistance range of 6–12 with an optimum at pH 9–10 and ability to hydrolyze both short and long acyl chain substrates. This new chimeric biocatalyst remained catalytically active after treatment with various organic solvents, inhibitors and surfactants. The GDEst-lip esterase/lipase also displayed high specific activity (600 U/mg) and one of the best catalytic character-istics (Vmax, Kcat, catalytic efficiency) and yield among all

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in this study analyzed lipolytic enzymes. Because of these features GDEst-lip biocatalyst has high potential to replace environmentally harmful chemical catalysts.

In this work the influence of additional domain on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic proper-ties was investigated for the first time. It was shown that usage of several fused domains can modulate the activity and physicochemical characteristics of target enzymes for industrial applications. Information presented in this work signifies that enzyme engineering by protein fusion is a powerful tool to improve biochemical and kinetic features of target industrially important enzymes.

Acknowledgements This work was supported by the MITA (Agency of Science, Innovation and Technology) program ‘‘Development of industrial biotechnology in Lithuania 2011–2013’’, project ‘‘Innova-tive tools for cosmetic industry (COSMETIZYM)’’, Grant No. MITA 31V-18.

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