Recombinant Esterase Research Articles

Photosynthetic production of enantioselective biocatalysts.

BackgroundGlobal resource depletion poses a dramatic threat to our society and creates a strong demand for alternative resources that do not compete with the production of food. Meeting this challenge requires a thorough rethinking of all steps of the value chain regarding their sustainability resource demand and the possibility to substitute current, petrol-based supply-chains with renewable resources. This regards also the production of catalysts for chemical synthesis. Phototrophic microorganisms have attracted considerable attention as a biomanufacturing platform for the sustainable production of chemicals and biofuels. They allow the direct utilization of carbon dioxide and do not compete with food production. Photosynthetic enzyme production of catalysts would be a sustainable supply of these important components of the biotechnological and chemical industries. This paper focuses on the usefulness of recombinant cyanobacteria for the photosynthetic expression of enantioselective catalysts. As a proof of concept, we used the cyanobacterium Synechocystis sp. PCC 6803 for the heterologous expression of two highly enantioselective enzymes.ResultsWe investigated the expression yield and the usefulness of cyanobacterial cell extracts for conducting stereoselective reactions. The cyanobacterial enzyme expression achieved protein yields of 3% of total soluble protein (%TSP) while the expression in E. coli yielded 6-8% TSP. Cell-free extracts from a recombinant strain expressing the recombinant esterase ST0071 from the thermophilic organism Sulfolobus tokodai ST0071 and arylmalonate decarboxylase from Bordetella bronchiseptica showed excellent enantioselectivity (>99% ee) and yield (>91%) in the desymmetrisation of prochiral malonates.ConclusionsWe were able to present the proof-of-concept of photoautotrophic enzyme expression as a viable alternative to heterotrophic expression hosts. Our results show that the introduction of foreign genes is straightforward. Cell components from Synechocystis did not interfere with the stereoselective transformations, underlining the usability of photoautotrophic organisms for the production of enzymes. Given the considerable commercial value of recombinant biocatalysts, cyanobacterial enzyme expression has thus the potential to complement existing approaches to use phototrophic organisms for the production of chemicals and biofuels.

Efficient production of l-menthol in a two-phase system with SDS using an immobilized Bacillus subtilis esterase

Abstract Background levo-Menthol is an important flavoring chemical, which can be prepared by enantioselective enzymatic hydrolysis of dl-menthyl esters. A recombinant esterase (BsE) cloned from Bacillus subtilis 0554 shows excellent enantioselectivity to dl-menthyl acetate and has been immobilized using cross-linked enzyme aggregates. Though BsE has relatively high substrate tolerance, the conversion of dl-menthyl acetate decreased sharply with the increase of substrate loading from 1 to 3 M in mono-aqueous system, which might be due to the severe inhibition of enzyme activity at extremely high load of substrate or product. In this work, enzymatic hydrolysis of dl-menthyl acetate with an extremely high load using the immobilized CLEA-BsE was investigated in an organic-aqueous biphasic system containing surfactant to establish a promising bioprocess for large-scale production of l-menthol. Results An efficient biphasic reaction system of pentanol-water containing sodium dodecyl sulfate (SDS) was developed for improving enantioselective hydrolysis of dl-menthyl acetate to produce l-menthol by immobilized BsE. Under the optimized reaction conditions, l-menthol was produced in >97% enantiomeric excess (ee) at a substrate load of up to 3.0 M with >40% conversion. Conclusions All the positive features demonstrate the potential applicability of the bioprocess for the large-scale production of l-menthol.

Characterization of a novel hormone-sensitive lipase family esterase from Rhizomucor miehei with tertiary alcohol hydrolysis activity

A novel esterase gene (designated RmEstB) from the thermophilic fungus Rhizomucor miehei was cloned and functionally expressed in Escherichia coli. Sequence analysis revealed a 960-bp open reading frame encoding a protein of 319 amino acids. The deduced protein sequence contained an HGGG motif, suggesting that the enzyme is a hormone-sensitive lipase (HSL) family esterase. It showed highest identity of 52% with the esterase from Pseudomonas mandelii. The recombinant esterase was purified to homogeneity at 5.1-fold purification with a recovery yield of 85%. The molecular mass of RmEstB was estimated to be 37kDa by SDS-PAGE. RmEstB was most active at pH 7.5 and 50°C. The enzyme was highly stable in the presence of 30% ethanol, methanol, acetone, isopropanol, dimethyl sulfoxide and acetonitrile. RmEstB showed a broad range of substrate specificities toward various p-nitrophenol (pNP) esters (C2–C10) and triglycerides (C2–C6), with the highest specific activities obtained for pNP acetate (255U/mg) and triacetin (1330U/mg), respectively. In addition, RmEstB efficiently catalyzed the hydrolysis of sterically hindered esters of tertiary alcohols. This study presents a novel fungal HSL family esterase with potential for some industrial applications.

Purification of tag-free recombinant fumonisin esterase FumD from Pichia pastoris culture supernatant for use as calibration standard for enzyme quantification

Purification of tag-free recombinant fumonisin esterase FumD from Pichia pastoris culture supernatant for use as calibration standard for enzyme quantification

Scale-Up of a Recombinant Pig Liver Esterase-Catalyzed Desymmetrization of Dimethyl Cyclohex-4-ene-cis-1,2-dicarboxylate

A recombinant isoenzyme of pig liver esterase was used for the highly enantioselective desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate. The selected recombinant esterase showed a significant advantage in enantioselectivity over the commonly used esterase from the mammalian source. The process was scaled up to yield 265 g of product with a simplified pH control, and the target molecule was obtained with an enantiopurity of >99.5% ee.

Erratum to: Cloning, Expression, and Characterization of a Recombinant Esterase from Cold-Adapted Pseudomonas mandelii

Erratum to: Cloning, Expression, and Characterization of a Recombinant Esterase from Cold-Adapted Pseudomonas mandelii

Nonspecific cleavage of proteins using graphene oxide

In this article, we report the intrinsic catalytic activity of graphene oxide (GO) for the nonspecific cleavage of proteins. We used bovine serum albumin (BSA) and a recombinant esterase (rEstKp) from the cold-adapted bacterium Pseudomonas mandelii as test proteins. Cleavage of BSA and rEstKp was nonspecific regarding amino acid sequence, but it exhibited dependence on temperature, time, and the amount of GO. However, cleavage of the proteins did not result in complete hydrolysis into their constituent amino acids. GO also invoked hydrolysis of p-nitrophenyl esters at moderate temperatures lower than those required for peptide hydrolysis regardless of chain length of the fatty acyl esters. Based on the results, the functional groups of GO, including alcohols, phenols, and carboxylates, can be considered as crucial roles in the GO-mediated hydrolysis of peptides and esters via general acid–base catalysis. Our findings provide novel insights into the role of GO as a carbocatalyst with nonspecific endopeptidase activity in biochemical reactions.

Increased thermal stability of cold-adapted esterase at ambient temperatures by immobilization on graphene oxide

In this study, the effect of graphene oxide (GO) on the thermal stability of a recombinant esterase from cold-adapted Pseudomonas mandelii, rEstKp, was investigated. The complex GO-rEstKp was formed by cross-linking. Both free rEstKp and GO-rEstKp complex showed similar optimum pH and temperatures. GO-rEstKp complex exhibited enhanced thermal stability at ambient temperatures than rEstKp, which prevented the denaturation of the enzyme by hydrophilic interactions. However, the catalytic efficiency of GO-rEstKp complex was lowered to approximately 40% of that of free rEstKp. This study provides an insight into the addition of GO for industrial applications of cold-adapted enzymes at ambient temperatures.

Separation of enantiopure m-substituted 1-phenylethanols in high space-time yield using Bacillus subtilis esterase

A recombinant Bacillus subtilis esterase (BsE) expressed in E. coli was found to exhibit excellent enantioselectivity (E was always greater than 100) towards m-substituted 1-phenylethanol acetates in the enantioselective hydrolysis reaction. An explanation for the high enantioselectivity observed towards these substrates was provided by molecular modeling. Moreover, the BsE also showed strong tolerance towards a high concentration of m-substituted 1-phenylethanol acetates (up to 1 M). Based on these excellent catalytic properties of BsE, a kind of m-substituted 1-phenylethanols, (R)-1-(3-chlorophenyl)ethanol, was efficiently synthesized in space-time yield of 920 g per L per day and 97% ee, indicating that the BsE was considered as a potentially ideal and promising biocatalyst for large-scale production of optically active m-substituted 1-phenylethanols.

Cloning, Expression, and Characterization of a Recombinant Esterase from Cold-Adapted Pseudomonas mandelii

A gene coding for the extracellular esterase (EstK) was cloned from the psychrotrophic bacterium Pseudomonas mandelii based on its partial amino acid sequence as determined by mass spectrometry. The entire open reading frame consisting of 1,011 bp was expressed in Escherichia coli as a soluble protein and purified by nickel-chelated affinity chromatography and Capto Q column chromatography. Here, we show that the 33-kDa recombinant EstK protein (rEstKsp) had a substrate preference for esters of short-chain fatty acids, especially, p-nitrophenyl acetate. Optimum activity of rEstKsp was at pH 8.5 and 40 °C. The esterase activity remained similar from a range of 4∼20 °C, but the maximum activity varied depending upon pH. With p-nitrophenyl acetate as the substrate, K (M) was 210 μM and k (cat) was 3.4 s(-1). Circular dichroism and fluorescence spectroscopy results revealed that rEstKsp had a predominantly α-helical structure and maintained its folded state at 4∼40 °C. Interestingly, the tertiary structure of rEstKsp was predicted based on the structures of other hyperthermophilic esterases. Our results demonstrated that both native and rEstKsp are active at low temperatures and have a unique substrate preference for p-nitrophenyl acetate.

Cloning and characterization of a thermostable carboxylesterase from inshore hot spring thermophile Geobacillus sp. ZH1

The gene (741 bp) encoding carboxylesterase from the thermophilic bacterium Geobacillus sp. ZHl was cloned and overexpressed in Escherichia coli. The purified recombinant protein presented a molecular mass of about 40 kDa by SDS-PAGE analysis. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding highest specific activity with p-nitrophenyl acetate. Among the p-nitrophenyl esters tested, the carboxylesterase presented preference for p-nitrophenyl caprylate, but hydrolyzed p-nitrophenyl butyrate more efficiently. When p-nitrophenyl butyrate was used as a substrate, the recombinant carboxylesterase exhibited highest activity at pH 8.0 and 60°C. Almost no decrease in esterase activity was observed at 60°C for 3 h, and over 40% of activity was still maintained after incubation at 90°C for 3 h. These results indicate that Geobacillus sp. ZH1 recombinant esterase was thermostable. The enzymatic activity was inhibited by the addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays a key role in the catalytic mechanism. Except SDS and xylene, this esterase showed stability toward other tested detergents and organic solvents. Cloning, expression, and biochemical characterization of Geobacillus sp. ZH1 carboxylesterase lay a good foundation for its structural characterization and industrial application.

Characterization of a new thermophilic and acid tolerant esterase from Thermotoga maritima capable of hydrolytic resolution of racemic ketoprofen ethyl ester

► The recombinant esterase Tm1160 could form ∼74 kDa dimers in solution. ► Tm1160 have high thermostability and high acid tolerance. ► Tm1160 could enantioselectively hydrolyze the racemic ketoprofen ethyl eater. A gene coding for a putative thermostable esterase (Tm1160) from the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli . The purified enzyme displayed optimal activity at 70 °C and had a half-life of 60 min at 90 °C. It was stable over a range of pHs from 5.0 to 7.5 with an optimum around 5.0–5.5. The enzyme was found to have high acid tolerance and maintained about 50% of its activity even after 60 min of treatment at pH 4.5 and 70 °C. Furthermore, the enzyme exhibited the highest specific activity with p -nitrophenyl butyrate (318 ± 7 s −1 mM −1 ). Under native conditions, Tm1160 forms a ∼74 kDa dimer in solution. In addition, the esterase Tm1160 could enantioselectively hydrolyze the racemic ketoprofen ethyl ester and with an enantiomeric excess (ee p ) of 91.4% at a conversion of 41.1%, which makes it as a promising biocatalyst for the chiral resolution of ( S )-ketoprofen.

Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties

Thermoalkalophilic esterase enzyme from Balçova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7M CaCl2 solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization.

Preparation, Characterization and Optimization of Chitosan Nanoparticles as Carrier for Immobilization of Thermophilic Recombinant Esterase

Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this study, thermophilic esterase enzyme was obtained using recombinant DNA technology and purified applying one-step His-Select HF nickel affinity gel. The synthesis of chitosan was achieved from chitin by deacetylation process and degree of deacetylation was calculated as 89% by elemental analysis. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. The physicochemical properties of the chitosan and chitosan nanoparticles were determined by several methods including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform Infrared Spectroscopy) and DLS (Dynamic Light Scattering). The morphology of chitosan nanoparticles was spherical and the nanospheres’ average diameter was 75.3 nm. The purified recombinant esterase was immobilized efficiently by physical adsorption onto chitosan nanoparticles and effects of various immobilization conditions were investigated in details to develope highly cost-effective esterase as a biocatalyst to be utilized in biotechnological purposes. The optimal conditions of immobilization were determined as follows; 1.0 mg/mL of recombinant esterase was immobilized on 1.5 mg chitosan nanoparticles for 30 min at 60°C, pH 7.0 under 100 rpm stirring speed. Under optimized conditions, immobilized recombinant esterase activity yield was 88.5%. The physicochemical characterization of enzyme immobilized chitosan nanoparticles was analyzed by SEM, FT-IR and AFM (Atomic Force Microscopy).

Enantioselective production of (S)-1-phenyl-1,2-ethanediol from dicarboxyesters by recombinant Bacillus subtilis esterase

Abstract The whole cells of recombinant Escherichia coli BL21 overexpressing a Bacillus subtilis esterase (BsE) were utilized to sequentially hydrolyze the dicarboxyester of 1-phenyl-1,2-ethanediol for production of (S)-1-phenyl-1,2-ethanediol (PED), exhibiting high hydrolytic activity, excellent regio- and enantioselectivities towards the dicarboxyester of PED. Among the dicarboxyesters with different acyl chains (e.g., acetyl, n-butyl, and n-hexyl), the best enantioselectivity (E = 176) was observed when PED diacetate was employed as the initial substrate. Various reaction conditions were systematically investigated for enantioselective hydrolysis of PED diacetate. Under the optimal reaction conditions, kinetic resolution of 100 mM PED diacetate resulted in 49% conversion within 1 h, affording (S)-PED in 96% ee. A 150-ml scale reaction was performed, affording (S)-PED in 49% yield and 95% ee. After recrystallization in chloroform, the optical purity of (S)-PED was improved up to >99% ee, with a total yield of 45%. These results imply that this recombinant esterase (BsE) is a potentially promising biocatalyst for bioproduction of (S)-PED, an important chiral building block with wide application in pharmaceutical industry and liquid-crystal display materials.

Immobilization of Bacillus subtilis esterase by simple cross-linking for enzymatic resolution of dl-menthyl acetate

A recombinant esterase (EC 3.1.1.1) cloned from Bacillus subtilis 0554 (BSE) was carrier-freely immobilized with the method of cross-linked enzyme aggregates. The conditions for preparing the cross-linked aggregates of BSE (CLA-BSE) were optimized, including the type and concentration of precipitants, and the concentration of cross-linker, and a simple and efficient procedure for preparing CLA-BSE was developed, consisting of a precipitation step with 0.5 g mL−1 (NH4)2SO4 and a cross-linking step with 60 mM glutaraldehyde for a period of 3 h as the cross-linking time. As a result, about 70% of the initial free BSE activity was incorporated into the CLA-BSE. The thermal stabilities of the immobilized enzyme at 30 °C and 50 °C were >360 and 14 times those of free BSE, respectively. More importantly, the operational stability of CLA-BSE was also considerably improved. In the kinetic resolution of dl-menthyl acetate to produce l-menthol with CLA-BSE gave eep > 94% at conversion of >40% and the CLA-BSE could be reused for 10 times with only about 8% reduction in activity. Therefore, the new biocatalyst immobilized through the methodology of CLEAs could significantly decrease the manufacturing cost of l-menthol and would be more beneficial for its practical applications.

Functional Proteomic Analysis of Rice Bran Esterases/Lipases and Characterization of a Novel Recombinant Esterase

An esterase from rice ( Oryza sativa ) bran was identified on two-dimensional gel using 4-methylumbelliferyl butyrate as a substrate. The esterase cDNA (870 bp) encoded a 289 amino acid protein (designated OsEST-b) and was expressed in Escherichia coli . The molecular weight of recombinant OsEST-b (rOsEST-b) was 27 kDa, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemical characterization demonstrated that rOsEST-b was active over a broad temperature range (optimum at 60 °C) and preferred alkaline conditions (optimum at pH 9.0). The rOsEST-b showed maximum activity toward p-nitrophenyl butyrate (C(4)) among various p-nitrophenyl esters (C(4)-C(18)), indicating that rOsEST-b is an esterase for short-chain fatty acids. The kinetic parameters under optimal conditions were K(m) = 27.03 μM, k(cat) = 49 s(-1), and k(cat)/K(m) = 1.81 s(-1) μM(-1). The activity of rOsEST-b was not influenced by ethylenediaminetetraacetic acid, suggesting that it is not a metalloenzyme. The amino acid sequence analysis revealed that OsEST-b had a conserved pentapeptide esterase/lipase motif but that the essential active site serine (GXSXG) was replaced by cysteine (C). These results suggest that OsEST-b is distinct from traditional esterases/lipases and is a novel lipolytic enzyme in rice bran.

Immobilization of a Recombinant Esterase from Lactobacillus plantarum on Polypropylene Accurel MP1000

A recombinant esterase from Lactobacillus plantarum was immobilized on hydrophobic support polypropylene Accurel MP1000 by adsorption. Adsorption efficiency was 83%, and the immobilized protein was 12.4 mg/g of support. Esterase activity was determined using p-nitrophenyl butyrate as substrate, and highest activities were observed at 50 °C for immobilized enzyme and 30 °C for free enzyme extract. Concerning thermal stability, after enzyme incubation at 80 °C for 30 min, immobilized and free enzyme retained 91% and 56% of initial activity, respectively. Immobilized enzyme presented lower V(max) and higher K(m) than free enzyme. Protein was not released from the support, and esterase activity increased after 3 cycles of reuse.

An efficient bioprocess for enzymatic production of l-menthol with high ratio of substrate to catalyst using whole cells of recombinant E. coli

A gene encoding an esterase of Bacillus subtilis ECU0554 previously isolated from soil was cloned and overexpressed in Escherichia coli BL21. The recombinant esterase (recBsE) showed the best enantioselectivity ( E > 100) towards dl-menthyl acetate, in contrast to dl-menthyl esters propionate and butyrate. A high ratio of substrate to catalyst (S/C-ratio, ≥50) was achieved in the kinetic resolution of dl-menthyl acetate by using whole cells of recombinant E. coli BL21. Some key parameters of the biocatalytic process, including amount of cosolvent, catalyst loading and substrate loading, were optimized. Compared with the process catalyzed by wild-type whole cells of B. subtilis ECU0554, the second-generation bioprocess using whole cells of recombinant E. coli BL21 afforded a 40-fold improvement in S/C-ratio and a 75-fold improvement in the volumetric productivity per biocatalyst loading. Moreover, the substrate loading was increased up to 200 g L −1 (∼1 M), the biocatalyst loading was reduced to 2.5 g L −1 and the space-time yield was improved from 54 g L −1 d −1 to 202 g L −1 d −1.

Heterologous expression of an esterase from Thermus thermophilus HB27 in Saccharomyces cerevisiae

In this work, a system for high-level secretion by Saccharomyces cerevisiae of the Thermus thermophilus HB27 putative esterase YP_004875.1 was constructed. The recombinant protein was purified and partially characterised. Its lipolytic activity dropped abruptly when the acyl chain length of the substrate increased from 12 to 18 carbon atoms, and variation of the reaction rate as function of substrate concentration followed Michaelis–Menten kinetics. These results suggested that the enzyme was an esterase. The recombinant enzyme was N-glycosylated and both the glycosylated and non-glycosylated forms showed activity. Compared to the native enzyme, thermal stability (half-life of 4.3 h at 85 °C) was higher, optimum temperature (40 °C) was lower and optimum pH (7.5–8.5) was similar. These characteristics support potential biotechnological applications of the recombinant esterase.