Recombinant Esterase Research Articles

Recombinant Esterase (BaCEm) Immobilized on Polyethyleneimine-Impregnated Mesoporous Silica SBA-15 Exhibits Outstanding Catalytic Performance.

A recombinant esterase, BaCEm, derived from Bacillus aryabhattai and heterologously expressed in Escherichia coli, was successfully immobilized on polyethyleneimine-impregnated mesoporous silica SBA-15. This immobilization utilized glutaraldehyde as a crosslinker. Optimal conditions were established with a PEI/SBA-15 ratio of 25% (w/w), a pH of 7.5, and a glutaraldehyde concentration of 0.5% (w/w), resulting in a loading capacity of 76.4mg/g, a recovery activity of 43.5%, and a specific activity of 7917 U/g for BaCEm. The immobilized BaCEm demonstrated high enantioselectivity, with an "E" value of 203.92, in the resolution assay of (R,S)-ethyl indoline-2-carboxylate. Notably, the immobilized enzyme, compared to its free counterpart, exhibited enhanced thermostability, maintaining 95.4% of its activity after 3h at 30°C. It also showed significant tolerance to organic solvents, retaining 48.4% and 28.7% residual activity in 10% v/v acetonitrile and acetone, respectively. Moreover, its storage stability was confirmed, with 68.5% residual activity preserved after 30days at 4°C. Remarkably, the immobilized BaCEm retained 58.1% of its activity after 10 reuse cycles, underscoring the potential of polyethyleneimine-impregnated mesoporous silica SBA-15 as an effective support for enzyme immobilization, promising for industrial applications.

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer

A fosmid library was constructed with the metagenomic DNA from the high-temperature sediment-rich water of the Albian aquifer (Algeria). Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. We identified a novel gene named AMWEst (1209 base pairs) encoding a protein of 402 amino acids with a predicted molecular weight of 43.44 kDa and conferring esterase activity. AMWEst was successfully overexpressed in the yeast mesophilic host Saccharomyces cerevisiae, and the expression system used proved to be efficient and produced sufficient activity for its biochemical characterization. Multiple sequence alignment indicated that AMWEst contained a conserved pentapeptide motif (Gly120-His121-Ser122-Gln123-Gly124). The optimum pH and temperature of the recombinant esterase AMWEst were 8 and 80 °C, respectively. Additionally, AMWEst showed higher activity towards short carbon substrates and showed maximum activity for p-nitrophenyl hexanoate (C6). Notably, AMWEst has a remarkable thermostability, and the enzyme retains almost maximum activity at 70 °C after incubation for 1 h. Moreover, enzyme activity was enhanced by high concentrations of SDS and Triton X-100 detergents.Key points• A novel thermostable esterase has been retrieved through functional metagenomics• The esterase is detergent-tolerant, which is attractive for some applications• The esterase can be expressed in a yeast mesophilic host to enhance its yield

A chemo-enzymatic transformation of linalyl acetate to estragole, a phenyl propanoid ether using recombinant esterase in acetone reaction system

The esterase gene from Bacillus subtilis E9 was expressed in E. coli BL21 (DE3) using the pTAC Bs-est recombinant plasmid and the enzyme was purified to determine its biocatalytic ability on the natural ester, linalyl acetate. The substrate affinity of the esterase enzyme was confirmed through docking studies on the interaction between the modelled esterase protein with the ligand. Recombinant esterase transformed linalyl acetate both in buffer and acetone containing reaction system to an alcohol, linalool through hydrolysis within four hours of incubation. This enzymatic hydrolysis involved the release of acetic acid, making the environment acidic. However, after 20 h of enzyme treatment, unlike that of aqueous reaction system, in acetone reaction system this acid condition favoured further chemical conversion of linalool to a cyclic phenyl propanoid ether, estragole. The presence of organic solvent-acetone in the reaction phase could have contributed to the structural modification of the enzyme, facilitating the hydrolysis of linalyl acetate to form linalool and further cyclization to estragole. The overall substrate conversion rate of linalyl acetate to the transformed products, linalool and estragole in acetone containing reaction system was 84 %.

An Esterase with Increased Acetone Tolerance from Bacillus subtilis E9 over Expressed in E. coli BL21 Using pTac Bs-est Vector.

Bacillus subtilis E9 was identified as a potential strain producing esterase. The gene coding esterase from B. subtilis E9 was amplified using esterase-specific primers and the sequence was translated in silico. The presence of conserved catalytic triad amino acid residues (His-Ser-Asp/Glu) confirmed the functional nature of the esterase enzyme. Docking studies conducted with modeled protein and the ligand p-nitrophenyl acetate showed that the amino acid residues interacting with the ligand were Ser77, His76, and Gly103. The gene coding for esterase from B. subtilis E9 was cloned into an assembled vector having Tac promoter (pTac), pUC origin of replication, Ni-Histidine residues, ampicillin cassette, and T7 terminator using Golden gate DNA assembly method. The generated pTac Bs-est (4598bp) recombinant plasmid was transformed and heterologously expressed in Escherichia coli BL21 (DE3) strain. The tagged recombinant protein was purified to yield 43.4% pure protein with specific activity of 772 U/mg. The purified recombinant protein was subjected to peptide sequencing and the identity was confirmed as esterase by peptide tandem mass fragmentation method using the LC-MS/MS analysis. The purified recombinant esterase was found to be organic solvent stable and tolerant up to 5days retaining around 95% residual activity in 30-90% v/v Acetone. The recombinant esterase expressed in our study was found to exhibit better organic solvent stability and tolerance than compared to the original bacterial esterase from B. subtilis E9, a property which could be explored in the biocatalytic and synthetic transformation reactions for industrial applications.

Heterologous expression, molecular studies and biochemical characterization of a novel alkaline esterase gene from Bacillus thuringiensis for detergent industry.

Present study was aimed to clone and express the esterase encoding gene from Bacillus thuringiensis in E. coli BL21. Purification of recombinant esterase enzyme was achieved up to 48.6 purification folds by ion exchange chromatography with specific activity of 126.36 U mg-1. Molecular weight of esterase enzyme was 29 kDa as measured by SDS-PAGE. Purified esterase enzyme showed stability up to 90% at 90 °C and remained stable in a wide pH range (8-11). Molecular docking strengthens the experimental results by showing the higher binding energy with p-NP-butyrate. Enzyme activity was found to be reduced by EDTA but enhanced in the presence of other metal ions. Enzyme activity was reduced with 1% SDS, PMSF, and urea but organic solvents did not show considerable impact on it even at higher concentrations. Purified recombinant esterase was also found to be compatible with commercial laundry detergents and showed very good stability (up to 90%). All these properties proved the esterase enzyme from B. thuringensis a significant addition in detergent industry.

Purification and refolding protocol for cold-active recombinant esterase AaSGNH1 from Aphanizomenon flos-aquae expressed as insoluble inclusion bodies

Microbial esterases are a highly desirable tool for numerous biosynthetic and biotechnological applications requiring ester bond cleavage. Once identified, microbial esterases are often produced recombinantly in Escherichia coli to enhance yield and ease of purification. In this study a polyhistidine-tagged SGNH esterase gene (AaSGNH1), originating from the cyanobacterium Aphanizomenon flos-aquae, was cloned into an over-expression plasmid and expressed in BL21(DE3) cells. The recombinant esterase enzyme was produced as inactive inclusion bodies which were insoluble in 8 M urea but readily solubilized by the detergent Empigen BB®. Crucially, the procurement of active enzyme required controlled removal of detergent during column chromatography and dialysis steps. The refolded esterase was characterized with respect to its ability to catalyze the cleavage of p-nitrophenol esters of different chain lengths (C2, C8, C16). In addition, the temperature and pH optima were determined and it was found that the enzyme was most active at low temperatures (5–15 °C) and under alkaline conditions (pH 8–10). It was found that the kinetic properties of AaSGNH1 were remarkably similar to other SGNH esterases described thereby validating that the protein was effectively refolded. Overall, this study provides a simple strategy for isolating cold-active recombinant esterase enzyme when expressed as inclusion bodies.

Thermoalkalophilic recombinant esterase entrapment in chitosan/calcium/alginate‐blended beads and its characterization

AbstractBACKGROUNDEsterases (EC 3.1.1.1), a class of hydrolases, degrade the ester bonds of lipids into alcohol and carboxylic acids and synthesize carboxylic ester bonds. They are used in a variety of biotechnological, industrial, environmental, and pharmaceutical applications due to their many valuable properties. Particularly, extremophilic esterases with many superior properties are of great interest for various reactions. Immobilization of enzymes may provide some advantages over free enzymes not only to improve the properties of enzymes but also to increase the reusability of biocatalyst in industrial applications. Therefore, many different immobilization applications for enzymes have been reported in various studies. To our knowledge, a thermophilic esterase has not so far been immobilized by entrapment using chitosan/calcium/alginate‐blended beads. Here, we reported the immobilization of thermoalkalophilic recombinant esterase by entrapment using chitosan/calcium/alginate‐blended beads, and then the entrapped esterase was characterized biochemically in details.RESULTSIn the present study, a thermophilic recombinant esterase was immobilized by entrapment in chitosan/calcium/alginate‐blended beads for the first time. The 0.5 mg mL−1 purified recombinant esterase was entrapped in 1% chitosan, 2% alginate, and 0.7 M CaCl2 blended beads. The results showed that immobilization yield and entrapment efficiency of the entrapped esterase were 69.5% and 80.4%, respectively. SEM micrograph showed that the surface of the beads resembled a mesh and very compact structures. Chitosan/calcium/alginate‐blended beads exhibited an 18.8% swelling ratio and had a moderate porous structure. The entrapment technique highly enhanced the thermostability of the esterase and shifted its optimum temperature from 65 to 80 °C. The immobilized esterase was very stable in a wide range of pH (8.5–11) displaying maximum activity at pH 9. ZnCl2 slightly increased the activity of immobilized esterase whereas several metal ions reduced the enzyme activity. When the enzyme was immobilized in chitosan/calcium/alginate‐blended beads, its Km increased about 2 times and Vmax value decreased almost 1.5 times. Immobilization allowed repeated uses of the esterase having good operational stability in a continuous process.CONCLUSIONThe results revealed that the immobilization of a thermophilic recombinant esterase by entrapment in chitosan/calcium/alginate‐blended beads exhibited considerably better compared to other immobilization processes with various entrapment strategies. © 2021 Society of Chemical Industry (SCI).

Properties of a Newly Identified Acetyl Esterase and Its Degradation of Diisobutyl Phthalate

AbstractPhthalic acid esters (PAEs) are attracting growing attention because of environmental pollution and their harms to mammals, and diisobutyl phthalate (DiBP) in PAEs is still little studied. Biodegradation is a fast, efficient and inexpensive treatment preferentially used to deal with the pollution of PAEs. In this study, the recombinant esterase Est824 was obtained by heterologous expression and purification. The optimal reaction temperature and pH of the enzyme are 40 °C and 8.0 respectively. The enzyme reserves activity above 70 % in metal ion solutions at varying concentrations and thus shows high metal ion resistance. In high‐concentration salt solutions, the enzyme activity remains above 50 %, indicating that the enzyme has strong salt tolerance. The response surface method was used to optimize the fermentation conditions. With the Box‐Behnken design, the esterase yield was 1003.54 U/L. The DiBP degradation effect of the enzyme was detected by HPLC, and the degradation rate was 100 %. Therefore, the enzyme has good industrial application prospects and may be a potential candidate for the degradation of PAEs.

Efficient Cocaine Degradation by Cocaine Esterase-Loaded Red Blood Cells.

Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37°C) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37°C. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability.

Isolation, Expression and Characterization of the Thermophilic Recombinant Esterase from Geobacillus thermodenitrificans PS01

Esterases are widely used in the food industry. Here, a new thermophilic bacterium, Geobacillus thermodenitrificans PS01, was isolated and the esterase-encoding gene est1 was cloned, sequenced, and recombinant expressed in Escherichia coli Tuner (DE3). The highest activity of recombinant Est1 was detected at pH 8.0, and 40°C and the extreme stability was observed at pH 6-9 over 30days at 4°C. In particular, Est1 can hydrolyze short- to medium-chain (C2-C10) triglycerides and p-nitrophenyl esters (C2-C12) and was not inhibited by most metal ions. Kinetic parameters of p-nitrophenyl butyrate hydrolysis under optimal conditions were determined: Km, 22.76μM; kcat, 10,415s-1; and kcat/Km, 457.53μM-1s-1. The outstanding specification of Est1 indicates its potential for use in industrial applications.

RECOMBINANT C1 ESTERASE INHIBITOR FOR SHORT-TERM PROPHYLAXIS IN PATIENTS WITH HEREDITARY ANGIOEDEMA

Introduction Patients with hereditary angioedema (HAE) are at risk for an acute attack after medical procedures. Short-term prophylaxis may minimize this risk. This study evaluated recombinant C1 esterase inhibitor (rhC1-INH) as short-term prophylaxis. Methods Patients with angioedema were treated with rhC1-INH prior to medical procedures/stressful life events; HAE attacks were recorded through 2 days and >2-7 days postprocedure. Results Fifty-one patients (median age, 44 years [range, 17-73 years]; 62.7% female; 92.2% HAE type 1) were included. A median rhC1-INH dose of 3075 IU (range, 2100-4200 IU) was administered as prophylaxis, median of 60 minutes prior, for 70 procedures (52.9% [n=37] dental [median, 60 minutes preprocedure]); 30.0% [n=21] surgical [median, 45 minutes preprocedure]; 15.7% [n=11] endoscopy [median, 30 minutes preprocedure, and 1.4% [n=1] stressful life event). Majority (n=48; 68.6%) of 70 cases had rhC1-INH administered 10-65 minutes preprocedure: 25 of 48 (52.1%) dental, 16 (33.3%) surgical, and 7 (14.6%) endoscopy. Nineteen (27.1%) cases involved long-term prophylaxis (danazol/tranexamic acid). Overall, 97.1% (68/70) of cases did not have an HAE attack within 2 days postprocedure; 91.4% (64/70) during >2-7 days postprocedure. For 2 attacks occurring within 2 days, rhC1-INH was administered 230 minutes and ≥24 hours preprocedure, respectively. No adverse events were reported. As a self-control group, 76.9% of 26 cases with no long-term/short-term prophylaxis preprocedure had an attack within 2 days postprocedure. Conclusions Short-term prophylaxis with rhC1-INH, administered within several hours preprocedure, was efficacious and safe in adolescents/adults and reduced the risk of an HAE attack postprocedure.

Presence of protein production enhancers results in significantly higher methanol-induced protein production in Pichia pastoris

BackgroundThe yeast Komagataella phaffii, better known as Pichia pastoris, is a commonly used host for recombinant protein production. Here expression vectors are reported that address the different steps of the transcription–translation–secretion pathway of heterologous protein production.ResultsTranscription and translation enhancing elements were introduced in an expression cassette for the production of recombinant Aspergillus niger feruloyl esterase A. The yield was increased by threefold as compared to the yield without these elements. Multiple copy strains were selected using a zeocin resistance marker in the expression cassette and showed another sixfold higher yield. Modification of the C-terminal amino acid sequence of the secretion signal did not significantly improve the production yield. Similar data were obtained for the production of another protein, recombinant human interleukin 8. Upscaling to fed-batch fermentation conditions resulted in a twofold increase for reference strains, while for strains with enhancing elements a tenfold improvement was observed.ConclusionsPichia pastoris is used for recombinant protein production in industrial fermentations. By addressing the transcription and translation of mRNA coding for recombinant protein, significant yield improvement was obtained. The yield improvement obtained under microscale conditions was maintained under fed-batch fermentation conditions. These data demonstrate the potential of these expression vectors for large scale application as improved production of proteins has major implications on the economics and sustainability of biocatalyst dependent production processes e.g. for the production of pharmaceuticals and for the bioconversions of complex molecules.

Characterization of two novel thermostable esterases from Thermoanaerobacterium thermosaccharolyticum

This paper first describes characterization of two thermostable esterases (ThLip1 and ThLip2) from the thermophilic bacterium Thermoanaerobacterium thermosaccharolyticum DSM 571. The recombinant esterase ThLip1 was active at 80 °C, pH 6.5 and maintained approx. 85% of original activity after 2 h incubation at 75 °C. Kinetic parameters, Km, Vmax and kcat/Km for 4-Nitrophenyl caprylate (pNPC) were 3.52 ± 0.47 mM, 191.18 ± 1.82 μmol min−1 mg−1 and 20.80 ± 0.07 mM−1 s−1, respectively. The purified recombinant esterase ThLip2 was optimally active at pH 6.5 and 75 °C and it was stable against a pH range of 6.0–8.0 possessing 2 h half-life at 80 °C. Kinetic experiments at 75 °C with pNPC as a substrate gave a Km of 3.37 mM, Vmax of 578.14 μmol min−1 mg−1and kcat of 231.2 s−1. The hydrolysis of linalyl acetate were carried out using ThLip1 and ThLip2 as catalyst, affording linalool yields over 140 mg/l in 10 h.

A Novel Cold Active Esterase from a Deep Sea Sponge Stelletta normani Metagenomic Library

Esterases catalyze the hydrolysis of ester bonds in fatty acid esters with short-chain acyl groups. Due to the widespread applications of lipolytic enzymes in various industrial applications, there continues to be an interest in novel esterases with unique properties. Marine ecosystem has long been acknowledged as a significant reservoir of microbial biodiversity and in particular of bacterial enzymes with desirable characteristics for industrial use, such as for example cold adaptation and activity in the alkaline pH range. We employed a functional metagenomic approach to exploit the enzymatic potential of one particular marine ecosystem, namely the microbiome of the deep sea sponge Stelletta normani. Screening of a metagenomics library from this sponge resulted in the identification of a number of lipolytic active clones. One of these encoded a highly, cold-active esterase 7N9, and the recombinant esterase was subsequently heterologously expressed in Escherichia coli. The esterase was classified as a type IV lipolytic enzyme, belonging to the GDSAG subfamily of hormone sensitive lipases. Furthermore the recombinant 7N9 esterase was biochemically characterized and and was found to be most active at alkaline pH (8.0) and displays salt tolerance over a wide range of concentrations. In silico docking studies confirmed the enzyme’s activity towards short-chain fatty acids while also highlighting the specificity towards certain inhibitors. Furthermore structural differences to a closely related mesophilic E40 esterase isolated from a marine sediment metagenomics library are discussed.

Recombinant esterase from Corynebacterium pseudotuberculosis in DNA and subunit recombinant vaccines partially protects mice against challenge.

We tested the efficacy of the esterase encoded by cp1002_RS09720 from Corynebacteriumpseudotuberculosis in recombinant subunit and DNA caseous lymphadenitis (CLA) vaccines. This target was predicted as one of the best CLA vaccine candidates by mature epitope density analysis. Gene cp1002_RS09720 was cloned into two different vectors (pAE for subunit vaccine and pTARGET for DNA vaccine). Four groups of 15 mice each were immunized with the recombinant esterase rCP09720 associated with aluminium hydroxide adjuvant (G1), pTARGET/cp09720 DNA vaccine (G2), a naked pTARGET (G3) or PBS as a negative control (G4). Immunization occurred in two doses intercalated by a 21 day interval. Twenty-one days after the last dose administration, animals were challenged with a virulent C. pseudotuberculosis MIC-6 strain. G1 showed high levels of IgG1 and IgG2a on days 21 and 42 post-immunization and a significant level of IFN-γ (P<0.05), suggesting a Th1 response. The protection levels obtained were 58.3 and 16.6 % for G1 and G2, respectively. The subunit vaccine composed of the recombinant esterase rCP09720 and Al(OH)3 is a promising antigenic formulation for use against CLA.

Characterization of EstQE, a new member of esterase family VIII from the quizalofop-P-ethyl-degrading bacterium Ochrobactrum sp. QE-9

Quizalofop-P-ethyl (QE) is a selective, systemic, post-emergence aryloxyphenoxy propanoate (AOPP) herbicide for the control of annual and perennial grassy weeds in a variety of crops. An efficient QE-degrading bacterial strain, QE-9, was isolated from a QE-manufacturing facility in China and identified as Ochrobactrum sp. A 4.0kb DNA fragment from QE-9 containing a full length esterase encoding gene was cloned and analyzed. Multiple sequence alignment showed that the esterase gene estqe encoded a 382 amino acid protein that contained the conserved S-X-X-K esterase motif and clustered within esterase family VIII, owning to a lack of beta-lactamase activity. Full-length estqe was amplified by PCR and cloned into plasmid pET-29a for functional expression in Escherichia coli BL21 (DE3). The purified recombinant esterase EstQE converted QE to quizalofop acid (QA) at an optimum temperature and pH of 45°C and 8.0, respectively. Enzyme activity was severely inhibited by Cu2+, whereas, Ca2+ and Mg2+ significantly increased enzyme activity to 142 and 122% of the control, respectively. The inhibitors PMSF, pCMB, and DEPC as well as detergent SDS strongly inhibited esterase activity. Furthermore, EstQE was capable of hydrolyzing a wide range of other AOPP herbicides, in the following order of catalytic efficiency, quizalofop-P-ethyl>fenoxaprop-P-ethyl>clodinafop-propargyl>cyhalofop-butyl>quizalofop-P-tefuryl>haloxyfop-P-methyl. These results suggest that EstQE is a potential candidate for remediation of AOPP herbicide-contaminated environments.

Biocatalytic Resolution of Rac-α-Ethyl-2-Oxo-Pyrrolidineacetic Acid Methyl Ester by Immobilized Recombinant Bacillus cereus Esterase.

A new esterase-producing strain (Bacillus cereus WZZ001) which exhibiting high hydrolytic activity and excellent enantioselectivity on rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester (R, S-1) has been isolated from soil sample by our laboratory. In this study, the stereoselective hydrolysis of (R, S-1) was performed using the recombinant Bacillus cereus esterase which expressed in Escherichia coli BL21 (DE3). Under the optimized conditions of pH 8.0, 35°C, and concentration of substrate 400mM, a successful enzymatic resolution was achieved with an e.e. s of 99.5% and conversion of 49%. Immobilization considerably increased the reusability of the recombinant esterase; the immobilized enzyme showed excellent reusability during 6cycles of repeated 2h reactions at 35°C. Thereby, it makes the recombinant B. cereus esterase a usable biocatalyst for industrial application.

Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6.

A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride.

The applicability of Impranil®DLN for gauging the biodegradation of polyurethanes

Polyurethane-based polymers and their eventual degradation products pervade modern society. One common method for determining whether a microorganism or protein can degrade this class of polymer is to qualitatively assess its ability to “clear” a polyester-polyurethane colloid branded Impranil®DLN (Impranil), whose formulation is proprietary. However, its colloidal state has ultimately made Impranil a precarious choice for determining if an organism or enzyme can degrade polyurethanes. In this work, the chemical hydrolysis products from Impranil using 0.1 M HCl or 0.1 M NaOH were identified and compared to the concentration of hydrolysis products formed using three commercial enzymes by proton nuclear magnetic spectroscopy (1H NMR) and Fourier-transform infrared spectroscopy (FT-IR). The differences in the integrated signal intensities from key 1H NMR signals were used to calculate the amount of Impranil that was hydrolyzed. These data were then correlated with the change in optical density of colloid containing reaction mixtures (termed as “clearing”). The enzymes (a Pseudomonas fluorescens recombinant esterase, Pseudomonas sp. lipase, and Bacillus sp. protease) showed significant esterase activities and partially-cleared, completely-cleared, or aggregated Impranil, respectively. However, only the Pseudomonas sp. lipase significantly degraded Impranil based on NMR and IR data. This study illustrates how Impranil can be used to quantitatively assess biodegradation rather than just be a qualitative “clearing” indicator of biodegradation.

Heterologous expression of a fungal sterol esterase/lipase in different hosts: Effect on solubility, glycosylation and production

Ophiostoma piceae secretes a versatile sterol-esterase (OPE) that shows high efficiency in both hydrolysis and synthesis of triglycerides and sterol esters. This enzyme produces aggregates in aqueous solutions, but the recombinant protein, expressed in Komagataella (synonym Pichia) pastoris, showed higher catalytic efficiency because of its higher solubility. This fact owes to a modification in the N-terminal sequence of the protein expressed in Pichia pastoris, which incorporated 4-8 additional amino acids, affecting its aggregation behavior. In this study we present a newly engineered P.pastoris strain with improved protein production. We also produced the recombinant protein in the yeast Saccharomyces cerevisiae and in the prokaryotic host Escherichia coli, corroborating that the presence of these N-terminal extra amino acids affected the protein's solubility. The OPE produced in the new P.pastoris strain presented the same physicochemical properties than the old one. An inactive form of the enzyme was produced by the bacterium, but the recombinant esterase from both yeasts was active even after its enzymatic deglycosylation, suggesting that the presence of N-linked carbohydrates in the mature protein is not essential for enzyme activity. Although the yield in S.cerevisiae was lower than that obtained in P.pastoris, this work demonstrates the importance of the choice of the heterologous host for successful production of soluble and active recombinant protein. In addition, S.cerevisiae constitutes a good engineering platform for improving the properties of this biocatalyst.