Organic Solvent-tolerant Enzymes Research Articles

Unusual long-term stability of enzymatic bioelectrocatalysis in organic solvents

Organic solvent tolerant enzymes are of tremendous importance for the pharmaceutical industry in search of high added-value chemicals. However, enzymes exhibit usually weak long-term activity and stability in various media. Here, we report a general strategy allowing stabilizing and keeping enzymes active in mixed-solvent systems upon immobilization on an electrode surface. It consists in the combination of an engineered solvent-tolerant enzyme, combined with fine-tuned osmium-based redox polymers where the concentration of osmium complex has been specifically tuned to minimize its deswelling, associated with the use of porous gold electrodes. This approach is validated with bilirubin oxidase as a model system. This copper enzyme is able to oxidize a wide range of substrates, combined with the reduction of O2 to water. While all other enzymatic systems irreversibly lose their activity and stability in the presence of 7.5 M methanol or below, this optimized enzymatic system stays functional in 12.5 M methanol with no loss in current density. It exhibits a half-life of more than 8 days, which is unprecedented in the literature. We show that this electrode can also operate in DMSO, dioxane, acetonitrile and acetone, thus opening up a very wide variety of applications in the field of bioelectrocatalysis and bioelectrosynthesis.

Different Effects of Salt Bridges near the Active Site of Cold-Adapted Proteus mirabilis Lipase on Thermal and Organic Solvent Stabilities

Organic solvent-tolerant (OST) enzymes have been discovered in psychrophiles. Cold-adapted OST enzymes exhibit increased conformational flexibility in polar organic solvents resulting from their intrinsically flexible structures. Proteus mirabilis lipase (PML), a cold-adapted OST lipase, was used to assess the contribution of salt bridges near the active site involving two arginine residues (R237 and R241) on the helix η1 and an aspartate residue (D248) on the connecting loop to the thermal and organic solvent stabilities of PML. Alanine substitutions for the ion pairs (R237A, R241A, D248A, and R237A/D248A) increased the conformational flexibility of PML mutants compared to that of the wild-type PML in an aqueous buffer. The PML mutants became more susceptible to denaturation after increasing the dimethyl sulfoxide or methanol concentration than after a temperature increase. Methanol was more detrimental to the structural stability of PML compared to dimethyl sulfoxide. These results suggest that direct interactions of dimethyl sulfoxide and methanol with the residues near the active site can have a destructive effect on the structure of PML compared with the global effect of heat on the protein structure. This study provides insight into the conformational changes within an OST enzyme with different effects on its thermal and organic solvent stabilities.

Haloferax volcanii as immobilised whole cell biocatalyst: new applications for halophilic systems

Enzyme-mediated synthesis of pharmaceutical compounds is a ‘green’ alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell–mediated biocatalysis by the halophilic archaeon H. volcanii.

Unravelling protein -organic solvent interaction of organic solvent tolerant elastase from Pseudomonas aeruginosa strain K crystal structure

The utilization of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages. However, an adaptation of enzyme towards organic solvent is unpredictable and not fully understood because of limited information on the organic solvent tolerant enzymes. To understand how the enzyme can adapt to the organic solvent environment, structural and computational approaches were employed. A recombinant elastase from Pseudomonas aeruginosa strain K was an organic solvent tolerant zinc metalloprotease was successfully crystallized and diffracted up to 1.39 Å. Crystal structure of elastase from strain K showed the typical, canonical alpha-beta hydrolase fold consisting of 10-helices (118 residues), 10- β-strands (38 residues) and 142 residues were formed other secondary structure such as loop and coil to whole structure. The elastase from Pseusomonas aeruginosa strain K possess His-140, His-144 and Glu-164 served as a ligand for zinc ion. The conserved catalytic triad was composed of Glu-141, Tyr-155 and His-223. Three-dimensional structure features such as calcium-binding and presence of disulphide-bridge contribute to the stabilizing the elastase structure. Molecular dynamic (MD) simulation of elastase revealed that, amino acid residues located at the surface area and disulphide bridge in Cys-30 to Cys-58 were responsible for enzyme stability in organic solvents.

Cloning, purification, and characterization of an organic solvent-tolerant chitinase, MtCh509, from Microbulbifer thermotolerans DAU221

BackgroundThe ability to use organic solvents in enzyme reactions offers a number of industrially useful advantages. However, most enzymes are almost completely inactive in the presence of organic solvents. Thus, organic solvent-tolerant enzymes have potential applications in industrial processes.ResultsA chitinase gene from Microbulbifer thermotolerans DAU221 (mtch509) was cloned and expressed in Escherichia coli BL21 (DE3). The molecular weight of the expressed MtCh509 protein was approximately 60 kDa, and it was purified by His-tag affinity chromatography. Enzymatic assays showed that the optimum temperature for MtCh509 chitinase activity was 55 °C, and the enzyme was stable for 2 h at up to 50 °C. The optimum pH for MtCh509 activity was in the sub-acidic range, at pH 4.6 and 5.0. The activity of MtCh509 was maintained in presence of 1 M salt, gradually decreasing at higher concentrations, with residual activity (20%) detected after incubation in 5 M salt. Some organic solvents (benzene, DMSO, hexane, isoamyl alcohol, isopropyl alcohol, and toluene; 10–20%, v/v) increased the reactivity of MtCh509 relative to the aqueous system. When using NAG3, as a substrate, MtCh509 produced NAG2 as the major product, as well as NAG4, demonstrating that MtCh509 has transglycosylation activity. The Km and Vmax values for MtCh509 using colloidal chitin as a substrate were 9.275 mg/mL and 20.4 U/mg, respectively. Thus, MtCh509 could be used in extreme industrial conditions.ConclusionThe results of the hydrolysate analysis and the observed increase in enzyme activity in the presence of organic solvents show that MtCh509 has industrially attractive advantages. This is the first report on an organic solvent-tolerant and transglycosylating chitinase from Microbulbifer species.

Characterization of esterase activity from an Acetomicrobium hydrogeniformans enzyme with high structural stability in extreme conditions.

The biotechnological and industrial uses of thermostable and organic solvent-tolerant enzymes are extensive and the investigation of such enzymes from microbiota present in oil reservoirs is a promising approach. Searching sequence databases for esterases from such microbiota, we have identified in silico a potentially secreted esterase from Acetomicrobium hydrogeniformans, named AhEst. The recombinant enzyme was produced in E. coli to be used in biochemical and biophysical characterization studies. AhEst presented hydrolytic activity on short-acyl-chain p-nitrophenyl ester substrates. AhEst activity was high and stable in temperatures up to 75°C. Interestingly, high salt concentration induced a significant increase of catalytic activity. AhEst still retained ~ 50% of its activity in 30% concentration of several organic solvents. Synchrotron radiation circular dichroism and fluorescence spectroscopies confirmed that AhEst displays high structural stability in extreme conditions of temperature, salinity, and organic solvents. The enzyme is a good emulsifier agent and is able to partially reverse the wettability of an oil-wet carbonate substrate, making it of potential interest for use in enhanced oil recovery. All the traits observed in AhEst make it an interesting candidate for many industrial applications, such as those in which a significant hydrolytic activity at high temperatures is required.

Organic solvent-tolerant, cold-adapted lipases PML and LipS exhibit increased conformational flexibility in polar organic solvents

Organic solvent-tolerant enzymes are generally stable in organic solvents, but the conformational changes of the enzymes in organic solvents have not been well studied. As cold-adapted enzymes exhibit increased conformational flexibility as compared with their mesophilic counterparts, we investigated the effects of polar organic solvents (dimethyl sulfoxide, methanol, and ethanol) on the flexibility and stability relationship in the organic solvent-tolerant, cold-adapted lipases PML and LipS. To our surprise, both PML and LipS exhibited increased conformational flexibility as organic solvent concentration increased. The conformational flexibility required for maximal enzymatic activity differed between PML and LipS, suggesting distinct conformations of PML and LipS in each organic solvent for maximal enzymatic activity. However, greater structural flexibility of PML and LipS in organic solvents was associated with lower thermal denaturation midpoints. Our study suggests that the effects of organic solvents on conformational flexibility are specific to cold-adapted enzymes displaying intrinsic conformational flexibility at low temperatures. Organic solvent-tolerant, cold-adapted enzymes could be used to investigate the flexibility and stability relationship in enzymes in many different organic solvents.

Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases

Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca2+-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65°C and retained ≥ 97% activity after incubation at 50°C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.

Influence of protein solution in nucleation and optimized formulation for the growth of ARM lipase crystal

ARM lipase is a thermostable and organic solvent tolerant enzyme which was highly purified prior to crystallization. The His-tagged ARM lipase was purified with immobilized metal affinity chromatography followed by anion-exchange chromatography. The effect of different salt concentrations on stability, solubility and crystal nucleation of the protein was studied. The highly purified and homogeneous ARM lipase with protein concentration of 2mg/mL was successfully crystallized by a sitting drop, vapor diffusion method with the use of 0.1M MES monohydrate pH 6.5 and 12% (v/v) polyethylene glycol (PEG) 20000 as precipitant. The crystallization conditions were optimized by changing the pH and concentration of the precipitant. The optimum crystallization condition was 2mg/mL ARM lipase in 0.1M Tris–HCl, 0.15M NaCl, pH 8.0 protein solution, crystallized using 0.1M Tris–HCl, pH 8.0 and 12% (v/v) PEG 20000 as precipitant.

Organic Solvent Tolerant Lipases and Applications

Lipases are a group of enzymes naturally endowed with the property of performing reactions in aqueous as well as organic solvents. The esterification reactions using lipase(s) could be performed in water-restricted organic media as organic solvent(s) not only improve(s) the solubility of substrate and reactant in reaction mixture but also permit(s) the reaction in the reverse direction, and often it is easy to recover the product in organic phase in two-phase equilibrium systems. The use of organic solvent tolerant lipase in organic media has exhibited many advantages: increased activity and stability, regiospecificity and stereoselectivity, higher solubility of substrate, ease of products recovery, and ability to shift the reaction equilibrium toward synthetic direction. Therefore the search for organic solvent tolerant enzymes has been an extensive area of research. A variety of fatty acid esters are now being produced commercially using immobilized lipase in nonaqueous solvents. This review describes the organic tolerance and industrial application of lipases. The main emphasis is to study the nature of organic solvent tolerant lipases. Also, the potential industrial applications that make lipases the biocatalysts of choice for the present and future have been presented.

Screening and isolation of an organic solvent tolerant-protease from Bacillus sp. JER02: Activity optimization by response surface methodology

Abstract Organic solvent-tolerant enzymes are becoming increasingly popular in synthetic biotechnology, due to their industrially attractive advantages over the aqueous solvents. Screening of organic solvent-tolerant microorganisms is a promising strategy for identification of novel enzymes that are naturally tolerant toward organic solvents. Crude proteases which have been shown to be active at high temperatures, alkaline pH and broad range of salt concentrations, are favorite proteases for industrial applications. In this study, Bacillus sp. JER02 was isolated from Gehver hot spring in Jiroft, Iran, and grown in medium supplemented with cyclohexane (30%), toluene (10%). Protease activity in the presence of different concentrations of organic solvents showed that, this enzyme was activated about 10% and 30% in the presence of 5% isopropanol and 10% DMF, respectively. In addition, this enzyme retained more than 90% of its initial activity after 1 h pre-incubation with 40% organic solvent at room temperature. Moreover, response surface methodology was applied to optimize the assay constituents of Bacillus sp. JER02 protease. The best condition for protease activity was achieved in buffer containing 8 mM NaCl, 4 mM MgSO4, at 54 °C, and pH8. The statistical optimization by RSM resulted in 2.5 fold increase in protease activity by Bacillus sp. JER02.

Solvent-Dependent Gating Motions of an Extremophilic Lipase from Pseudomonas aeruginosa

Understanding how organic solvent-stable proteins can function in anhydrous and often complex solutions is essential for the study of the interaction of protein and molecular immiscible interfaces and the design of efficient industrial enzymes in nonaqueous solvents. Using an extremophilic lipase from Pseudomonas aeruginosa as an example, we investigated the conformational dynamics of an organic solvent-tolerant enzyme in complex solvent milieux. Four 100-ns molecular dynamics simulations of the lipase were performed in solvent systems: water, hexane, and two mixtures of hexane and water, 5% and 95% (w/w) hexane. Our results show a solvent-dependent structural change of the protein, especially in the region that regulates the admission of the substrate. We observed that the lipase is much less flexible in hexane than in aqueous solution or at the immiscible interface. Quantified by the size of the accessible channel, the lipase in water has a closed-gate conformation and no access to the active site, while in the hexane-containing systems, the lipase is at various degrees of open-gate state, with the immiscible interface setup being in the widely open conformation ensembles. The composition of explicit solvents in the access channel showed a significant influence on the conformational dynamics of the protein. Interestingly, the slowest step (bottleneck) of the hexane-induced conformational switch seems to be correlated with the slow dehydration dynamics of the channel.

Purification and characterization of trans-3-(4-methoxyphenyl) glycidic acid methyl ester hydrolyzing lipase from Pseudomonas aeruginosa

Enantiospecific lipase was purified from Pseudomonas aeruginosa MTCC 5113 and it was used for the hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate, a key intermediate in the synthesis of cardiovascular drug, diltiazem. Enzyme from broth supernatant was precipitated with acetone and purified by anion exchange and gel filtration chromatography. The purified lipase was a homogenous protein having a molecular weight of 59.4 kDa as determined by SDS-PAGE. Isoelectric point was found to be approximately 5.5 after 2D electrophoresis. This organic solvent tolerant enzyme was found to be active in presence of EDTA, Tween-80 and β-mercaptoethanol whereas sodium dodecyl sulphate and dithiothreitol inhibited its activity. The K m and V max of the enzyme were 50 mM and 27.1 μmol/min mg, respectively using p-nitrophenyl palmitate as a substrate. The activity of lipase was confirmed by (±)-MPGM hydrolysis and zymography.