Improve Enzyme Stability Research Articles

Liposome-supported enzymatic peritoneal dialysis.

Compared to hemodialysis, peritoneal dialysis represents a more straightforward and less invasive alternative, though current solutions are not as effective. Herein, the feasibility of liposome-supported enzymatic peritoneal dialysis (LSEPD) is explored to increase the functionality of peritoneal dialysis for the model indication acute alcohol poisoning. Enzyme-loaded liposomes (E-Liposomes) containing alcohol metabolizing enzymes, alcohol oxidase and catalase, are developed and their invitro and invivo performances investigated. The E-Liposomes outperform the free enzymes in stability, overcoming the thermal instability of alcohol oxidase and enhancing the invitro ethanol elimination, which is further accelerated by hydrogen peroxide, due to the rapid generation of oxygen by catalase. Compared to the free enzymes, the E-Liposomes exhibit reduced systemic exposure and organ distribution. In a rodent ethanol intoxication model, LSEPD enhances ethanol metabolism as evidenced by an increased acetaldehyde production, ethanol's primary metabolite. In conclusion, LSEPD presents an innovative platform to temporarily enhance xenobiotic metabolism, in view of the improved enzyme stability and peritoneal retention.

Surfactants Facilitating Carbonic Anhydrase Enzyme-Mediated CO2 Absorption into a Carbonate Solution.

Carbonic anhydrase (CA) enzyme-mediated absorption processes are regarded as promising alternatives to the conventional amine-based process for CO2 capture because of their low energy penalty and low risk of causing secondary pollution. The activity and stability of the CA enzyme are crucial to reducing the equipment and operating costs of the enzyme-mediated process. This work investigated three cationic and nonionic surfactants to improve the activity and stability of a technical-grade CA enzyme in a 20 wt % potassium carbonate solution. Experimental results revealed that the impact of the surfactants on the CA enzyme depended on their properties. For example, the cationic surfactant significantly enhanced the activity of CA enzyme but adversely affected enzyme stability. However, in the presence of the cationic surfactant after 30 days at 50 °C, the activity of the CA enzyme still outperformed that of CA without added surfactant. The nonionic surfactant significantly improved enzyme stability. Furthermore, the addition of surfactants within a critical micelle concentration of 1.0 did not distinctly influence the gas-liquid mass transfer, indicating that surfactant-enzyme interaction was responsible for the observed variations in the activity and stability of the tested enzyme.

Immobilization of Trametes versicolor laccase on different PGMA‐based polymeric microspheres using response surface methodology: Optimization of conditions

ABSTRACTEnzymatic immobilization is a versatile alternative to improve enzyme stability and enable its reuse. In this study, the change in the immobilized enzyme properties due to the type of functional group on the carrier was evaluated. For that, monodisperse polymeric microspheres with two functional groups widely used in laccase immobilization—oxirane [poly(glycidyl methacrylate) (PGMA)] and hydrazide—were synthesized by dispersion polymerization to covalently immobilize the laccase Trametes versicolor. Using a response surface methodology, laccase immobilization was optimized for each microsphere type. As a result, laccase immobilization on PGMA carriers appears to broaden the pH and temperature ranges, storage stability, and reusability compared to free and hydrazide enzymes. These aforementioned characteristics indicate that PGMA microspheres could act as an ideal support for enzyme immobilization in biotechnological applications. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45249.

Effect of immobilization rate and enzyme crowding on enzyme stability under different conditions. The case of lipase from Thermomyces lanuginosus immobilized on octyl agarose beads

Abstract Lipase from Thermomyces lanuginosus (TLL) was immobilized on octyl agarose (OC). Three different TLL-OC biocatalysts were prepared: one lowly loaded using a low enzyme concentration, one fully loaded using a low enzyme concentrations, and a final one using a large excess of enzyme at a higher concentration. The activities after immobilization increased (180%), although diffusion limitations reduced the hyperactivation of the fully loaded preparations (140%). The stabilities of both preparations using low enzyme concentrations were similar under all studied conditions discounting the diffusional limitations of the biocatalyst. However, the biocatalyst prepared using a large concentration of enzyme was less stable that the other preparations at pH 7.0, more stable at pH 5.0 and with a similar stability at pH 9.0. Adding 3 M NaCl, the stability of the fully loaded preparations significantly increased; while the lowly loaded preparation slightly improved enzyme stability. This produced that the biocatalyst prepared under using high enzyme concentration become significantly more stable than the other two TLL preparations. Glycerin increased immobilized TLL stability, in this case all OC-TLL preparations became with similar stabilities. Results show that the TLL concentration during immobilization may greatly affect TLL properties, perhaps due to altering enzyme packing.

Novel Magnetic Cross-Linked Cellulase Aggregates with a Potential Application in Lignocellulosic Biomass Bioconversion.

The utilization of renewable biomass resources to produce high-value chemicals by enzymatic processes is beneficial for alternative energy production, due to the accelerating depletion of fossil fuels. As immobilization techniques can improve enzyme stability and reusability, a novel magnetic cross-linked cellulase aggregate has been developed and applied for biomass bioconversion. The cross-linked aggregates could purify and immobilize enzymes in a single operation, and could then be combined with magnetic nanoparticles (MNPs), which provides easy separation of the materials. The immobilized cellulase showed a better activity at a wider temperature range and pH values than that of the free cellulase. After six cycles of consecutive reuse, the immobilized cellulase performed successful magnetic separation and retained 74% of its initial activity when carboxylmethyl cellulose (CMC) was used as the model substrate. Furthermore, the structure and morphology of the immobilized cellulase were studied by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Moreover, the immobilized cellulase was shown to hydrolyze bamboo biomass with a yield of 21%, and was re-used in biomass conversion up to four cycles with 38% activity retention, which indicated that the immobilized enzyme has good potential for biomass applications.

Optimization of the coating of octyl-CALB with ionic polymers to improve stability and decrease enzyme leakage

Lipase B from Candida antarctica (CALB) immobilized on octyl-agarose (OC) was submitted to coating with polyethylenimine (PEI) and dextran sulfate (DS). Using lowly loaded enzyme preparations, the properties of OC-CALB preparations hardly improved, suggesting too large the distance between enzyme molecules. However, using OC-CALB preparations with maximum loading, CALB stability was greatly improved in different conditions after PEI coating. Moreover, the CALB release from the OC support in the presence of detergents, or during thermal or organic solvent inactivations was greatly reduced after this treatment (PEI plus DS coating). The results pointed that the main positive effect of this coating could be derived from the physical intermolecular crosslinking of the CALB molecules with the polymers that reduce the enzyme desorption from the support. The coating of OC-CALB-PEI with DS only produced a minimal improvement on enzyme performance. Even though the enzyme release was much more difficult after physical crosslinking, all enzyme molecules could be released from the OC support combining an ionic detergent (SDS), high buffer concentration, pH 3 and 45 °C, while using the OC-CALB just 2% SDS at pH 7 and 25 °C was enough to release all enzyme. The support could be reused several cycles. Thus, this strategy permitted to greatly reduce the enzyme desorption during operation and to improve enzyme stability while keeping the enzyme immobilization reversibility.

Improved enzyme properties upon glutaraldehyde cross-linking of alginate entrapped xylanase from Bacillus licheniformis

Enzyme immobilization is an exciting alternative to improve the stability of enzymatic processes and economic viability in terms of reusability. In the current study, purified xylanase from B. licheniformis Alk-1 was immobilized within glutaraldehyde activated calcium alginate beads and characterized in respect of free enzyme. Immobilization increases the optimum pH and temperature of entrapped and cross-linked enzyme from pH=8.0 to 9.0 and 50–60°C. The kinetics parameter of immobilized (cross-linked) enzyme showed an increase in Km (from 4.36mg/mL to 5.38mg/mL) and decrease in Vmax (from 383 IU/mg/min to 370 IU/mg/min). Immobilization increases the optimum reaction time for xylan degradation of immobilized xylanase from 15 to 30min when compare to free form. The storage stability study suggested that the immobilized enzyme retains 80% of its original activity at 4°C after 30days compared to free enzyme (5%). Further, immobilization improved enzyme stability in presence of different additives. The immobilized (cross-linked) enzyme also exhibited adequate recycling efficiency up to five reaction cycles with 37% retention activity. The finding of this study suggests improvement of overall performance of immobilized xylanase in respect to free form and can be used to make a bioreactor for various applications such as poultry feed preparations.

Controlled Aggregation and Increased Stability of β-Glucuronidase by Cellulose Binding Domain Fusion.

Cellulose-binding domains (CBDs) are protein domains with cellulose-binding activity, and some act as leaders in the localization of cellulosomal scaffoldin proteins to the hydrophobic surface of crystalline cellulose. In this study, we found that a CBD fusion enhanced and improved soluble β-glucuronidase (GusA) enzyme properties through the formation of an artificially oligomeric state. First, a soluble CBD fused to the C-terminus of GusA (GusA-CBD) was obtained and characterized. Interestingly, the soluble GusA-CBD showed maximum activity at higher temperatures (65°C) and more acidic pH values (pH 6.0) than free GusA did (60°C and pH 7.5). Moreover, the GusA-CBD enzyme showed higher thermal and pH stabilities than the free GusA enzyme did. Additionally, GusA-CBD showed higher enzymatic activity in the presence of methanol than free GusA did. Evaluation of the protease accessibility of both enzymes revealed that GusA-CBD retained 100% of its activity after 1 h incubation in 0.5 mg/ml protease K, while free GusA completely lost its activity. Simple fusion of CBD as a single domain may be useful for tunable enzyme states to improve enzyme stability in industrial applications.

Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation. Stronger Adsorption of Polymers/Unfolded Protein Composites.

Lipases from Candida antarctica (isoform B) and Rhizomucor miehei (CALB and RML) have been immobilized on octyl-agarose (OC) and further coated with polyethylenimine (PEI) and dextran sulfate (DS). The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed). The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 °C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme.

Reversible sol-gel-sol medium for enzymatic optical biosensors.

In this paper we for the first time report a reversible sol-gel-sol approach to obtain optical enzymatic biosensors with improved enzyme stability and good sensitivity by using desktop inkjet printing. The developed technique is based on the bio-inorganic inks allowing for a sol-gel-sol transition of the inorganic matrix: from liquid ink to a solid alumina matrix with entrapped enzymes and a subsequent color response due to the enzymatic reaction upon the resuspension of the matrix. This approach improves the stability of the enzymes entrapped in the porous inorganic matrix, and at the same time maintains a high sensitivity of the biomolecules, whose facile release is ensured by the gel-sol transition. Rheological parameters of the developed bio-inorganic ink are highly adjustable making it suitable for the deposition on different surfaces by inkjet printing. The potential and utility of this approach is demonstrated by a successful production of optical biosensors for glucose and uric acid.

Physical crosslinking of lipase from Rhizomucor miehei immobilized on octyl agarose via coating with ionic polymers

Abstract Lipase from Rhizomucor miehei (RML) was immobilized on octyl-agarose (OC) at different loadings. Using low enzyme loadings (1/7 of the maximum loading), the incubation of the enzyme with polyethylenimine (PEI) increased the resistance to enzyme desorption in the presence of Triton X-100. However, more than 10% of the enzyme activity could be released from the OC-RML-PEI. The same treatment using fully loaded biocatalyst reduced the enzyme desorption to less than 5%. Further treatment with dextran sulfate (DS) of the PEI treaded immobilized enzyme fully avoids the enzyme desorption even in presence of a Triton X-100 concentration higher than that required for the complete enzyme release from OC-RML. This treatment produced a high stabilization of OC-RML in thermal or organic solvent inactivations, reducing the enzyme release under these drastic conditions. Nevertheless, the support could be recovered by incubation under adequate conditions, and reused in several adsorption/desorption cycles. Thus, the strategy permitted to avoid enzyme desorption, very likely by physical intermolecular crosslinking improving enzyme stability, while still maintaining the reversibility of the immobilization.

ChemInform Abstract: Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities

AbstractReview: [82 refs.

Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities.

Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed.

Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain.

Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. In this study, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine with glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined, including TEM-1, CTX-M-14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutant were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to that of the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48, and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. Additionally, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intrahelical hydrogen bond network compared to those of the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. These findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the requirements for optimal stability.

Specific Enzymatic Halogenation-From the Discovery of Halogenated Enzymes to Their Applications In Vitro and In Vivo.

During the last 20 years, focus has shifted from haloperoxidases to flavin-dependent and non-heme-iron halogenases because of their proven involvement in the biosynthesis of halogenated metabolites in different organisms and the regioselectivity of their reactions. During the first 10-12 years, the main research topics were the detection of halogenases as well as the elucidation of three-dimensional structures and reaction mechanisms. This Review mainly deals with studies on halogenating enzymes published between 2010 and 2015. It focusses on the elucidation of the involvement of halogenating enzymes in halometabolite biosynthesis, application of halogenases in in vivo and in vitro systems, in vivo modification of biosynthetic pathways in bacteria and plants, improvement of enzyme stability, broadening of substrate specificity, and the combination of biocatalysis with chemical synthesis to produce new compounds.

Intense PEGylation of Enzyme Surfaces: Relevant Stabilizing Effects.

This chapter describes the physicochemical coating of the surface of immobilized enzymes with a dense layer of polyethylene glycol (PEG) to improve enzyme stability. One hypothesis is that a dense, viscous, polar PEG layer around the enzyme would enhance enzyme thermal stability, while still providing access to the active site. PEG groups were attached by using aldehyde-dextran polymers, the dextran polymers are in turn attached to the enzyme surface that have been enriched with excess primary amino groups. The enzymes themselves were initially attached onto porous solids such that they may be separated easily from the reaction mixtures for easy downstream processing and that they may be recycled to reduce the cost of the biocatalyst. The hierarchical modification of enzyme surface with three different sublayers, under chemical design, provided a rational control at several structural levels. Few methods for increasing the number of amino groups on the surface of the enzyme are described: (a) chemical amination of carboxyl residues and (b) coating of the enzyme surface with cationic polymers containing a high percentage of primary amines. Reliable protocols for the PEGylation of four different enzymes are described here. For example, lipases from Thermomyces lanuginosa, Candida antarctica B, and Rhizomucor miehei attached to octyl sepharose and chemically modified via PEGylation are stabilized from 7- to 50-fold when compared to the stability of the corresponding unmodified enzyme. A derivative of endoxylanase from Trichoderma reesei, immobilized by multipoint covalent attachment on glyoxyl agarose, is stabilized by 50-fold. Very likely, the PEG layer generated a dense, high viscosity medium surrounding the enzyme surface and this increase in viscosity around the enzyme microenvironment resists distortion of enzyme structure by heat or other denaturing agents.

The Preparation of Bioimprinted Whole-cell Biocatalyst and Its Application in Bioconversion of Biodiesel

Biodiesel has attracted considerable attention as an environmentally friendly alternative fuel. Lipase is the most popular enzyme for biodiesel production and immobilization has been deployed to improve enzyme stability and reusability. Exploitation of high activity lipase is the key point for biodiesel production. Whole-cell biocatalysts have been applied in the biosynthesis of biodiesel and bioimprinting is a promising approach for enzyme performances improvement. In this study, based on the <em>S. cerevisiae</em> cell-surface display system with $\alpha$-agglutinin as anchor, a whole-cell biocatalyst of codon-optimized <em>Rhizopus oryzae</em> lipase was constructed and bioimprinted with oleic acid, gaining 5-fold increase on enzymatic activity in the alcoholysis of soybean oil to biodiesel. Moreover, the conversion of FAME was up to 95.45±2.73% after a 27-h reaction at 60°C. Our results indicated that combining bioimprinting with yeast display technique to prepare whole-cell biocatalyst could result in potential enzymes for bioconversion of biodiesel in organic solvents.

Reduced graphene oxide enhances horseradish peroxidase stability by serving as radical scavenger and redox mediator

Graphene-based nanomaterials have been widely studied as high-performance matrices for enzyme immobilization and in the development of biosensors. Surface O-functionalities of graphene induce changes in chemical reactivity and electronic conductivity of nanomaterials and may interfere with enzymatic processes; however, the mechanisms are not fully understood. We compare the effects of three commercially available graphene-based nanomaterials, namely a graphene, a graphene oxide (GO), and a reduced graphene oxide (RGO), on the activity/stability of horseradish peroxidase. Both graphene and GO significantly reduced enzyme stability by altering enzyme conformation, which was evidenced by circular dichroism spectroscopy. However, RGO improved enzyme stability up to 7-fold. This increased stability was attributed to the capability of RGO to quench superoxide radicals, which were primarily responsible for the enzyme deactivation. The basal plane of RGO, mainly through quinone moieties, may act as a redox mediator to facilitate enzymatic turnover. Overall, the radical scavenging plus redox mediating capabilities of RGO suggest the potential for graphene-based nanomaterials to enhance enzyme engineering and enzyme-based sensors.

Evaluation of divinylsulfone activated agarose to immobilize lipases and to tune their catalytic properties

Divinylsulfone (DVS) activated agarose beads have been used to immobilize several lipases, those from Pseudomonas flourescens, Rhizomucor miehei, and Thermomyces lanuginosus (TLL), as well as the artificial chimeric phospholipase Lecitase Ultra. The best results in terms of activity recovery and immobilization yield were achieved using TLL. This enzyme could be immobilized at pH from 5 (with poor yield) to pH 10 (with 100% yield). The incubation of the immobilized enzymes for 72h at pH 10 before the blocking step (using ethylenediamine) improved the enzyme stability whatever the immobilization pH value, but the stabilization achieved in each case depended on the immobilization pH value, and also on the inactivation conditions.The enzyme activities versus different substrates were very dependent on the immobilization protocol and the conditions of activity determination. That way, TLL immobilized at pH 5 on DVS activated support was the most active versus methyl mandelate, even more active than TLL immobilized on octyl-agarose (between 3 and 7 fold factors depending on the pH of measure). Using ethyl hexanoate the most active preparation was the octyl-TLL preparation. The most active among the enzymes immobilized in DVS-activated supports was that just immobilized at pH 5 if the activity was determined at pH 7 or 8.5, while at pH 5 the most active was that enzyme but after incubation at pH 10.The results show that this support may be very useful for tuning lipase properties just by altering the first immobilization pH value, and that the further incubation at pH 10 improved enzyme stability, and in some cases, even increased activity.

Immobilization of thermostable nucleoside phosphorylases on MagReSyn® epoxide microspheres and their application for the synthesis of 2,6-dihalogenated purine nucleosides

Immobilization of enzymes has been considered as an efficient approach to facilitate enzyme recovery and to improve biocatalyst stability. However, multimeric nucleoside phosphorylases, useful for the synthesis of modified nucleosides, encounter several challenges to their immobilization, including requirement for high enzymatic load, and poor retention of enzyme activity. In this study, multimeric enzymes pyrimidine nucleoside phosphorylase (PyNP) and purine nucleoside phosphorylase (PNP), from Thermus thermophilus and Geobacillus thermoglucosidasius, respectively, were successfully immobilized on the magnetic beads with cross-linked polyethyleneimine and epoxide functional groups, resulting in high enzyme loading (up to 0.4 and 1.3g per g dry beads), high enzyme activity maintenance (41% and 83% under the highest enzyme loading), and improved enzyme stability. The screening of the immobilization conditions showed that binding buffer pH, enzyme loading amount, binding temperature and binding time are important factors for the immobilization yield. The application of the immobilized enzymes in the synthesis of 2,6-dihalogenated purine nucleosides achieved with high substrate conversion (78.5–85.5%) as well as high productivity (1.5–2.0gL−1h−1).