Water-in-ionic liquid microemulsion-based organogels as novel matrices for enzyme immobilization

The use of water-in-ionic liquid microemulsion-based organogels (w/IL MBGs) as novel supports for the immobilization of lipase B from Candida antarctica and lipase from Chromobacterium viscosum was investigated. These novel lipase-containing w/IL MBGs can be effectively used as solid phase biocatalysts in various polar and non-polar organic solvents or ILs, exhibiting up to 4.4-fold higher esterification activity compared to water-in-oil microemulsion-based organogels. The immobilized lipases retain their activity for several hours at 70°C, while their half life time is up to 25-fold higher compared to that observed in w/IL microemulsions. Fourier-transform infrared spectroscopy data indicate that immobilized lipases adopt a more rigid structure, referring to the structure in aqueous solution, which is in correlation with their enhanced catalytic behavior observed.     

An automated method for measuring the operational stability of biocatalysts with carbonic anhydrase activity

The operational stability of an enzyme can be quantified by its half-life, or the length of time after which 50% of its original activity has degraded. Ideally, continuous methods for measuring half-lives are preferred but they can be expensive and relatively low throughput. Batch methods, while simple, cannot be used for all enzymes. For example, batch reactions can be difficult when there is a gas phase reactant or when there is significant product or substrate inhibition. Here we describe a repeated-batch method for measuring the half-life of carbonic anhydrase (CA)-based biocatalysts by automated periodic switching between a forward and reverse reaction. This method is inexpensive and can be multiplexed for high-throughput analysis of enzyme variants. Several purified CA enzymes as well as whole-cell biocatalysts with engineered CA activity were evaluated with this method. The results indicate a significant increase in operational stability is achieved upon immobilization of CA in the cellular periplasm of Escherichia coli.     

One-step enzyme extraction and immobilization for biocatalysis applications

An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.     

Sucrose phosphorylase as cross-linked enzyme aggregate: Improved thermal stability for industrial applications

Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60°C to avoid microbial contamination. Cross-linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17°C higher than that of the soluble enzyme. Furthermore, the immobilized enzyme displays an exceptional thermostability, retaining all of its activity after 1 week incubation at 60°C. Recycling of the biocatalyst allows its use in at least ten consecutive reactions, which should dramatically increase the commercial potential of its glycosylating activity.     

Bacterial cellulose as carrier for immobilization of laccase: Optimization and characterization

Bacterial cellulose (BC) has attracted attention as a new functional material due to its excellent mechanical strength, tridimensional nanostructure, high purity, and increased water absorption, compared to plant cellulose. In this work, commercial laccase was immobilized on BC and the influence of enzyme concentration, contact time, and pH was optimized toward the recovery activity of immobilized laccase. This optimization was carried out using a 3³ experimental design and response surface methodology. Enzyme concentration played a critical role in laccase immobilization. Under optimized conditions (0.15 μL L^?1 of enzyme concentration, 4.8 h of contact time, pH 5.4), the predicted and experimental response were equal to 47.88 and 49.30%, respectively. The thermal stability of the immobilized laccase was found to increase notably at 60 and 70°C presenting stabilization factor equal to 1.79 and 2.11, respectively. The immobilized laccase showed high operational stability, since it retained 86% of its initial activity after seven consecutive biocatalytic cycles of reaction with 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid). Kinetic studies showed that the values of Michaelis–Menten constant and maximum reaction rate decreased upon immobilization (9.9‐ and 1.6‐fold, respectively). Globally, the use of immobilized laccase on BC offers an interesting tool for industrial biocatalytic applications.     

Development of a novel immobilization method for enzymes from hyperthermophiles

Peptide tags containing tyrosines (Y-tag) were introduced at the C-terminus of a hyperthermophilic enzyme, alkaline phosphatase from Pyrococcus furiosus (PfuAP). Immobilization of the recombinant PfuAPs onto water-in-oil-in-water (W/O/W) type microcapsules was performed by an in situ polymerization method. All the recombinant PfuAPs prepared in this study were quantitatively immobilized onto microcapsules. The PfuAP-immobilized microcapsules showed no significant loss of enzymatic activity until the 5th round of assays. This result implies that the recombinant PfuAPs were covalently immobilized onto microcapsules. Immobilized PfuAP tagged with a Y-tag having the sequence GGYYY exhibited approximately a twofold higher catalytic activity compared with the wild-type PfuAP. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A fluorescence spectroscopy assay for real-time monitoring of enzyme immobilization into mesoporous silica particles

Mesoporous silica particles are used as support material for immobilization of enzymes. Here we investigated a fluorescence-based assay for real-time monitoring of the immobilization of lipase, bovine serum albumin, and glucose oxidase into micrometer-sized mesoporous silica particles. The proteins are labeled with the dye epicocconone, and the interaction with the particles is observed as an increase in emission intensity of the protein–dye conjugates that can be quantified if correcting for a comparatively slow photobleaching. The immobilization occurs in tens of minutes to hours depending on particle concentration and type of protein. In the limit of excess particles over proteins, the formation of the particle–protein complexes can be described by a single exponential growth for all three investigated proteins, and the fitted pseudo-first-order rate constant increases linearly with particle concentration for each protein type. The derived second-order rate constant k varies with the protein hydrodynamic radius according to k ∼ R_H^{−4.70±0.01}, indicating that the rate-limiting step at high particle concentrations is not the diffusional encounter between proteins and particles but rather the entry into the pores, consistent with the hydrodynamic radii of the three proteins being smaller but comparable to the pore radius of the particles.     

Broadening the synthetic potential of disaccharide phosphorylases through enzyme engineering

In recent years, disaccharide phosphorylases have attracted increasing attention as promising biocatalysts for the production of glycosylated compounds. These enzymes make use of a glycosyl phosphate as donor substrate, which is much cheaper than the nucleotide-activated donors required by glycosyl transferases. Unfortunately, the number of available donor specificities is rather limited, and typically only allow the transfer of either a glucosyl or a galactosyl residue. In addition, most phosphorylases have a strong preference for carbohydrate acceptors, and can thus only be used for the synthesis of saccharide chains. The engineering of their substrate specificity thus is of significant value to broaden the range of products that can be obtained. Furthermore, the stability of some phosphorylases will also need to be improved to allow their commercial exploitation in a variety of industrial processes. In this review, several strategies for the engineering of these parameters are discussed and illustrated with some recent successes.     

Novel auto-inducing expression systems for the development of whole-cell biocatalysts

Novel expression systems for the development of whole-cell biocatalysts were generated. Their novelty consists both in the host, Pseudomonas putida, and in the ability to auto-induce the expression of genes of interest at the exhaustion of the carbon source used for the biomass growth. The auto-induction relies on new expression vectors developed in this study and based on the activator TouR from Pseudomonas sp. OX1, which was shown to mediate the activation of target promoters in an effector-independent growth-phase-dependent manner when the carbon source is exhausted at the onset of the stationary phase. We validated the suitability of these expression systems through the production of (S)-styrene oxide by the styrene monooxygenase from Pseudomonas fluorescens ST. The yields of epoxides produced by these biocatalysts in flask experiments showed to be as efficient as those currently available based on inducible Escherichia coli systems. In addition, a larger scale of biomass production showed no reduction of biocatalysis efficiency. Therefore, the systems developed in this study constitute a valid alternative to current expression systems to use in bioconversion processes.     

Glucose oxidase enzyme immobilized porous silica for improved performance of a glucose biosensor

High activity of glucose oxidase (GOD) enzyme (immobilized in porous silica particles) is desirable for a better glucose biosensor. In this work, effect of pore diameter of two porous hosts on enzyme immobilization, activity and glucose sensing was compared. The hosts were amine functionalized: (i) microporous silica (NH₂-MS) and (ii) mesoporous silica (NH₂-SBA-15). Based on whether the dimension of GOD is either larger or smaller than the pore diameter, GOD was immobilized on either external or internal surface of NH₂-MS and NH₂-SBA-15, with loadings of 512.5 and 634 mg/g, respectively. However, GOD in NH₂-SBA-15 gave a higher normalized absolute activity (NAA), which led to an amperometric sensor with a larger linear range of 0.4–13.0 mM glucose. In comparison, GOD in NH₂-MS had a lower NAA and a smaller linear range of 0.4–3.1 mM. In fact, the present GOD-NH₂-SBA-15 electrode based sensor was better than other MS and SBA-15 based electrodes reported in literature. Thus, achieving only a high GOD loading (as in NH₂-MS) does not necessarily give a good sensor performance. Instead, a host with a relatively larger pore than enzyme, together with optimized electrode composition ensures the sensor to be functional in both hyper- and hypoglycemic range.     

Lipases: Useful biocatalysts for the preparation of pharmaceuticals

Biocatalysis offers a clean and ecological way to perform chemical processes, in mild reaction conditions and with high degree of selectivity. The use of enzymes, specially lipases, in organic solvents proves an excellent methodology for the preparation of single-isomer chiral drugs. This review covers some general aspects and representative examples of the use of lipases for the enantioselective or regioselective preparation of alcohol and amine intermediates in the synthesis of pharmaceuticals.     

Enantioselective synthesis of amines via reductive amination with a dehydrogenase mutant from Exigobacterium sibiricum: Substrate scope,co-solvent tolerance and biocatalyst immobilization

In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance. Kinetics as well as inhibition effects were also studied and the suitability of this method for synthetic purpose was demonstrated with acetophenone as a model substrate. Even at an elevated substrate concentration of 50?mM, excellent conversion was achieved. In addition, the impact of water-miscible co-solvents was examined, and good activities were found when using DMSO of up to 30% (v/v). Furthermore, a successful immobilization of the EsLeuDH-DM was demonstrated utilizing a hydrophobic support and a support for covalent binding, respectively, as a carrier.     

Encapsulation of glucose oxidase microparticles within a nanoscale layer-by-layer film: immobilization and biosensor applications

We report on an immobilization strategy utilizing layer-by-layer encapsulated microparticles of enzymes within a nanoscale polyelectrolyte film. Encapsulation of glucose oxidase (GOD) microparticles was achieved by the sequential adsorption of oppositely charged polyelectrolytes onto the GOD biocrystal surface. The polyelectrolyte system polyallylamine/polystyrene sulfonate was used under high salt conditions to preserve the solid state of the highly water soluble GOD biocrystals during the encapsulation process. The resulting polymer multilayer capsule of about 15 nm wall thickness is permeable for small molecules (glucose), but non-permeable for macromolecules thus preventing the enzyme from leakage and at the same time shielding it from the outer environment e.g., from protease or microbial activity. Decrease of the buffer salt concentration leads to the dissolution of the enzyme under formation of μ-bioreactors. The spherical μ-bioreactors are bearing an extremely high loading of biocompound per volume. Encapsulated GOD was subsequently used to construct a biosensor by nanoengineered immobilisation of μ-bioreactor capsules onto an electrode surface. The presented approach demonstrates a general method to encapsulate highly soluble solid biomaterials and an immobilization strategy with the potential to create highly active thin and stable films of biomaterial.     

Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges

Cross-linked enzyme aggregate (CLEA) technology has been regarded as an effective carrier-free immobilization method. This method is very attractive due to its simplicity and robustness, as well as for the possibility of using the crude enzyme extract and the opportunity to co-immobilize multiple different enzymes. The resulting CLEAs generally exhibit high catalyst productivities, improved storage and operational stability and are easy to recycle. Nowadays, although the technology has been applied to various enzymes, some undesirable properties have limited its further application. To overcome these limitations, novel strategies have been developing in recent years. This mini-review focuses on process optimization, new improved strategies and the latest advances on CLEAs technology.     

Formulation of organic and inorganic hydrogel matrices for immobilization of β‐glucosidase in microfluidic platform

The aim of this study was to formulate silica and alginate hydrogels for immobilization of β‐glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate–polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized β‐glucosidase was loaded into glass–silicon–glass microreactors and catalysis of 4‐nitrophenyl β‐d ‐glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.     

Cross-linked enzyme aggregates (CLEAs): A novel and versatile method for enzyme immobilization (a review)

The key to obtaining optimum performance of an enzyme is often a question of devising an effective method for its immobilization. This review describes a novel, versatile and effective methodology for enzyme immobilization, namely, as cross-linked enzyme aggregates (CLEAs). The method is exquisitely simple - involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules - and amenable to rapid optimization. It is applicable to a wide variety of enzymes, including cofactor-dependent oxidoreductases and lyases, and affords stable, recyclable catalysts with high retention of activity, sometimes higher than that of the free enzyme it was derived from. The enzyme does not need to be of high purity. Indeed, the methodology is essentially a combination of purification and immobilization in one step. The technique is also applicable to the preparation of combi-CLEAs, containing two or more enzymes, for use in one-pot, multi-step syntheses. For example, an oxynitrilase/nitrilase combi-CLEA was used for the one-pot synthesis of (S)-mandelic acid from benzaldehyde, in high yield and enantiomeric purity.     

The state-of-the-art strategies of protein engineering for enzyme stabilization

Enzymes generated by natural recruitment and protein engineering have greatly contribute in various sets of applications. However, their insufficient stability is a bottleneck that limit the rapid development of biocatalysis. Novel approaches based on precise and global structural dissection, advanced gene manipulation, and combination with the multidisciplinary techniques open a new horizon to generate stable enzymes efficiently. Here, we comprehensively introduced emerging advances of protein engineering strategies for enzyme stabilization. Then, we highlighted practical cases to show importance of enzyme stabilization in pharmaceutical and industrial applications. Combining computational enzyme design with molecular evolution will hold considerable promise in this field.     

Cross-linked enzyme aggregates (CLEAs): A novel and versatile method for enzyme immobilization (a review)

The key to obtaining optimum performance of an enzyme is often a question of devising an effective method for its immobilization. This review describes a novel, versatile and effective methodology for enzyme immobilization, namely, as cross-linked enzyme aggregates (CLEAs). The method is exquisitely simple – involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules – and amenable to rapid optimization. It is applicable to a wide variety of enzymes, including cofactor-dependent oxidoreductases and lyases, and affords stable, recyclable catalysts with high retention of activity, sometimes higher than that of the free enzyme it was derived from. The enzyme does not need to be of high purity. Indeed, the methodology is essentially a combination of purification and immobilization in one step. The technique is also applicable to the preparation of combi-CLEAs, containing two or more enzymes, for use in one-pot, multi-step syntheses. For example, an oxynitrilase/nitrilase combi-CLEA was used for the one-pot synthesis of (S)-mandelic acid from benzaldehyde, in high yield and enantiomeric purity.     

Reaction of Trigonopsis variabilis d-amino acid oxidase with 2,6-dichloroindophenol: kinetic characterisation and development of an oxygen-independent assay of the enzyme activity

2,6-Dichloroindophenol (DCIP) is shown to be utilised efficiently as electron acceptor replacing dioxygen in the reaction of Trigonopsis variabilis d-amino acid oxidase (TvDAO) with d-methionine as the substrate. The specificity constant for DCIP reduction at 30 °C is one-twelfth that of oxygen conversion into hydrogen peroxide. Time course analysis of simultaneous consumption of DCIP and dioxygen, recorded on-line by absorption and non-invasive fluorescence quenching, respectively, pinpoints the preferential utilisation of dioxygen; and reveals a maximum DCIP conversion rate that is independent of the initial concentration of dioxygen. A robust direct assay of TvDAO activity has been developed that does not require anaerobic reaction conditions. It was down-scaled to microtitre plate format and overcomes practical limitations of other assays due to the low affinity of TvDAO for dioxygen (K_m ≈ 0.7 mmol L⁻¹).     

Design of β‐galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β‐galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro‐mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120‐fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β‐galactosidase immobilized on glyoxyl‐activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl‐activated silica was similar to that observed in macroporous agarose‐glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml^?1, 290 UI g^?1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.