Design Of Biocatalysts Research Articles

Preparation, activity and structure of cross-linked enzyme aggregates (CLEAs) with nanoparticle.

Cross-linked enzyme aggregates (CLEAs) have emerged as an interesting biocatalyst design for enzyme immobilization. However, the commercialization of CLEAs is often hampered by their shortcomings, such as poor-controlled particle size, low activity and sticky characteristic. In order to overcome these drawbacks, five nanoparticles (NPs) (nano-TiO2, nano-MgO, nano-Ni, nano-Cu and nano-Fe3O4) were used to improve CLEAs activity and structure in this study. Results showed that moderate dosage of nano-TiO2 addition increased CLEAs activity, and the most increment was 15.2% relative to CLEAs without NPs. This was possibly due to more channels and smaller size particle of CLEAs after nano-TiO2 addition. Moreover, nano-TiO2 addition not only decreased fluorescence intensity but also caused the red shift in tryptophan (Trp) and tyrosine (Typ) residues. Nano-TiO2 addition decreased Km and increased Vmax of CLEAs. These changes were benefit to the activity and structure of CLEAs. However, addition of other four NPs (nano-MgO, nano-Ni, nano-Cu and nano-Fe3O4) did not increase CLEAs activity and even decrease CLEAs activity due to less amorphous cavities and larger or discrete particle size than CLEAs without NPs. In addition, FT-IR results showed that NPs addition increases the content of regular structure of CLEAs, and causes a partial transformation of β-turn into β-sheet. This study showed that it was potential to improve CLEAs performance by nano-TiO2 addition.

Maximizing the stability of metabolic engineering-derived whole-cell biocatalysts.

A systematic and powerful knowledge-based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case-by-case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole-cell biocatalysts. They are still largely missing for whole-cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole-cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole-cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.

Design of a lipase-nano particle biocatalysts and its use in the kinetic resolution of medicament precursors

Superparamagnetic iron oxide nanoparticles (Fe3O4) were prepared by the co-precipitation method and functionalized with 3-amino-propyltriethoxysilane (APTES) or branched-polyethylenimine (PEI). After that, two parallels methods to immobilize the lipase from Thermomyces lanuginosus (TLL) were performed: the first one by ionic exchange and the second one by covalent attachment after the functionalization of the support with glutaraldehyde (GA). X-ray powder diffraction, magnetometry and infrared spectroscopy analysis were used to characterize the TLL preparations. These analyses showed that all samples presented superparamagnetic properties even after the immobilization procedure. The SPMN (superparamagnetic nanoparticle) @APTES covalent preparation had around 450min of half-life time at pH 7.0 and 70°C while that of the free enzyme was 46min. These biocatalysts were evaluated in the kinetic resolution of rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate in different co-solvents (acetonitrile, isopropanol, ethyl ether and tetrahydrofuran). The best results were for the enzyme/substrate ratio of 2:1, in the presence of the ethyl ether or THF (20% v/v both) at 30°C during 24h with the SPMN@PEI-TLL biocatalyst. The conversion attained was 50% and the enantiomeric excess of the product was 99%. The new SPMN support are an excellent strategy to easy recovery of the biocatalyst by applying a magnetic field.

Revealing the Origin of the Efficiency of the De Novo Designed Kemp Eliminase HG-3.17 by Comparison with the Former Developed HG-3.

The design of new biocatalysts is a goal in biotechnology to improve the rate, selectivity and environmental impact of industrial chemical processes. In this regard, the use of computational techniques has provided valuable assistance in the design of new enzymes with remarkable catalytic activity. In this paper, hybrid QM/MM molecular dynamics simulations have allowed insights to be gained on the origin of the limited efficiency of a computationally designed enzyme for the Kemp elimination; the HG-3. Comparison of results derived from this enzyme with those of a more evolved protein containing additional point mutations, HG-3.17, rendered important information that should be taken into account in the design of new enzymes. For this Kemp eliminase reaction, higher reactivity has been demonstrated to be related to a better electrostatic preorganisation of an environment that creates a more favourable electrostatic potential for the reaction to proceed. The limitations of HG-3 can be related to a lack of flexibility, a not well-fitted active site, and a lack of protein electrostatic preorganisation, which decrease the reorganisation around the oxyanion hole.

Oriented Coimmobilization of Oxidase and Catalase on Tailor-Made Ordered Mesoporous Silica.

Mesoporous silica materials are promising carriers for enzyme immobilization in heterogeneous biocatalysis applications. By tailoring their pore structural framework, these materials are designable for appropriate enzyme binding capacity and internal diffusivity. To supply O2 efficiently to solid-supported immobilized enzymes represents a core problem of heterogeneously catalyzed oxidative biotransformations. In this study, therefore, we synthesized and compared three internally well-ordered and two amorphous silica materials as enzyme carriers, each of those with pore sizes of ≥10 nm, to enable the coimmobilization of d-amino-acid oxidase (79 kDa) and catalase (217 kDa). Both enzymes were fused to the silica-binding module Zbasic2 to facilitate their selective and oriented immobilization directly from crude protein mixtures on native silica materials. Analyzing the effects of varied pore architecture and internal surface area on the performance of the immobilized bienzymatic system, we showed that a uniform pore structural framework was beneficial for enzyme loading (≥70 mg protein/g carrier), immobilization yield (≥90%), surface and pore volume filling without hindered adsorption, and catalytic effectiveness (≥60%) of the coimmobilizate. Using the best carrier LP-SBA-15, we obtained a solid oxidase-catalase preparation with an activity of 2000 μmol/(min g_material) that was recyclable and stable during oxidation of d-methionine. These results affirm a strategy of optimizing immobilized O2-dependent enzymes via tunable internal structuring of the silica material used as carrier. They thus make a significant advance toward the molecular design of heterogeneous oxidation biocatalysts on mesoporous silica supports.

Constrained evolution of a bispecific enzyme: lessons for biocatalyst design.

One of the central goals of protein design and engineering is to be able to accurately predict the effects of a mutation on stability and activity. However, the genetic context into which mutations are introduced can lead to complex interactions between the mutation and other amino acids and unpredictable, non-additive, effects. This phenomenon is known as intramolecular epistasis and has been shown to restrict evolutionary paths through laboratory directed evolution experiments and ancestral protein reconstruction, but has rarely been studied at a quantitative level in naturally evolving enzymes. Atrazine-specific and atrazine/ametryn bispecific triazine hydrolases (TrzN) have evolved in different bacterial strains over the past fifty years in response to the presence of the synthetic herbicides atrazine and ametryn. Here, we have investigated all 24 evolutionary trajectories that are possible from monofunctional to bispecific TrzN isoforms in terms of activity, stability, expression and structure. The results reveal that half of these trajectories are unviable due to inactive intermediates, with only 1/24 trajectories exhibiting consistent improvement in bispecificity. The most viable path requires the mutation of Gln241 to Glu241 first, which increases activity 3-fold with atrazine and 105-fold with ametryn, which is further optimised in subsequent evolutionary steps. The epistatic interactions between mutations, involving control of the pKa of catalytic residues, the thermostability of the protein, and soluble expression are shown to be responsible for the bottlenecks in this evolutionary landscape. This comprehensive analysis of the evolution of bispecificity highlights the importance of epistasis in protein engineering and evolution, which makes identifying the correct sequence in which to combine mutations extremely important.

Asymmetric synthesis of (S)-phenylacetylcarbinol – closing a gap in C–C bond formation

By the combination of biocatalyst design and reaction engineering, the so far not stereoselectively accessible (S)-phenylacetylcarbinol could be enzymatically synthesized with product concentrations >48 g L−1 and an enantiomeric excess up to 97%.

Enzyme Engineering for In Situ Immobilization.

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

An Efficient, Recyclable, and Stable Immobilized Biocatalyst Based on Bioinspired Microcapsules-in-Hydrogel Scaffolds.

Design and preparation of high-performance immobilized biocatalysts with exquisite structures and elucidation of their profound structure-performance relationship are highly desired for green and sustainable biotransformation processes. Learning from nature has been recognized as a shortcut to achieve such an impressive goal. Loose connective tissue, which is composed of hierarchically organized cells by extracellular matrix (ECM) and is recognized as an efficient catalytic system to ensure the ordered proceeding of metabolism, may offer an ideal prototype for preparing immobilized biocatalysts with high catalytic activity, recyclability, and stability. Inspired by the hierarchical structure of loose connective tissue, we prepared an immobilized biocatalyst enabled by microcapsules-in-hydrogel (MCH) scaffolds via biomimetic mineralization in agarose hydrogel. In brief, the in situ synthesized hybrid microcapsules encapsulated with glucose oxidase (GOD) are hierarchically organized by the fibrous framework of agarose hydrogel, where the fibers are intercalated into the capsule wall. The as-prepared immobilized biocatalyst shows structure-dependent catalytic performance. The porous hydrogel permits free diffusion of glucose molecules (diffusion coefficient: ∼6 × 10(-6) cm(2) s(-1), close to that in water) and retains the enzyme activity as much as possible after immobilization (initial reaction rate: 1.5 × 10(-2) mM min(-1)). The monolithic macroscale of agarose hydrogel facilitates the easy recycling of the immobilized biocatalyst (only by using tweezers), which contributes to the nonactivity decline during the recycling test. The fiber-intercalating structure elevates the mechanical stability of the in situ synthesized hybrid microcapsules, which inhibits the leaching and enhances the stability of the encapsulated GOD, achieving immobilization efficiency of ∼95%. This study will, therefore, provide a generic method for the hierarchical organization of (bio)active materials and the rational design of novel (bio)catalysts.

Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies

BackgroundIn order to perform selective CH functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)2PhenA (bpy=2,2′-bipyridine and PhenA=5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective CH bond hydroxylation of several substrates. MethodsA combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway. ResultsThe crystal structure of the sL407C-1 enzyme was solved in the open and closed conformations revealing a through-space electron transfer pathway involving highly conserved, F393 and Q403, residues. Several mutations of these residues (F393A, F393W or Q403W) were introduced to probe their roles in the overall reaction. Transient absorption measurements confirm rapid electron transfer as heme reduction is observed in all four hybrid enzymes. Compared to the parent sL407C-1, photocatalytic activity was negligible in the dF393A-1 enzyme while 60% increase in activity with total turnover numbers of 420 and 90% product conversion was observed with the dQ403W-1 mutant. ConclusionsIn the sL407C-1 enzyme, the photosensitizer is ideally located to rapidly deliver electrons, using the naturally occurring electron transfer pathway, to the heme center in order to activate molecular dioxygen and sustain photocatalytic activity. General significanceThe results shed light on the design of efficient light-driven biocatalysts and the approach can be generalized to other members of the P450 superfamily.

Towards sustainable biofuel production: Design of a new biocatalyst to biodiesel synthesis from waste oil and commercial ethanol

The sustainable technologies to produce alternative energies as biofuel with focus on the harnessing of renewable sources and waste biomass is gradually gaining ground. Although the biodiesel is a very attractive biofuel its production from vegetable oils competes with the feed generation with the consequent socio-economic costs involved. Therefore, the reuse of waste oils appears as an alternative highly promising. However the high content of free fatty acid and water in this raw material difficult its use in the conventional processes employed in the actuality. In this work, we successfully develop hybrid catalysts based in Pseudomonas fluorescens lipase immobilized over Ca and Na modified mesoporous SBA-15 supports. The physic-chemical properties of the supports were determined by Small-angle X-ray Scattering (SAXS), Transmission Electron Microscopy (TEM), Scanning Electron Microscope (SEM), Infrared Spectroscopy (IR), Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP) and N2 adsorption measurements. These hybrid catalysts were capable to process waste oils, and even, commercial ethanol (96%) obtained from a fermentative process An optimum activity, with around 90% of FAEE yield, was achieved with lipase immobilized on Ca modified SBA-15 using 4% of water respect to oil, 1:4 oil/ethanol ratio, 400 mg/g of enzyme immobilized, at 37 °C and 180 oscillations/min. This catalyst could contribute to development of a more environmentally and economically viable process to biodiesel production.

Lipase coated clusters of iron oxide nanoparticles for biodiesel synthesis in a solvent free medium

Methyl or ethyl esters of long chain fatty acids are called biodiesel. Biodiesel is synthesized by the alcoholysis of oils/fats. In this work, lipase from Thermomyces lanuginosus was precipitated over the clusters of Fe3O4 nanoparticles. This biocatalyst preparation was used for obtaining biodiesel from soybean oil. After optimization of both immobilization conditions and process parameters, complete conversion to biodiesel was obtained in 3h and on lowering the enzyme amount, as little as 1.7U of lipase gave 96% conversion in 7h. The solvent free media with oil:ethanol (w/w) of 1:4 and 40°C with 2% (w/w) water along with 20% (w/w) silica (for facilitating acyl migration) were employed for reaching this high % of conversion. The biocatalyst design enables one to use a rather small amount of lipase. This should help in switching over to a biobased production of biodiesel.

Dual bioimprinting of Thermomyces lanuginosus lipase for synthesis of biodiesel

Use of biodiesel as an alternative to non-renewable sources of energy has become an attractive option in recent years. The enzymatic synthesis of biodiesel by transesterification of fats/oils with an alcohol is a much more sustainable route than the chemical method. However, cost effectiveness of the enzymatic route is a major barrier in its commercialization. In this work, a high activity biocatalyst design of Thermomyces lanuginosus lipase is made by dually bioimprinting it with substrate and a surfactant (which is believed to open up the lid covering the active site of the lipase) during precipitation of the lipase in organic solvent. When the lipase was bioimprinted with only the surfactants, 28U of the enzyme/g of oil could yield 99% biodiesel from soybean oil in about 4h. However, when dually bioimprinted even very low enzyme load 1.4U/g of oil, yielded 99% biodiesel within 48h.

Structural and Dynamical Aspects of Electrostatic Interactions by Applying Aspherical Atom Model in HIV-1 Protease

Accurate approximation of electrostatic energies is of major importance for our understanding protein energetics in computer-aided drug design as well as for the design of novel biocatalysts and protein therapeutics. In this study, we applied new method, based on an aspherical atom model build from University at Buffalo Database (UBDB), for estimating electrostatic interactions in HIV protease (HIV-PR). The method is much more accurate than classically used force fields. HIV-PR, an aspartyl protease enzyme involved in human immunodeficiency virus (HIV) replication, is an important target for drug design strategies to combat acquired immune deficiency syndrome (AIDS). Most of the currently used HIV-PR inhibitors in HIV treatment have been prone to suffer from the mutations associated with drug resistance. Therefore, it is necessary to search for potent alternatives helping to overcome the resistance. The active site of PR protein is formed by the dimerization of the two monomers and covered by two glycine rich, antiparallel beta-hairpins flaps. In recent study, it has been shown that monomeric HIV-PR is relatively stable as compared to its dimeric form. It is also reported that mutations at or near the monomer-monomer interface shift the monomer-dimer equilibrium to inactive monomeric form, which is now of much interest in the contexts of drug design. Such observation can be confirmed by applying molecular dynamics simulation providing ample insights into both biological and technical aspect of macromolecular conformation and dynamics at atomistic level. So it's worth to investigate the multi-conformers of HIV-PR existing due to various mutations by combining MD simulation with the UBDB derived electron density for high resolution structures ≈0.8-1.00 A. The research may lead to the discovery of a general rule of conserved electrostatic interaction that stabilizes the HIV-PR dimer.

Nanotechnology Applications for Food and Bioprocessing Industries

Nanoscience is an emerging technology today with great application in different fields. Biocatalysts design, identification of different bacterial strains, monitoring the quality of food by different biosensors, food packaging with smart systems, active, intelligent and nano-encapsulation of bioactive food compounds are examples of some of these applications in the food industry. In this paper, some topics related to the potential of nanotechnology in the food industry are updated. In addition, some concerns about nanotechnology application in this popular industry are also discussed.

Whole-Cell Biocatalysts for Stereoselective C-H Amination Reactions.

Enantiomerically pure chiral amines are ubiquitous chemical building blocks in bioactive pharmaceutical products and their synthesis from simple starting materials is of great interest. One of the most attractive strategies is the stereoselective installation of a chiral amine through C-H amination, which is a challenging chemical transformation. Herein we report the application of a multienzyme cascade, generated in a single bacterial whole-cell system, which is able to catalyze stereoselective benzylic aminations with ee values of 97.5%. The cascade uses four heterologously expressed recombinant enzymes with cofactors provided by the host cell and isopropyl amine added as the amine donor. The cascade presents the first example of the successful de novo design of a single whole-cell biocatalyst for formal stereoselective C-H amination.

Computational Studies of Candida Antarctica Lipase B to Test Its Capability as a Starting Point To Redesign New Diels-Alderases.

The design of new biocatalysts is a target that is receiving increasing attention. One of the most popular reactions in this regard is the Diels-Alder cycloaddition because of its applications in organic synthesis and the absence of efficient natural enzymes that catalyze it. In this paper, the possibilities of using the highly promiscuous Candida Antarctica lipase B as a protein scaffold to redesign a Diels-Alderase has been explored by means of theoretical quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations. Free energy surfaces have been computed for two reactions in the wild-type and in several mutants with hybrid AM1/MM potentials with corrections at M06-2X/MM level. The study of the counterpart reactions in solution has allowed performing comparative analysis that render interesting conclusions. Since the dienophile anchors very well in the oxyanion hole of all tested protein variants, the slight electronic changes from reactant complex to the transition state suggest that mutations should be focused in favoring the formation of reactive conformations of a reactant complex that, in turn, would reduce the energy barrier.

Probing the roles of two tryptophans surrounding the unique zinc coordination site in lipase family I.5.

A unique zinc domain found in all of the identified members of the lipase family I.5 is surrounded by two conserved tryptophans (W61 and W212). In this study, we investigated the role of these hydrophobic residues in thermostability and thermoactivity of the lipase from Bacillus thermocatenulatus (BTL2) taken as the representative of the family. Circular dichroism spectroscopy revealed that the secondary structure of BTL2 is conserved by the tryptophan mutations (W61A, W212A, and W61A/W212A), and that W61 is located in a more rigid and less solvent exposed region than is W212. Thermal denaturation and optimal activity analyses pointed out that zinc induces thermostability and thermoactivity of BTL2, in which both tryptophans W61 and W212 play contributing roles. Molecular explanations describing the roles of these tryptophans were pursued by X-ray crystallography of the open form of the W61A mutant and molecular dynamics simulations which highlighted a critical function for W212 in zinc binding to the coordination site. This study reflects the potential use of hydrophobic amino acids in vicinity of metal coordination sites in lipase biocatalysts design.

ChemInform Abstract: Advanced Characterization of Immobilized Enzymes as Heterogeneous Biocatalysts

AbstractReview: 211 refs.

About the role of typical spacer/crosslinker on the design of efficient magnetic biocatalysts based on nanosized magnetite

The immobilization of Candida antarctica lipase B (CALB) was carried out using glutaraldehyde (GLUT) and/or 3-aminopropyl-triethoxisilane (APTS). The aim of this work was to elucidate the role of these crosslinkers/functionalizers on the efficiency of the prepared nanosized catalysts in solvent-free oleic acid esterification.A series of biocatalysts were prepared in presence or absence of GLUT and APTS. The impact of the amount of initial CALB was also explored. An experimental design was utilized to study the variables that maximize biocatalyst activity.A strong dependence of enzymatic activity with the nominal amount of GLUT as well as the final protein/CALB loading was found. Nominal quantity of APTS did not affect catalyst⿿s activity when used in combination with GLUT. Additional studies demonstrated that stability during storage was mainly dependent on the enzyme loading. The optimum biocatalyst was reused six cycles without mass loss. Biocatalyst⿿s performance decreased with reuse. Mechanisms justifying these results were proposed.The role of GLUT and APTS on stability during storage and on differences between initial enzymatic activity and the performance in the reaction after two months was discussed. The problem of mixed interaction of CALB (covalent bonding plus simple adsorption) was carefully addressed to explain leaching of the lipase.Leaching and stability on storage should be included in the analysis of modifiers impact when support⿿s modifiers are used. The fresh and stored biocatalyst enzymatic activity has to be addressed looking at the practical aspects of implementation in technological settings.