Immobilization of Lipoxygenase,Catalase, and Lipase for a Reactor Design Targeting Linoleic Acid Hydroperoxidation

Hydroperoxy-9Z,11E-octadecadienoic acid (13-HPODE) can be obtained from safflower oil in an enzyme cascade utilizing lipase, lipoxygenase (LOX), and catalase for in situ oxygen generation. The application of immobilized enzymes may open a new path to a cost-efficient production of 13-HPODE, which is used for the synthesis of green note aroma hexanal. Ten immobilization supports are compared for immobilization of lipoxygenase-1 from Glycine max (LOX-1) and oxirane-based Immobead 150 P proves to be best with a maximum LOX-1 activity of 22 470 U g⁻¹. The immobilizate is successfully recycled in eight consecutive batches and maintains full activity over a period of 16 h using a 3D-printed column reactor. Catalase from Micrococcus lysodeikticus and LOX-1 are co-immobilized on Immobead 150 P allowing a constant production of 13-HPODE for up to six cycles with a maximum product conversion of 45% and a 13-regioselectivity of 83% . In this two-enzyme system with H₂O₂-dosage, foam generation is significantly reduced. Co-immobilization of LOX-1 and lipaseis possible; however, rapid lipase deactivation occurs. Therefore, a two-reactor approach with oil hydrolysis in the first reactor is proposed. Immobilized lipases from C. rugosa are suitable for safflower oil hydrolysis and maintain full activity over ten hydrolysis cycles. Practical applications: Linoleic acid hydroperoxide (13-HPODE) is the starting material for the synthesis of the green note aroma compound hexanal. The byproduct of the hydroperoxide splitting is ω-oxododecenoic acid, which is currently not employed industrially. The bifunctional oxodocecenoic acid is interesting as precursor for the synthesis of polymer building blocks. Simple one-step derivatization of the oxo-group can yield suitable C12 monomers such as dicarboxylic acids, ω-amino acids, or ω-hydroxy acids. Cost-efficiency is a key parameter to incorporate these new biobased building blocks for polymer applications. In this approach, immobilized enzymes are used for the synthesis of 13-HPODE starting from safflower oil with in situ oxygen generation to prevent excessive foam formation. A two-reactor concept is designed to circumvent hydroperoxide-induced lipase deactivation. Direct comparison of both batch and continuous process is performed and provides information for the implementation of the enzyme cascade and the design of an optimized reactor system.     

Asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester by undifferentiated cells of white turnip in phosphate buffer/organic solvent

Ethyl (S)-3-hydroxy-3-phenylpropionate was synthesized by asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester with undifferentiated cells of white turnip in phosphate buffer/organic solvent. The conversion increased with the LogP _oct of organic solvent increase. The phosphate buffer (0.2 mol/L, pH 7.0)/dodecane was selected as optimum medium for reduction. The optimal content of dodecane in medium is 10% (v). The conversion decreased with initial substrate concentration increase. Addition of more biomass of plant cells and 10% ethanol as co-substrate can improve conversion. The plant cells can be reused well for three times. The enantiomeric excess of ethyl (S)-3-hydroxy-3-phenylpropionate reached 100% with 1% allyl bromide as inhibitor.     

A Peroxygenase-Alcohol Dehydrogenase Cascade Reaction to Transform Ethylbenzene Derivatives into Enantioenriched Phenylethanols

In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)- or (S)-phenylethanols using the (R)-selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)-selective ADH from Rhodococcus ruber (ADH-A). In a one-pot two-step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity.     

Stereodivergent Biocatalytic Formal Reduction of α-Angelica Lactone to (R)- and (S)-γ-Valerolactone in a One-Pot Cascade

The formal asymmetric and stereodivergent enzymatic reduction of α-angelica lactone to both enantiomers of γ-valerolactone was achieved in a one-pot cascade by uniting the promiscuous stereoselective isomerization activity of Old Yellow Enzymes with their native reductase activity. In addition to running the cascade with one enzyme for each catalytic step, a bifunctional isomerase-reductase biocatalyst was designed by fusing two Old Yellow Enzymes, thereby generating an unprecedented case of an artificial enzyme catalyzing the reduction of nonactivated C=C bonds to access (R)-valerolactone in overall 41 % conversion and up to 91 % ee. The enzyme BfOYE4 could be used as single biocatalyst for both steps and delivered (S)-valerolactone in up to 84 % ee and 41 % overall conversion. The reducing equivalents were provided by a nicotinamide recycling system based on formate and formate dehydrogenase, added in a second step. This enzymatic system provides an asymmetric route to valuable chiral building blocks from an abundant bio-based chemical.     

Coevolution of the Activity and Thermostability of an ϵ-Keto Ester Reductase for Better Synthesis of an (R)-α-Lipoic Acid Precursor

In this work, we have identified a significantly improved variant (S131Y/Q252I) of the natural ϵ-keto ester reductase CpAR2 from Candida parapsilosis for efficiently manufacturing (R)-8-chloro-6-hydroxyoctanoic acid [(R)-ECHO] through co-evolution of activity and thermostability. The activity of the variant CpAR2_S131Y/Q252I towards the ϵ-keto ester ethyl 8-chloro-6-oxooctanoate was improved to 214 U mg⁻¹—from 120 U mg⁻¹ in the case of the wild-type enzyme (CpAR2_WT)—and the half-deactivating temperature (T₅₀, for 15 min incubation) was simultaneously increased by 2.3 °C in relation to that of CpAR2_WT. Consequently, only 2 g L⁻¹ of lyophilized E. coli cells harboring CpAR2_S131Y/Q252I and a glucose dehydrogenase (GDH) were required in order to achieve productivity similar to that obtained in our previous work, under optimized reaction conditions (530 g L⁻¹ d⁻¹). This result demonstrated a more economical and efficient process for the production of the key (R)-α-lipoic acid intermediate ethyl 8-chloro-6-oxooctanoate.     

Expanding Synthetic Applications of Δ1-Piperidine-2-carboxylate/Δ1-pyrroline-2-carboxylate Reductase from Pseudomonas syringae (DpkAPsyrin). Biocatalytic Asymmetric Synthesis of (S,E)-2-hydroxy-4-arylbut-3-enoic Acid Derivatives

Chiral 2-hydroxy-4-arylbut-3-enoic acid derivatives are important precursors for the synthesis of angiotensin converting enzyme (ACE) inhibitors, such as enalapril, lisinopril, cilapril or benazepril. In this work, we take advantage of the unexplored promiscuous ketoreductase activity of Δ¹-piperidine-2-carboxylate/Δ¹-pyrroline-2-carboxylate reductase from Pseudomonas syringae pv. tomato DSM 50315 (DpkA_Psyrin) for the synthesis of (S,E)-2-hydroxy-4-arylbut-3-enoic acids. The strategy was designed as an enzymatic cascade comprising an aldol condensation between pyruvate with aryl aldehydes, catalyzed by the trans-o-hydroxybenzylidene pyruvate hydratase-aldolase from Pseudomonas putida (HBPA_Pputida), for the construction of carbon scaffold, and an ensuing asymmetric reduction of the carbonyl group catalyzed by DpkA_Psyrin. The enzymatic cascade provided quantitative conversions, with global isolated yields between 57–85%. A total of nine structurally diverse (S,E)-2-hydroxy-4-arylbut-3-enoic acids were prepared in ee between 87–99%.     

An l-amino acid electrode for monitoring beer fermentation

A membrane covered amperometric l-amino acid electrode is described, employing l-amino acid oxidase immobilized on a Pt disc electrode with rabbit albumin and glutaraldehyde. The electrode response to a range of l-amino acids and a theoretical treatment for the rate determining step are presented. Results are also given for the application of the electrode in monitoring beer fermentations. Appropriate amino acid utilisation is vital for both yeast cell growth and beer flavour development.List of symbols A electrode area - D diffusion coefficient - e reduced enzyme concentration - e total enzyme concentration - F Faraday constant - i electrode current - i_D l/A - I l/k_ME - j flux - L thickness of the electrolyte layer - L _M thickness of the membrane - k_cat rate constant for enzyme/substrate reaction - k rate constant for electrode reaction - k_ME electrochemical rate constant for the enzyme reaction - k_S mass transfer rate constant for substrate in membrane - K membrane constant - K _s partition coefficient of substrate in membrane - K_M Michaelis constant - n number of electrons S substrate - Ii_D/[S] - y (^–1 – 1)/[S]     

Co-Immobilization of Rhizopus oryzae and Candida rugosa Lipases onto mMWCNTs@4-arm-PEG-NH2—A Novel Magnetic Nanotube–Polyethylene Glycol Amine Composite—And Its Applications for Biodiesel Production

This article describes the successful synthesis of a novel nanocomposite of superparamagnetic multi-walled nanotubes with a four-arm polyethylene glycol amine polymer (mMWCNTs@4-arm-PEG-NH₂). This composite was then employed as a support for the covalent co-immobilization of Rhizopus oryzae and Candida rugosa lipases under appropriate conditions. The co-immobilized lipases (CIL-mMWCNTs@4-arm-PEG-NH₂) exhibited maximum specific activity of 99.626U/mg protein, which was 34.5-fold superior to that of free ROL, and its thermal stability was greatly improved. Most significantly, CIL-mMWCNTs@4-arm-PEG-NH₂ was used to prepare biodiesel from waste cooking oil under ultrasound conditions, and within 120 min, the biodiesel conversion rate reached 97.64%. This was due to the synergy effect between ROL and CRL and the ultrasound-assisted enzymatic process, resulting in an increased biodiesel yield in a short reaction time. Moreover, after ten reuse cycles, the co-immobilized lipases still retained a biodiesel yield of over 78.55%, exhibiting excellent operational stability that is attractive for practical applications. Consequently, the combined use of a novel designed carrier, the co-immobilized lipases with synergy effect, and the ultrasound-assisted enzymatic reaction exhibited potential prospects for future applications in biodiesel production and various industrial applications.     

Novel BQCA- and TBPB-Derived M1 Receptor Hybrid Ligands: Orthosteric Carbachol Differentially Regulates Partial Agonism

Recently, investigations of the complex mechanisms of allostery have led to a deeper understanding of G protein-coupled receptor (GPCR) activation and signaling processes. In this context, muscarinic acetylcholine receptors (mAChRs) are highly relevant due to their exemplary role in the study of allosteric modulation. In this work, we compare and discuss two sets of putatively dualsteric ligands, which were designed to connect carbachol to different types of allosteric ligands. We chose derivatives of TBPB [1-(1′-(2-tolyl)-1,4′-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one] as M₁-selective putative bitopic ligands, and derivatives of benzyl quinolone carboxylic acid (BQCA) as an M₁ positive allosteric modulator, varying the distance between the allosteric and orthosteric building blocks. Luciferase protein complementation assays demonstrated that linker length must be carefully chosen to yield either agonist or antagonist behavior. These findings may help to design biased signaling and/or different extents of efficacy.     

Mannich reaction in water using acidic ionic liquid as recoverable and reusable catalyst

The task-specific room-temperature ionic liquid (TSIL) N, N, N-trimethyl-N-butanesulfonic acid ammonium hydrogen sulfate [TMBSA]HSO₄ was synthesized as a cheap and recyclable catalyst for one-pot three-component Mannich reaction in water. Sixteen β-amino carbonyl compounds were obtained in good yields under the mild conditions. The products could simply be separated from the catalyst/water, and the catalyst could be reused at least 7 times without noticeably decreasing the catalytic activity.      

Biguanide/Pd(OAc)2 immobilized on magnetic nanoparticle as a recyclable catalyst for the heterogeneous Suzuki reaction in aqueous media

Immobilization of Pd(II) ions on magnetite nanoparticle (MNP) has been simply achieved through a surface modification of Fe₃O₄ nanoparticle with a biguanide. This surface-modified nanoparticle was characterized by various techniques such as TEM, XRD, VSM, TGA, elemental analyzer, atomic absorption spectroscopy, N₂ adsorption–desorption (BET and BJH), and FT-IR. This nanosolid exhibits excellent catalytic activity for heterogeneous Suzuki reaction in aqueous media, and could be easily separated by an external magnet and reused for several times. TEM study of the recovered catalyst showed the preservation of the support core–shell structure and the good dispersion of the produced palladium nanoparticles.     

Immobilization of polyphenol oxidase on carboxymethylcellulose hydrogel beads: preparation and characterization

Carboxymethylcellulose (CMC) beads were prepared by a liquid curing method in the presence of trivalent ferric ions, and epicholorohydrin was covalently attached to the CMC beads. Polyphenol oxidase (PPO) was then covalently immobilized onto CMC beads. The enzyme loading was 603 µg g⁻¹ bead and the retained activity of the immobilized enzyme was found to be 44%. The K_m values were 0.65 and 0.87 mM for the free and the immobilized enzyme, and the V_max values were found to be 1890 and 760 U mg⁻¹ for the free and the immobilized enzyme, respectively. The optimum pH was 6.5 for the free and 7.0 for the immobilized enzyme. The optimum reaction temperature for the free enzyme was 40 °C and for the immobilized enzyme was 45 °C. Immobilization onto CMC hydrogel beads made PPO more stable to heat and storage, implying that the covalent immobilization imparted higher conformational stability to the enzyme. © 2000 Society of Chemical Industry     

Invertase immobilized on spacer‐arm attached poly(hydroxyethyl methacrylate) membrane: Preparation and properties

Microporous poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane was prepared by UV‐initiated photopolymerization. The spacer arm (i.e., hexamethylene diamine) was attached covalently and then invertase was immobilized by the condensation reaction of the amino groups of the spacer arm with carboxyl groups of the enzyme in the presence of carbodiimides. The values of the Michael's constant K_m of invertase were significantly larger (ca. 2.5 times) upon immobilization, indicating decreased affinity by the enzyme for its substrate, whereas V_max was smaller for the immobilized invertase. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 70°C the half times for the activity decay were 12 min for the free enzyme and 41 min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1685–1692, 2000     

Utilization of rice hull ash as a support material for immobilization of Candida cylindracea lipase

Rice hull ash was heated in a muffle furnace at 700°C for 2 h and metallic oxides were leached with 10% sulfuric acid. The acid-activated ash was then examined for immobilization of Candida cylindracea lipase. Immobilization was carried out by direct addition of the enzyme solution to the activated ash suspended in hexane. The immobilized lipase retained 30% of its hydrolytic activity, but thermal stability was greatly increased. Half-lives of the immobilized enzyme at 50, 60, and 70°C were 45, 17, and 4 min, respectively. Optimal pH of the immobilized enzyme was 7.2. The apparent K_m and V_max for olive oil were 41 mM and 99.5 μmol/h-mg solid, respectively.     

Chitosaneous hydrogel beads for immobilizing neutral protease for application in the preparation of low molecular weight chitosan and chito‐oligomers

Enzyme hydrolysis with immobilized neutral protease was carried out to produce low molecular weight chitosan (LMWC) and chito‐oligomers. Neutral protease was immobilized on (CS), carboxymethyl chitosan (CMCS), and N‐succinyl chitosan (NSCS) hydrogel beads. The properties of free and immobilized neutral proteases on chitosaneous hydrogel beads were investigated and compared. Immobilization enhanced enzyme stability against changes in pH and temperature. When the three different enzyme supports were compared, the neutral protease immobilized on CS hydrogel beads had the highest thermal stability and storage stability, and the enzyme immobilized on NSCS hydrogel beads had the highest activity compared to those immobilized on the other supports, despite its lower protein loading. Immobilized neutral protease on all the three supports had a higher K_m (Michaelis‐Menten constant) than free enzyme. The V_max (maximum reaction velocity) value of neutral protease immobilized on CS hydrogel beads was lower than the free enzyme, whereas the V_max values of enzyme immobilized on CMCS and NSCS hydrogel beads were higher than that of the free enzyme. Immobilized neutral protease on CS, CMCS, and NSCS hydrogel beads retained 70.4, 78.2, and 82.5% of its initial activity after 10 batch hydrolytic cycles. The activation energy decreased for the immobilization of neutral protease on chitosaneous hydrogel beads. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 3743–3750, 2006     

A Multi-enzyme Cascade for the Biosynthesis of AICA Ribonucleoside Di- and Triphosphate

AICA (5′-aminoimidazole-4-carboxamide) ribonucleotides with different phosphorylation levels are the pharmaceutically active metabolites of AICA nucleoside-based drugs. The chemical synthesis of AICA ribonucleotides with defined phosphorylation is challenging and expensive. In this study, we describe two enzymatic cascades to synthesize AICA derivatives with defined phosphorylation levels from the corresponding nucleobase and the co-substrate phosphoribosyl pyrophosphate. The cascades are composed of an adenine phosphoribosyltransferase from Escherichia coli (EcAPT) and different polyphosphate kinases: polyphosphate kinase from Acinetobacter johnsonii (AjPPK), and polyphosphate kinase from Meiothermus ruber (MrPPK). The role of the EcAPT is to bind the nucleobase to the sugar moiety, while the kinases are responsible for further phosphorylation of the nucleotide to produce the desired phosphorylated AICA ribonucleotide. The selected enzymes were characterized, and conditions were established for two enzymatic cascades. The diphosphorylated AICA ribonucleotide derivative ZDP, synthesized from the cascade EcAPT/AjPPK, was produced with a conversion up to 91 %. The EcAPT/MrPPK cascade yielded ZTP with conversion up to 65 % with ZDP as a side product.     

Hidden Specificities in Enzyme Catalysis: Structural Basis of Substrate Structure-Selectivity Relationship of a Ketoreductase

Enzymes often convert both physiological and non-physiological substrates with high stereoselectivity; yet, for some enzymes, opposite product chirality is observed. A possible explanation is the existence of hidden specificities becoming apparent when non-physiological substrates confer different substrate–enzyme interactions than the physiological substrate. To test this hypothesis, a series of α-methylated β-keto esters were converted with Tyl-KR1, a ketoreductase from polyketide synthesis in Streptomyces fradiae. The conversions of six substrates with different physicochemical properties exhibited enantioselectivities ranging from 84 % ee for R,R to 84 % ee for S,S, yet high and uniform diastereoselectivity (anti, d.r.>9:1). The exchange of a single atom, namely an oxygen ester instead of a thioester, led to almost complete loss of enantioselectivity (<5 % ee). An additional S,S-selective binding mode as a hidden specificity in Tyl-KR1 has been identified through molecular modeling and site-directed mutagenesis.     

Synthesis and Biological Evaluation of CF3Se-Substituted α-Amino Acid Derivatives

Several CF₃Se-substituted α-amino acid derivatives, such as (R)-2-amino-3-((trifluoromethyl)selanyl)propanoates ( 5 a / 6 a ), (S)-2-amino-4-((trifluoromethyl)selanyl)butanoates ( 5 b / 6 b ), (2R,3R)-2-amino-3-((trifluoromethyl)selanyl)butanoates ( 5 c / 6 c ), (R)-2-((S)-2-amino-3-phenylpropanamido)-3-((trifluoromethyl)selanyl)propanoates ( 11 a / 12 a ), and (R)-2-(2-aminoacetamido)-3-((trifluoromethyl)selanyl)propanoates ( 11 b / 12 b ), were readily synthesized from natural amino acids and [Me₄N][SeCF₃]. The primary in vitro cytotoxicity assays revealed that compounds 6 a , 11 a and 12 a were more effective cell growth inhibitors than the other tested CF₃Se-substituted derivatives towards MCF-7, HCT116, and SK-OV-3 cells, with their IC₅₀ values being less than 10 μM for MCF-7 and HCT116 cells. This study indicated the potentials of CF₃Se moiety as a pharmaceutically relevant group in the design and synthesis of novel biologically active molecules.     

Thermo-responsive block copolymer micelle-supported (S)-α,α-diphenylprolinol trimethylsilyl ether for asymmetric Michael addition of nitroalkenes and aldehydes in water

An amphiphilic block copolymer PNIPAM₅₃-b-(PS₃₀-co-P4AMS₁₀) was facilely prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. The immobilization of (S)-α,α-diphenylprolinol trimethylsilyl ether onto the block copolymers was then performed through using copper-catalyzed alkyne-azide cycloaddition (CuAAC), and the generating amphiphilic diblock copolymer supported chiral catalyst PNIPAM₅₃-b-(PS₃₀-co-P4AMS₁₀)/proTMS was self-assembled into micelles with regular diameters about 50 nm in aqueous solution. The micellar catalyst was further used for the asymmetric Michael reaction between propanal and trans-β-nitrostyrene in water. Using only 1 mol% micellar catalyst, the corresponding Michael addition products could be obtained in good yields and high enantioselectivities as well as good diastereoselectivities. In addition, this micellar catalyst could be reused at least for four times. Moreover, the micellar catalyst could be applied for the asymmetric addition reaction of 4-chlorocinnamyl aldehyde and nitromethane, and thus constructing the baclofen pharmaceutical intermediate.     

Computational Design of Enantiocomplementary Epoxide Hydrolases for Asymmetric Synthesis of Aliphatic and Aromatic Diols

The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso-epoxides. Three substrates of different sizes were targeted: cis-2,3-butene oxide, cyclopentene oxide, and cis-stilbene oxide. Most of the 28 designs tested were active and showed the predicted enantioselectivity. Excellent enantioselectivities were obtained for the bulky substrate cis-stilbene oxide, and enantiocomplementary mutants produced (S,S)- and (R,R)-stilbene diol with >97 % enantiomeric excess. An (R,R)-selective mutant was used to prepare (R,R)-stilbene diol with high enantiopurity (98 % conversion into diol, >99 % ee). Some variants displayed higher catalytic rates (k_cat) than the original enzyme, but in most cases K_M values increased as well. The results demonstrate the feasibility of computational design and screening to engineer enantioselective epoxide hydrolase variants with very limited laboratory screening.