Effects of water on enzyme performance with an emphasis on the reactions in supercritical fluids

Enzymes require a certain level of water in their structures in order to maintain their natural conformation, allowing them to deliver their full functionality. Furthermore, as a modifier of the solvent, up to a certain level, water can modify the solvent properties such as polarity/polarizability as well as the solubility of the reactants and the products. In addition, depending on the type of the reaction, water can be a substrate (e.g., in hydrolysis) or a product (e.g., in esterolysis) of the enzymatic reaction, influencing the enzyme turnover in different ways. It is found that regardless of the type of reaction, the functionality of enzyme itself is maximum at an optimum level of water, beyond which the enzyme performance is declined due to the loss in enzyme stability. Furthermore, mass transfer limitations caused by pathway blockage and/or by reduced solubilities of the reactants and/or products can also affect the enzyme performance at higher water levels. Controlling water content of ingoing CO2 and substrates as well as precise management of enzyme support and salt hydrates are important strategies to adjust water level in reaction media, especially in supercritical environments.     

Acidolysis of sardine oil by lipase to concentrate eicosapentaenoic and docosahexaenoic acids in glycerides

Lipoprotein lipase from Pseudomonas sp. was the best enzyme to concentrate eicosapentaenoic and docosahexaenoic acids (EPA and DHA) in sardine oil by acidolysis reaction, and acetone was more effective than n-hexane as a solvent for dissolving the reactants and concentrating the two fatty acids. The water concentration in the reaction mixture was a decisive factor governing the enrichment of EPA and DHA and the yield of glycerides. EPA and DHA were more concentrated, but the yield of glycerides decreased, when the water concentration was increased gradually. Thus, the concentration rates of both the fatty acids were low with 0.25% water, although a considerable amount of diglyceride was detectable in the reaction products. The effect of reaction temperature was very slight with the use of acetone; however, the ratio DHA/EPA increased when the temperature was lowered in the presence of n-hexane. When acidolysis was performed at 25°C for 1 h, using 10,000 units of lipase per g of the reactants, the total percentage of EPA and DHA reached 65% in the glycerides and the recoveries of the two acids were 87.4 and 81.3%, respectively, based on the contents in the original sardine oil. The relationship of the enzyme substrate specificity to the reaction results was also investigated.     

Enzymatic Production of Alkyl Esters Through Lipase-Catalyzed Transesterification Reactions in Organic Solvents: Solvent Effects and Prediction Capabilities of Equilibrium Conversions

The solvent effect on the equilibrium position of the transesterification reaction of hexanol with ethyl acetate catalyzed by a lipase has been investigated in a variety of non-polar and polar solvents - and binary mixtures. The results obtained indicate that the solvent effect on the equilibrium conversion is very small as compared to that for the direct esterification reactions.

Equilibrium conversions were then predicted using the equilibrium constant for the reaction obtained from Gibbs free energy of formation information for reactants and products in combination with the UNIFAC activity coefficient model. A solvent independent equilibrium conversion was obtained, which was in good agreement with the observed average value for all solvents. This indicates that UNIFAC provides satisfactory estimates of the activity coefficients but its group contribution structure does not allow the prediction of the small differences in conversion among the solvents examined.

Finally plots of these conversions versus the solvent octanol/water partition coefficient or the solubility of water in the solvent, that provide the correct trend in direct esterification reactions, did not achieve the same for transesterification.     

Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products

Background aimsSuccessful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me₂SO).MethodsIn the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santoña, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me₂SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality.ResultsLimbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me₂SO.ConclusionsResults showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.     

Enzymatic Reactions in Non-Conventional Media: Prediction of Solvent Water Content for Optimum Water Activity

Most enzymes provide their optimum performance at a given water activity (aw), which is generally solvent independent. For a given organic liquid solvent at a specific temperature or for a supercritical solvent at a specific temperature and pressure this corresponds to a water concentration in which water has the desired activity. We present here a methodology for predicting this water concentration thus reducing substantially the amount of experimental work needed to find the optimum solvent with respect to equilibrium conversion.

If the enzyme optimum water activity is known, the methodology predicts the required water content in the solvent to achieve this aw value. If, in addition, the enzyme water activity curve is available, this methodology provides the total water that must be added to the system (enzyme plus solvent) so that a specific water activity can be obtained.

The same methodology can also be applied to predict the effect of the total water content of the system (initial or initial plus produced) on the water activity values. It is shown that: (a) for esterification reactions taking place in hydrophobic organic solvents, the produced water can lead to a substantial change in water activity, but not for less hydrophobic solvents; (b) introduction of dry CO₂ into a system, pre-equilibrated to a certain water activity at atmospheric pressure, can lead to a substantial decrease in the water activity especially at temperatures just above the critical one of the solvent and pressures larger than that.     

Reaction equilibrium for lipase-catalyzed condensation in organic solvent systems

Lipase-catalyzed condensation in an organic solvent is useful for the syntheses of esters. To reasonably design and optimize the reaction conditions, knowledge of the reaction equilibrium is required. The interaction of water with other reactants and the quantitative predictions for adsorption of water by a desiccant are discussed. The solvent effects on the reaction equilibrium are also elucidated in mixtures of nitrile and tert-alcohol.     

Operational concept for the improved synthesis of (R)-3,3'-furoin and related hydrophobic compounds with benzaldehyde lyase

Biphasic reaction systems for enzyme catalysis are an elegant way to overcome limited solubility and stability of reactants and facilitate continuous processes. However, many synthetically useful enzymes are not stable in biphasic systems of water and organic solvent. The entrapment in polymer beads of polyvinyl alcohol has been shown to enable the stable operation of enzymes unstable in conventional biphasic reaction systems. We report the extension of this concept to continuous operation in a fluidised bed reactor. The enzyme benzaldehyde lyase was used for the continuous synthesis of enantiopure (R)-3,3'-furoin. The results show enhanced stability with half-life times under operation conditions of more than 100 h, as well as superior enzyme utilisation in terms of productivity. Furthermore, racemisation and oxidation of the product could be successfully prevented under the non-aqueous and inert reaction conditions.     

Laccase grafted membranes for advanced water filtration systems: a green approach to water purification technology

Purpose: Conventional wastewater treatment technologies are not good enough to completely remove all endocrine disrupting compounds (EDCs) from the water. Membrane separation systems have emerged as an attractive alternative to conventional clarification processes for waste and drinking water. Coupling of a membrane separation process with an enzymatic reaction has opened up new avenues to further enhance the quality of water. This review article deliberates the feasibility of implementing enzymatic membrane reactors has been deliberated.

Materials and methods: A comprehensive study of conventional water treatment technologies was carried out and their shortcomings were pointed out. Research findings from the leading groups working on enzyme grafted membrane based water purification were summarized. This review also comprehends the patent documents pertinent to the technology of enzyme grafted membranes for water purification.

Results: Immobilization of an enzyme on a membrane improves the performance of membrane filtration, and processes for the treatment of polluted water. Research has started exploring the potential for laccase enzymes because it can catalyze the oxidation of a wide range of substrates, structurally comparable to EDCs, by a radical-catalyzed reaction mechanism, with corresponding reduction of oxygen to water in an electron transfer process. Further, in the presence of certain mediators, the substrate range of laccases can be further enhanced to non-aromatic substrates.

Conclusions: Removal of EDCs by laccase cross-linked enzyme aggregates in fixed-bed reactors or fluidized-bed reactors and laccase immobilized ultrafiltration (LIUF) membranes are proving their worth in water purification technology. The major operational issues with the use of LIUF membranes are enzyme instability in real wastewater and membrane fouling. In view of the above-stated characteristics, laccases are considered as the most promising enzyme for a greener and less expensive water purification technology.     

接种功能内生细菌Diaphorobacter sp.Phe15减少蔬菜亚细胞菲积累的体外试验研究

[目的]土壤中的多环芳烃(polycyclic aromatic hydrocarbons, PAHs)可被蔬菜根系吸收并在可食部分积累进而通过食物链威胁人群健康。接种功能内生细菌能有效减低蔬菜中PAHs的积累,而关于其对蔬菜亚细胞组分中PAHs积累的影响却鲜有报道。[方法]采用体外实验,研究了接种具有菲降解功能的菌株Diaphorobacter sp. Phe15对空心菜茎叶亚细胞组分中菲积累的影响及PAHs代谢相关酶活性的响应。[结果]接种Phe15可以可加速空心菜茎叶亚细胞中菲的降解,显著削减空心菜亚细胞组分中菲的含量,接菌后空心菜亚细胞组分中菲降解率达90%以上。此外,接种功能菌Phe15可以影响空心菜亚细胞组分中PAHs代谢相关酶系的活性,空心菜亚细胞水平POD、PPO、C230活性整体得到提高,且酶系活性与空心菜体内菲积累呈负相关关系。[结论]接种具有菲降解功能的菌株Phe15增加了空心菜亚细胞水平PAHs代谢相关酶系活性,进而降低空心菜体内菲的积累,研究结果为利用功能内生细菌削减蔬菜中多环芳烃污染提供了一定的参考和理论依据。     

Synthesis and characterization of poly(d,l-lactic acid) via enzymatic ring opening polymerization by using free and immobilized lipase

Abstract

Polylactic acid is an interesting biodegradable and bioabsorbable material, and is produced from lactic acid, either by the direct polycondensation of lactic acid or via the ring-opening polymerization (ROP) of lactide. A future target of it is to improve some of the polyester properties for specific biomedical applications. The biocatalytic ROP of lactide is attractive as a route to polymer synthesis due to its lack of toxic reactants, mild reaction requirements, and recyclability of immobilized enzyme. Therefore, the use of immobilized enzymes is also being investigated.

The aim of this work was to develop a methodology to synthesize high molecular weight polylactic acid via enzymatic ROP method using free enzyme and Candida antarctica lipase B (CALB) immobilized onto chitin and chitosan. The efficiency of the two approaches has been compared, with polymerization kinetics and resulting products fully characterized by FT-IR, NMR, DSC, XRD, and TGA analyses.     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

Studies on the synthesis of short-chain geranyl esters catalysed by Fusarium oxysporum esterase in organic solvents

A novel esterase isolated from Fusarium oxysporum was investigated for the synthesis of short-chain esters of geraniol by alcoholysis and direct esterification reactions in organic solvents. The enzyme was used as a dried powder (i.e., not immobilized). The reaction parameters affecting the enzyme behavior such as the nature of organic solvent and acyl donor, the concentration of substrates and the water activity of the system were studied. High yields (80–90%) were obtained by both approaches (alcoholysis and direct esterification) at low values of water activity (a_w=0.11) in n-hexane. The enzyme retain its catalytic activity even after fifth reuse in n-hexane at a_w=0.11, demonstrating its stability and efficiency under the conditions of this study.     

Solvent Structure and Enzyme Activity

Abstract

Enzymes are fluctuating particles in thermal equilibrium with their solvent environment. A variety of models of enzyme action have postulated selective excitation of enzyme vibrational modes or triggering of correlated motion of catalytic groups through collisions with solvent particles as the basis of catalytic activity. Solvent composition and structure are expected to influence such interactions. Solutes such as p-dioxane, t-butanol, and tetraalkylammonium chlorides are known to be strong perturbants of the structure of water. However, when the kinetic parameters of two enzymes, carboxypeptidase A and α-chymotrypsin, were examined carefully in aqueous mixtures containing these solutes, no significant influence of solvent structure or mass composition on the catalytic rate constant was found. The results indicate, furthermore, that, within the low viscosity limit, fluctuations in enzyme structure that are responsible for activated processes in the catalytically rate limiting step appear not to be significantly influenced by dynamic processes in the bulk solvent.     

Effects of domain connection and disconnection on the yields of in-plane bimolecular reactions in membranes.

It has recently been shown (Vaz, W.L.C., E.C.C. Melo, and T.E. Thompson. 1989. Biophys. J. 56:869-875; 1990. Biophys. J. 58:273-275) that in lipid bilayer membranes in which ordered and disordered phases coexist, the ordered phase can form a two-dimensional reticular structure that subdivides the coexisting disordered phase into a disconnected domain structure. Here we consider theoretically the yields of bimolecular reactions between membrane-localized reactants, when both the reactants and products are confined to the disordered phase. It is shown that compartmentalization of reactants in disconnected domains can lead to significant reductions in reaction yields. The reduction in yield was calculated for classical bimolecular processes and for enzyme-catalyzed reactions. These ideas can be used to explain certain experimental observations.     

DFT study of the mechanism of manganese quercetin 2,3-dioxygenase: quest for origins of enzyme unique nitroxygenase activity and regioselectivity

Quercetin 2,3-dioxygenase (QDO) is an enzyme which accepts various transition metal ions as cofactors, and cleaves the heterocyclic ring of quercetin with consumption of dioxygen and release of carbon monoxide. QDO from B. subtilis that binds Mn(II) displays an unprecedented nitroxygenase activity, whereby nitroxyl (HNO) is incorporated into quercetin cleavage products instead of dioxygen. Interestingly, the reaction proceeds with high regiospecificity, i.e., nitrogen and oxygen atoms of HNO are incorporated into specific fragments of the cleavage product. A nonenzymatic base-catalyzed reaction, which occurs in pH above 7.5, yields the same reaction products. Herein, we report results of quantum chemical studies on the mechanisms of the nitroxygenase reaction of Mn-QDO. Density functional method with dispersion correction (B3LYP-D3) was applied to the Mn-QDO active site model and the reactants of the nonenzymatic reaction. Co(II)- and Fe(II)-variants of the active site were also considered. Analysis of reaction energy profiles suggests that the regiospecificity of the reaction is an inherent property of the reactants, whereas the unique reactivity of Mn-QDO, as opposed to Co- or Fe-QDO that do not catalyze nitroxygenation, stems from weak HNO binding and lack of strong preference for coordination of HNO through the nitrogen atom. Moreover, the enzyme activates quercetin through deprotonation and the proton acceptor—Glu69 needs to reorient for the reaction to proceed.     

Computer-assisted design for atenolol prodrugs for the use in aqueous formulations

Based on stability studies on the drugs atenolol and propranolol and some of their derivatives it is believed that increasing the lipophilicity of the drug will lead to an increase in the stability of its aqueous solutions and will provide a prodrug system with the potential for releasing atenolol in a controlled manner. Using DFT theoretical calculations we have calculated an intramolecular acid catalyzed hydrolysis in nine maleamic (4-amino-4-oxo-2butenoic) acids (Kirby’s N-alkylmaleamic acids), 1–9. The DFT calculations confirmed that the acid-catalyzed hydrolysis mechanism in these systems involves: (1) a proton transfer from the hydroxyl of the carboxyl group to the adjacent amide carbonyl carbon, (2) an approach of the carboxylate anion toward the protonated amide carbonyl carbon to form a tetrahedral intermediate; and (3) a collapse of the tetrahedral intermediate into products. Furthermore, DFT calculations in different media revealed that the reaction rate-limiting step depends on the reaction medium. In aqueous medium the rate-limiting step is the collapse of the tetrahedral intermediate whereas in the gas phase the formation of the tetrahedral intermediate is the rate-limiting step. Furthermore, the calculations establish that the acid-catalyzed hydrolysis efficiency is largely sensitive to the pattern of substitution on the carbon-carbon double bond. Based on the experimental t_1/2 (the time needed for the conversion of 50% of the reactants to products) and EM (effective molarity) values for processes 1–9 we have calculated the t_1/2 values for the conversion of the two prodrugs to the parental drug, atenolol. The calculated t_1/2 values for ProD 1–2 are predicted to be 65.3 hours and 11.8 minutes, respectively. Thus, the rate by which atenolol prodrug undergoes cleavage to release atenolol can be determined according to the nature of the linker of the prodrug (Kirby’s N-alkylmaleamic acids 1–9).     

The addition reactions of germylenoid H<Subscript>2</Subscript>GeAlCl<Subscript>3</Subscript> with ethylene: a theoretical investigation

Theoretical calculations using the M062X and QCISD methods were performed on the addition reactions of the aluminum germylenoid H₂GeAlCl₃ with ethylene. The most two stable structures of germylenoid H₂GeAlCl₃, i.e., the p-complex and three-membered ring structures, respectively, were employed as reactants. The calculated results indicate that, for the p-complex, H₂GeAlCl₃ there are two pathways, I and II, of which path I involves just one transition state, while path II involves two transition states between reactants and products. Comparing the reaction barrier heights of path I (44.6 kJ mol^?1) and II (37.6 kJ mol^?1), the two pathways are competitive, with similar barriers under the same conditions, while for the three-membered ring structure, another two pathways, III and IV, also exist. Path III has one transition state; however, in path IV, two transition states exist. By comparing their barrier heights, path III (barrier height 39.2 kJ mol^?1) could occur more easily than path IV (barrier height 92.8 kJ mol^?1). Considering solvent effects on these addition reactions, the PCM model and CH₂Cl₂ solvent were used in calculations, and the calculated results demonstrate that CH₂Cl₂ solvent is unfavorable for the reactions, except for path II. In CH₂Cl₂ solvent, paths II and III are more favorable than paths I and IV.     

Enzymatic preparation of food-grade l-α-glycerylphosphorylcholine from soy phosphatidylcholine or fractionated soy lecithin

l -α-Glycerylphosphorylcholine (l -α-GPC) is a biosynthetic precursor for the neurotransmitter acetylcholine in humans, making it a useful as a cognitive enhancer for treating patients with stroke and dementia, including Alzheimer's disease. The aim of this study was to prepare l -α-GPC via Novozym 435 (an immobilized Candida antarctica lipase B)-catalyzed hydrolysis of soy phosphatidylcholine or a fractionated soy lecithin, from which triacylglycerols were completely removed, followed by food-grade solvent extraction of l -α-GPC from the reaction products. The reaction was performed in n-hexane–water biphasic media in a stirred-batch reactor. Phosphatidylcholine was completely hydrolyzed to l -α-GPC under optimal conditions: temperature, 55°C; water content, 100 wt% of the substrate weight; enzyme loading, 10 wt% of the substrate weight; and reaction time of 6 hr (for soy phosphatidylcholine) or 8 hr (for fractionated soy lecithin). Water-soluble fractions of the reaction products containing 98.6 area% l -α-GPC (from soy phosphatidylcholine) or 52.4 area% glycerophosphodiesters, including l -α-GPC (from fractionated soy lecithin), were obtained after phase separation of the media. The resulting products would be suitable for use as food-grade cognitive enhancers because of the use of enzymatic reaction and food-grade solvent extraction.     

Solvent tolerant Pseudomonads as a source of novel lipases for applications in non-aqueous systems

Abstract

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are ubiquitous biocatalysts known to catalyze the hydrolysis of water insoluble triglycerides in aqueous medium and carry out the reverse reaction (synthesis) under organic solvent rich medium. Microbial lipases have received a great deal of attention in the field of food technology, pharmaceutical sciences, chemical and detergent industries due to their stability, selectivity, mild operation conditions and broad substrate specificity. Despite these advantages, low activity and stability displayed in organic medium has restricted their commercial application in organic synthesis. Researchers have explored alternative ways to modify the enzymes making them suitable for use in non-conventional media. In this context, harvesting lipases from “Solvent Tolerant Microbes” has recently become an attractive approach. These microbes are able to grow in the presence of high concentrations of organic solvents, generally known to have detrimental effect on microorganisms. Such microbes survive through novel adaptation mechanisms and secretion of solvent stable enzymes having efficient functionality in solvent-rich media. These enzymes could be useful for bioconversion in non-conventional media. In the current review, this approach is described with an emphasis on characteristics, applications and genetic aspect of lipases from the genus Pseudomonas.     

A kinetic study on the Novozyme 435‐catalyzed esterification of free fatty acids with octanol to produce octyl esters

Octyl esters can serve as an important class of biolubricant components replacing their mineral oil counterparts. The purpose of the current work was to investigate the enzymatic esterification reaction of free fatty acids (FFA, from waste cooking oil) with octanol in a solvent‐free system using a commercial lipase Novozyme 435. It was found that the esterificaton reaction followed the Ping‐pong bi‐bi kinetics with no inhibition by substrates or products within the studied concentration range. The maximum reaction rate was estimated to be 0.041 mol L^?1 g^?1 h^?1. Additionally, the stability of Novozyme 435 in the current reaction system was studied by determining its activity and final conversion of FFA to esters after 12 successive utilizations. Novozyme 435 exhibited almost 100% enzyme activity up to 7 cycles of reaction and gradually decreased (by 5%) thereafter. The kinetic parameters evaluated from the study shall assist in the design of reactors for large‐scale production of octyl esters from a cheap biomass source. The enzyme reusability data can further facilitate mass production by curtailing the cost of expensive enzyme consumption. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1494–1499, 2015