Kinetics and modelling of an alcoholysis reaction catalyzed by cutinase immobilized on NaY zeolite

Fusarium solani pisi recombinant cutinase was immobilized by adsorption on NaY zeolite. The kinetics of the alcoholysis reaction of butyl acetate with hexanol in isooctane catalyzed by cutinase immobilized on NaY zeolite, was studied. The reaction kinetics is suggested to follow a Ping-Pong bi–bi mechanism in which competitive inhibition by excess of alcohols has been identified. No evidence of any significant external diffusional limitation has been detected. The time validation of the model was successfully achieved simultaneously for 15 experimental product evolutions in a batch stirred tank reactor (BSTR) for different initial reactant concentrations.     

Operational stabilities of different chemical derivatives of Novozym 435 in an alcoholysis reaction

Industrial use of Novozym 435 in synthesis of structured lipids and biodiesel via alcoholysis is limited by mass transfer effects of the glycerides through immobilized enzymes and its low operational stability under operation conditions. To better understand this, differently modified Novozym 435 preparations, differing in their surface nature and in their interactions with reactants, have been compared in the alcoholysis of Camelina sativa oil. The three modifications performed have been carried out under conditions where all exposed groups of the enzyme have been modified. These modifications were: 2,4,6-trinitrobenzensulfonic acid (Novo-TNBS), ethylendiamine (Novo-EDA) and polyethylenimine (Novo-PEI). Changes in their operational performance are analyzed in terms of changes detected by scan electron microscopy in the support morphology.The hydrophobic nature of the TNBS accelerates the reaction rate; t-ButOH co-solvent swells the macroporous acrylic particles of Lewatit VP OC 1600 in all biocatalysts, except in the case of Novo-PEI. This co-solvent only increases the maximal conversions obtained at 24 h using the modified biocatalysts. t-ButOH reduces enzyme inactivation by alcohol and water. In a co-solvent system, these four biocatalysts remain fully active after 14 consecutive reaction cycles of 24 h, but only Novo-TNBS yields maximal conversion before cycle 5. Some deposits on biocatalyst particles could be appreciated during reuses, and TNBS derivatization diminishes the accumulation of product deposits on the catalyst surface. Most particles of commercial Novozym^® 435 are broken after operation for 14 reaction cycles. The broken particles are fully active, but they cause problems of blockage in filtration operations and column reactors. The three derivatizations studied make the matrix particles more resistant to rupture.     

Two new dialkoxycarbonylated nucleosides obtained through a regioselective enzymatic alcoholysis

Two new compounds, 2′,3′-di-O-ethoxycarbonyluridine and 2′,3′-di-O-ethoxycarbonylinosine, were obtained through a Candida antarctica lipase B catalysed regioselective ethanolysis of the corresponding trialcoxycarbonylated nucleosides with benzyl alcohol in 1,4-dioxane at 30°C.     

An enzymatic strategy for site-specific immobilization of functional proteins using microbial transglutaminase

A novel strategy for site-specific immobilization of recombinant proteins was investigated using microbial transglutaminase (MTG). Alkaline phosphatase (AP) was selected as a model protein and tagged with a short peptide (MKHKGS) at the N-terminus to provide a reactive Lys residue for MTG. On the other hand, casein, a well-known substrate for MTG, was chemically attached onto a polyacrylic resin to provide reactive Gln residues for the enzymatic immobilization of the recombinant AP. As a result, we succeeded in MTG-mediated functional immobilization of the recombinant AP onto casein-coated polyacrylic resin. It was found that the immobilized AP prepared using MTG exhibited much higher specific activity than that prepared by chemical modification. Moreover, enzymatic immobilization gave an immobilized formulation with higher stability upon repeated use than that obtained by physical adsorption. Use of this ability of MTG in posttranslational protein modification will provide us with a benign, site-specific immobilization method for functional proteins.     

Zeolites as new chromatographic carriers for proteins--easy recovery of proteins adsorbed on zeolites by polyethylene glycol

Zeolites are able to adsorb proteins on their surface and might be suitable as a new type of chromatographic carrier material for proteins and for their conjugates (Matsui et al., Chem. Eur. J. 7 (2001) 1555-1560). Interestingly, maximum adsorption was observed at the isoelectric point (pI) of each protein. The current study was performed to investigate the desorption of proteins from the zeolites at pI. Proteins adsorbed to zeolites could be desorbed at pI by polyethylene glycol (PEG), but not by conventional eluents. The eluted proteins still retained their activities. The zeolite Na-BEA was an especially good composite for desorption by PEG. Using this method for the adsorption and desorption of proteins at pI, we succeeded in separating various proteins. The application of zeolites to biochemistry and biotechnology is also discussed.     

Lipase-catalysed preparation of acyl derivatives of the germacranolide cnicin

Several acyl derivatives of cnicin were obtained through lipase-catalysed acylation and alcoholysis reactions. In most reactions lipases showed a regioselective behaviour affording only one product. Longer chain acyl derivatives were prepared at lower temperature than the used in lipase-catalysed reactions, to preclude side products formation. The enzymatic approach let to prepare a family of novel acetyl and fatty acid derivatives of cnicin which are not obtainable following traditional organic synthetic procedures.     

Effect of water on lipase NS81006-catalyzed alcoholysis for biodiesel production

Biodiesel production catalyzed by free lipase has been drawing attention for its lower cost and faster reaction rate compared to immobilized lipase. It has been found that free lipase NS81006 could efficiently catalyze alkyl esters production and a certain amount of water is demonstrated to be necessary for the catalytic process. The effect of water content on liquid lipase NS81006-mediated methanolysis and ethanolysis for biodiesel production was first explored respectively in this paper. It was found that with water content ranging from 3% to 10% (based on oil weight), there was no significant difference in the final alkyl ester yield either in NS81006-mediated methanolysis or ethanolysis process, while the quality of biodiesel varied obviously. The acid value as well as the contents of monoglyceride and diglyceride were much lower in the lower water-containing system. With the water content decreasing from 10% to 3%, the acid value reduced from 8.24 to 4.89 mg KOH/g oil, and the content of MAG and DAG dropped to 0.31 and 0.22, from 0.62 and 0.74, respectively. Lipase could maintain rather good stability with proper alcohol adding strategy and the gradual reduction in biodiesel yield in the repeated uses resulted from the accumulation of by-product glycerol. The continuous running of lipase-mediated methanolysis of waste cooking oil was successfully realized at 30L reactor and a final methyl ester yield of over 90% could be obtained.     

Cofactor regeneration for sustainable enzymatic biosynthesis

Oxidoreductases are attractive catalysts for biosynthesis of chiral compounds and polymers, construction of biosensors, and degradation of environmental pollutants. Their practical applications, however, can be quite challenging since they often require cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These cofactors are generally expensive. Efficient regeneration of cofactors is therefore critical to the economic viability of industrial-scale biotransformations using oxidoreductases. The chemistry of cofactor regeneration is well known nowadays. The challenge is mostly regarding how to achieve the regeneration with immobilized enzyme systems which are preferred for industrial processes to facilitate the recovery and continuous use of the catalysts. This has become a great hurdle for the industrialization of many promising enzymatic processes, and as a result, most of the biotransformations involving cofactors have been traditionally performed with living cells in industry. Accompanying the rapidly growing interest in industrial biotechnology, immobilized enzyme biocatalyst systems with cofactor regeneration have been the focus for many studies reported since the late 1990s. The current paper reviews the methods of cofactor retention for development of sustainable and regenerative biocatalysts as revealed in these recent studies, with the intent to complement other reviewing articles that are mostly regeneration chemistry-oriented. We classify in this paper the methods of sustainable cofactor regeneration into two categories, namely membrane entrapment and solid-attachment of cofactors.     

Strategies of enzyme immobilization on nanomatrix supports and their intracellular delivery

Abstract

Enzymes are one of the foundations and regulators for all major biological activities in living bodies. Hence, enormous efforts have been made for enhancing the efficiency of enzymes under different conditions. The use of nanomaterials as novel carriers for enzyme delivery and regulating the activities of enzymes has stimulated significant interests in the field of nano-biotechnology for biomedical applications. Since, all types of nanoparticles (NPs) offer large surface to volume ratios, the use of NPs as enzyme carriers affect the structure, performance, loading efficiency, and the reaction kinetics of enzymes. Hence, the immobilization of enzymes on nanomatrices can be used as a useful approach for direct delivery of therapeutic enzymes to the targeted sites. In other words, NPs can be used as advanced enzyme delivery nanocarriers. In this paper, we present an overview of different binding of enzymes to the nanomaterials as well as different types of nanomatrix supports for immobilization of enzymes. Afterwards, the enzyme immobilization on nanomaterials as a potential system for enzyme delivery has been discussed. Finally, the challenges associated with the enzyme delivery using nano matrices and their future perspective have been discussed.

Communicated by Ramasamy H. Sarma     

Error thresholds for self- and cross-specific enzymatic replication

The information content of a non-enzymatic self-replicator is limited by Eigen's error threshold. Presumably, enzymatic replication can maintain higher complexity, but in a competitive environment such a replicator is faced with two problems related to its twofold role as enzyme and substrate: as enzyme, it should replicate itself rather than wastefully copy non-functional substrates, and as substrate it should preferably be replicated by superior enzymes instead of less-efficient mutants. Because specific recognition can enforce these propensities, we thoroughly analyze an idealized quasispecies model for enzymatic replication, with replication rates that are either a decreasing (self-specific) or increasing (cross-specific) function of the Hamming distance between the recognition or “tag” sequences of enzyme and substrate. We find that very weak self-specificity suffices to localize a population about a master sequence and thus to preserve its information, while simultaneous localization about complementary sequences in the cross-specific case is more challenging. A surprising result is that stronger specificity constraints allow longer recognition sequences, because the populations are better localized. Extrapolating from experimental data, we obtain rough quantitative estimates for the maximal length of the recognition or tag sequence that can be used to reliably discriminate appropriate and infeasible enzymes and substrates, respectively.     

Enzyme immobilization: an update

Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immo-bilized enzyme systems allows an easy recovery of both enzymes and products, multiple re-use of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This paper is a review of the recent literatures on enzyme immobilization by various techniques, the need for immobilization and different applications in industry, covering the last two decades. The most recent papers, patents, and reviews on immobilization strategies and application are reviewed.     

Concentration of docosahexaenoic and eicosapentaenoic acids by enzymatic alcoholysis with different acyl-acceptors

The aim of this work was to produce docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) enriched acylglycerols by alcoholysis of tuna and sardine oils, respectively, using isobutanol and 1-butanol as acyl-acceptors. The alcoholysis reactions were catalyzed by lipases Lipozyme® TL IM from Thermomyces lanuginosus and lipase QLG® from Alcaligenes sp., because these lipases have shown selectivity towards DHA and EPA, respectively. Studies were made to determine the influence of reaction time, alcohol/oil molar ratio, lipase amount and temperature. In the optimized conditions for the alcoholysis of tuna and sardine oils catalyzed by Lipozyme TL IM and lipase QLG, respectively, the DHA and EPA contents were trebled (from 22 to 69% for DHA, and from 19 to 61% for EPA). The stability of both lipases was also determined. Although Lipozyme TL IM is much more stable in isobutanol than in ethanol, with the former the conversion attained after four reaction cycles was about 40% of the initial conversion. In similar conditions, the conversion obtained with lipase QLG was about 88% of the initial conversion. In addition, the separation of DHA enriched acylglycerols and isobutyl esters from an alcoholysis reaction was studied by liquid–liquid fractionation using the ethanol–water–hexane biphasic system. The DHA enriched acylglycerols obtained were 97.6% pure (64.4% DHA).     

Improvement of enzymatic performance of Asclepias curassavica L. proteases by immobilization. Application to the synthesis of an antihypertensive peptide

The aim of this work was to study different immobilization strategies on silica supports in order to obtain robust biocatalysts from latex proteases of Asclepias curassavica L., a South American native plant. Immobilized enzyme performance was evaluated under harsh reaction conditions such as the synthesis of the antihypertensive peptide N-α-CBZ-Val-Gly-OH.Proteases from A. curassavica, named asclepain, were immobilized (0.51–5.56 mg of protein/ g of support) in non-functionalized silica (S), in glyoxyl-silica (GS) and in octyl-glyoxyl-silica (OGS), by adsorption, and multi-point covalent attachment on mono and hetero-functional supports, respectively, under previously determined optimal immobilization conditions. Immobilization yields were expressed as activity yield (Y_a) and protein yield (Y_p).Asclepain-OGS showed the highest Y_a (178 ± 1.62 %) meaning an expressed activity 1.8 times higher than the offered activity, while Y_p was 75 ± 0.4 %. Y_a for asclepain-S and -GS were 64 ± 1.45 % and 16 ± 0.37 %, respectively. Best results were attributed to the ability of OGS support to guide the enzyme before covalent attachment, increasing its reactivity. Asclepain-OGS led to product yield of 95.5 ± 0.14 %, five times higher than soluble asclepain in the synthesis of N-α-CBZ-Val-Gly-OH, after 3 h in 30 % methanol in 0.1 M Tris-HCl buffer pH 8.     

Electrochemical investigation of immobilized hemoglobin: redox chemistry and enzymatic catalysis

Hb entrapped in the Konjak glucomannan (KGM) film could transfer electrons directly to an edge-plane pyrolytic graphite (EPG) electrode, corresponding to the redox couple of Fe(III)/Fe(II). The redox properties of Hb, such as formal potential, electron transfer rate constant, the stability of the redox state of protein and redox Bohr effect, were characterized by cyclic voltammetry and square wave voltammetry. The stable Hb-KGM/EPG gave analytically useful electrochemical catalytic responses to oxygen, hydrogen peroxide and nitrite.     

Isolation of Rat Pituitary Prolactin Isohormones Differing in Charge,Size, and Specific Immunological Activity

A method has been described for the isolation of three differently charged isohormones of rat prolactin from a discard fraction obtained after extraction of gonadotropins, thyrotropin and growth hormone from homogenized frozen pituitaries. The procedure involved extraction at pH 9.8, ammonium sulphate fractionation, molecular sieve chromatography on Sephadex G-100, and column electrophoresis in agarose suspension. The purification was monitored by radioimnunoassays and the recovered components were all found to possess a specific immunoactivity exceeding that of the standard preparation (RP-1) supplied by the NIAMDD, Bethesda, U.S.A. Increased acidity among these isohormones was found to be paralleled by significantly decreased immunopotency. Each component showed biological activity in radioreceptor assay.

A high degree of purity of the isolated components was shown by analytical electrophoresis in polyacrylamide gel. Sodium dodecyl sulphate electrophoresis in the same medium showed no size heterogeneity and yielded a value of approximately 25 000 for the molecular weight of the isohormones.

In addition a large form of prolactin, suggested to represent a dimer, was isolated by a further extraction step (pH 10.5) followed by molecular sieve chromatography on Sephadex G-100 and electrophoresis in agarose suspension. The large form was associated with both biopotency and immuno-potency. The electrophoresis resolved the prolactin activity into three or four immunoactive components. This pleomorphism of the large prolactin was confirmed by analytical polyacrylamide gel electrophoresis.

Amino acid analyses revealed a close similarity between the three monomers and the major dimeric form of the hormone.     

Economic comparison of enzymatic reactors and advanced oxidation processes applied to the degradation of phenol as a model compound

Abstract

Some micropollutants present in wastewaters are barely removed in sewage treatment plants. In many cases a post-treatment process based on separation and/or oxidation has to be applied. The aim of this study was the technical and economic comparison of enzymatic technologies with other advanced oxidation processes (AOPs) for the degradation of phenol. Batch and continuous enzymatic reactors, using free and immobilized manganese peroxidase (MnP, EC 1.11.1.13), were considered. Continuous degradation of phenol in an enzymatic membrane reactor was shown to be the fastest process and degradation in a continuous reactor with immobilized enzyme involved the lowest consumption of enzyme. However, the immobilization process increased the enzyme cost 100-fold. A continuous enzymatic membrane reactor gave high degradation efficiency and may be a viable technology for phenol removal when compared with other AOPs from both technical and economic points of view.     

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports

In this work, we have used supports activated with m-amino-phenylboronic groups to “reversibly” immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.     

Effects of pressure and temperature on enzymatic reactions in supercritical fluids

Supercritical fluids (SCFs) are receiving increasing attention as reaction media because they permit higher reaction rates compared with the conventional solvents. The ease of manipulating the physical properties of the SCFs enables easier control of the reaction conditions and easier solvent removal after the reaction. This review focuses on effects of pressure, temperature and the properties of the SCFs, on enzymatic reactions. Phase behavior, reaction rate and activation volume in SCFs are discussed. Within the ranges of pressure (10-40 MPa) and temperature (35-60 degrees C) that typically characterize the supercritical region, an increase in pressure and/or a decrease in temperature lead to a decrease in the enzyme turnover because the diffusion coefficients of the substrates migrating to the active sites of enzymes are affected.     

The importance of the amide bond nearest the thiol group in enzymatic reactions of coenzyme A

Analogues of coenzyme A (CoA) and of CoA thioesters have been prepared in which the amide bond nearest the thiol group has been modified. An analogue of acetyl-CoA in which this amide bond is replaced with an ester linkage was a good substrate for the enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with K(m) values 2- to 8-fold higher than those of acetyl-CoA and V(max) values from 14 to >80% those of the natural substrate. An analogue in which an extra methylene group was inserted between the amide bond and the thiol group showed less than 4-fold diminished binding to the three enzymes but exhibited less than 1% activity relative to acetyl-CoA with carnitine acetyltransferase and no measurable activity with the other two enzymes. Analogues of several CoA thioesters in which the amide bond was replaced with a hemithioacetal linkage exhibited no measurable activity with the appropriate enzymes. The results indicate that some aspects of the amide bond and proper distance between this amide and the thiol/thioester moiety are critical for activity of CoA ester-utilizing enzymes.     

Multi-faceted strategy based on enzyme immobilization with reactant adsorption and membrane technology for biocatalytic removal of pollutants: A critical review

In the modern era, the use of sustainable, environmentally friendly alternatives for removal of recalcitrant pollutants in streams resulting from industrial processes is of key importance. In this context, biodegradation of phenolic compounds, pharmaceuticals and dyes in wastewater by using oxidoreductases offers numerous benefits. Tremendous research efforts have been made to develop novel, hybrid strategies for simultaneous immobilization of oxidoreductase and removal of toxic compounds. The use of support materials with the options for combining enzyme immobilization with adsorption technology focused on phenolic pollutants and products of biocatalytic conversion seems to be of particular interest. Application of enzymatic reactors based on immobilized oxidoreductases for coupling enzyme-aided degradation and membrane separation also attract still growing attention. However, prior selection of the most suitable support/sorbent material and/or membrane as well as operational mode and immobilization technique is required in order to achieve high removal efficiency. Thus, in the framework of this review, we present an overview of the impact of support/sorbent material on the catalytic properties of immobilized enzymes and sorption of pollutants as well as parameters of membranes for effective bioconversion and separation. Finally, future perspectives of the use of processes combining enzyme immobilization and sorption technology as well as application of enzymatic reactors for removal of environmental pollutants are discussed.