Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6 h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40-60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6?h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40–60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

Biocatalytic polyester acrylation--process optimization and enzyme stability

An OH-functional polyester has been acrylated via transesterification of ethyl acrylate, catalyzed by Candida antarctica lipase B (CalB) in two different preparations: Novozym 435 and immobilized on Accurel MP1000. The batch process resulted in incomplete acrylation as well as severe degradation of the polyester. A high degree of acrylation was achieved by optimization through the application of low pressure (15 kPa), continuous inflow of ethyl acrylate and continuous distillation to evaporate the by-product, ethanol. The enzyme preparations displayed good stability with half-lives of 180 and 324 h for Novozym 435 and CalB/MP1000, respectively. This translates into product yields of 3600 and 6200 times the weight of the catalyst, indicating that the enzyme will have a marginal impact on the total process cost.     

提高微生物脂肪酶产量、活性和稳定性的方法研究

微生物脂肪酶是一类广泛应用于诸多工业领域的生物催化剂。提高微生物脂肪酶的产量、活性和稳定性,增强产品的市场竞争力,一直是微生物脂肪酶研究的重点和热点。本文从产脂肪酶菌株的改造、脂肪酶基因的改良、脂肪酶发酵工程和脂肪酶后期处理等四个方面概述了提高微生物脂肪酶产量、活性和稳定性的方法,以期为微生物脂肪酶的规模化工业生产提供方法性指导。     

Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg^-1 h^-1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.     

Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg^?1 h^?1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.     

Enzymatic hydrolysis of lignocellulosic materials: II. Experimental investigations of theoretical hydrolysis--process models for an increased enzyme recovery

Stream pretreatment of wheat straw solubilized most of the xylan present. Xylose and other sugars were recovered by washing the substrate with water but only a minor part (34%) was monomeric. Treatment of this solutions with celulases and hemicellulases improved the yield of monomeric sugars to 69%, the main product being xylose. Some xylose was also obtained during enzymatic hydrolysis of the solid substrate although the pretreatment step contributed 64% (mean value) of total xylose formed. A reference model, No. 1, and two other models, Nos. 2 and 4, described in the first part of this article series (this issue) have been studied experimentally and results confirm the theoretical conclusions. An uninterrupted hydrolysis over a given time period leads to a lower degree of saccharification than when hydrolysate is withdrawn several times. Saccharification is also favored if the residue is removed at a late stage, i.e., at the end of the 24 h hydrolysis cycle. Extended recirculation of the enzymes during a 4 x 24-h experimental period gave the following average yields of saccharification on a 24-h basis: 65% (Reference), 73% (Model 2), and 79% (Model 4). It is concluded that enzyme recovery with model 4 is 70% or more, while the Reference and Model 2 attain a lower level of recovery. The design of an improved hydrolysis model is also discussed.     

Application of iterative robust model‐based optimal experimental design for the calibration of biocatalytic models

The aim of model calibration is to estimate unique parameter values from available experimental data, here applied to a biocatalytic process. The traditional approach of first gathering data followed by performing a model calibration is inefficient, since the information gathered during experimentation is not actively used to optimize the experimental design. By applying an iterative robust model‐based optimal experimental design, the limited amount of data collected is used to design additional informative experiments. The algorithm is used here to calibrate the initial reaction rate of an ω‐transaminase catalyzed reaction in a more accurate way. The parameter confidence region estimated from the Fisher Information Matrix is compared with the likelihood confidence region, which is not only more accurate but also a computationally more expensive method. As a result, an important deviation between both approaches is found, confirming that linearization methods should be applied with care for nonlinear models. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1278–1293, 2017     

Unexpected impact of esterification on the antioxidant activity and (photo)stability of a eumelanin from 5,6‐dihydroxyindole‐2‐carboxylic acid

To inquire into the role of the carboxyl group as determinant of the properties of 5,6‐dihydroxyindole melanins, melanins from aerial oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) and its DHICA methyl ester (MeDHICA) were comparatively tested for their antioxidant activity. MALDI MS spectrometry analysis of MeDHICA melanin provided evidence for a collection of intact oligomers. EPR analysis showed g‐values almost identical and signal amplitudes (ΔB) comparable to those of DHICA melanin, but spin density was one order of magnitude higher, with a different response to pH changes. Antioxidant assays were performed, and a model of lipid peroxidation was used to compare the protective effects of the melanins. In all cases, MeDHICA melanin performed better than DHICA melanin. This capacity was substantially maintained following exposure to air in aqueous buffer over 1 week or to solar simulator over 3 hr. Different from DHICA melanin, MeDHICA melanin was proved to be fairly soluble in different water‐miscible organic solvents, suggesting its use in dermocosmetic applications.     

Bioreactor productivity and media cost comparison for different intensified cell culture processes

Process intensification in biomanufacturing has attracted a great deal of interest in recent years. Manufacturing platform improvements leading to higher cell density and bioreactor productivity have been pursued. Here we evaluated a variety of intensified mammalian cell culture processes for producing monoclonal antibodies. Cell culture operational modes including fed‐batch (normal seeding density or high seeding density with N‐1 perfusion), perfusion, and concentrated fed‐batch (CFB) were assessed using the same media set with the same Chinese Hamster Ovary (CHO) cell line. Limited media modification was done to quickly fit the media set to different operational modes. Perfusion and CFB processes were developed using an alternating tangential flow filtration device. Independent of the operational modes, comparable cell specific productivity (fed‐batch: 29.4 pg/cell/day; fed‐batch with N‐1 perfusion: 32.0 pg/cell/day; perfusion: 31.0 pg/cell/day; CFB: 20.1 – 45.1 pg/cell/day) was reached with similar media conditions. Continuous media exchange enabled much higher bioreactor productivity in the perfusion (up to 2.29 g/L/day) and CFB processes (up to 2.04 g/L/day), compared with that in the fed‐batch processes (ranging from 0.39 to 0.49 g/L/day), largely due to the higher cell density maintained. Furthermore, media cost per gram of antibody produced from perfusion was found to be highly comparable with that from fed‐batch; and the media cost for CFB was the highest due to the short batch duration. Our experimental data supports the argument that media cost for perfusion process could be even lower than that in a fed‐batch process, as long as sufficient bioreactor productivity is achieved. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:867–878, 2017     

Generating favorable nano-environments for thermal and solvent stabilization of immobilized beta-galactosidase.

beta-Galactosidase (Escherichia coli) was immobilized through its thiol groups on thiolsulfinate-agarose gel. After enzyme immobilization, different nano-environments were generated by reacting the excess of gel-bound thiolsulfinate moieties with 2-mercaptoethanesulfonic acid (S-gel), glutathione (G-gel), cysteamine (C-gel), and mercaptoethanol (M-gel). Concerning thermal stability at 50 degrees C, the G-gel and the M-gel derivatives were the most stable with residual activity values of 67% and 45%, respectively. The stability in several solvent systems was studied: ethyl acetate (1.6% vol/vol), ethylene glycol (50% vol/vol), and 2-propanol (50% vol/vol). In ethyl acetate, both the M-gel and S-gel were highly stabilized; the time required for activity to decay to 80% of the initial activity was increased 29-fold for the M-gel and 20-fold for the S-gel with respect to the soluble enzyme. The G-gel was the least stable of all the derivatives. The different behaviors of the derivatives in thermal and solvent stability studies suggest that each nano-environment contributes differently to the enzyme stability, depending on the denaturing conditions. Therefore, it may be possible to tailor the matrix surface to maximize enzyme stability in particular applications.     

Novel Magnetic Cross‐Linked Lipase Aggregates for Improving the Resolution of (R,S)‐2‐octanol

Novel magnetic cross‐linked lipase aggregates were fabricated by immobilizing the cross‐linked lipase aggregates onto magnetic particles with a high number of ‐NH₂ terminal groups using p‐benzoquinone as the cross‐linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross‐linked lipase aggregates were achieved. The magnetic cross‐linked lipase aggregates were able to efficiently resolve (R, S)‐2‐octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross‐linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross‐linking. These results provide a great potential for industrial applications of the magnetic cross‐linked lipase aggregates. Chirality 27:199–204, 2015. © 2014 Wiley Periodicals, Inc.     

A two-step enzymatic resolution process for large-scale production of (S)- and (R)-ethyl-3-hydroxybutyrate

An efficient two-step enzymatic process for production of (R)- and (S)-ethyl-3-hydroxybutyrate (HEB), two important chiral intermediates for the pharmaceutical market, was developed and scaled-up to a multikilogram scale. Both enantiomers were obtained at 99% chemical purity and over 96% enantiomeric excess, with a total process yield of 73%. The first reaction involved a solvent-free acetylation of racemic HEB with vinylacetate for the production of (S)-HEB. In the second reaction, (R)-enriched ethyl-3-acetoxybutyrate (AEB) was subjected to alcoholysis with ethanol to derive optically pure (R)-HEB. Immobilized Candida antarctica lipase B (CALB) was employed in both stages, with high productivity and selectivity. The type of butyric acid ester influenced the enantioselectivity of the enzyme. Thus, extending the ester alkyl chain from ethyl to octyl resulted in a decrease in enantiomeric excess, whereas using bulky groups such as benzyl or t-butyl, improved the enantioselectivity of the enzyme. A stirred reactor was found unsuitable for large-scale production due to attrition of the enzyme particles and, therefore, a batchwise loop reactor system was used for bench-scale production. The immobilized enzyme was confined to a column and the reactants were circulated through the enzyme bed until the targeted conversion was reached. The desired products were separated from the reaction mixture in each of the two stages by fractional distillation. The main features of the process are the exclusion of solvent (thus ensuring high process throughput), and the use of the same enzyme for both the acetylation and the alcoholysis steps. Kilogram quantities of (S)-HEB and (R)-HEB were effectively prepared using this unit, which can be easily scaled-up to produce industrial quantities.     

A novel approach for enhancing the catalytic efficiency of a protease at low temperature: Reduction in substrate inhibition by chemical modification

The alkaline protease, savinase was chemically modified to enhance the productivity of the enzyme at low temperatures on a complex polymeric protein (azocasein) substrate. At 5 and 15°C, savinase modified with ficol or dextran hydrolyzed fivefold more azocasein than the unmodified savinase. Kinetic studies showed that the catalytic improvements are associated with changes in uncompetitive substrate inhibition with K_i values of modified savinases sixfold higher than the unmodified savinase. Modeling of small‐angle scattering data indicates that two substrate molecules bind on opposing sides of the enzyme. The combined kinetic and structural data indicate that the polysaccharide modifier sterically blocks the allosteric site and reduces substrate inhibition. In contrast to the properties of cold‐active enzymes that generally manifest as low activation enthalpy and high flexibility, this study shows that increased activity and productivity at low temperature can be achieved by reducing uncompetitive substrate inhibition, and that this can be achieved using chemical modification with an enzyme in a commercial enzyme‐formulation. Biotechnol. Bioeng. 2009;103: 676–686. © 2009 Wiley Periodicals, Inc.     

A pseudo steady‐state model for galacto‐oligosaccharides synthesis with β‐galactosidase from Aspergillus oryzae

A pseudo steady‐state model for the kinetically controlled synthesis of galacto‐oligosaccharides (GOS) with Aspergillus oryzae β‐galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta‐oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl–enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl–enzyme complex is higher than that of the free enzyme. Biotechnol. Bioeng. 2011;108: 2270–2279. © 2011 Wiley Periodicals, Inc.     

A monolithic lipase reactor for biodiesel production by transesterification of triacylglycerides into fatty acid methyl esters

An enzymatic reactor with lipase immobilized on a monolithic polymer support has been prepared and used to catalyze the transesterification of triacylglycerides into the fatty acid methyl esters commonly used for biodiesel. A design of experiments procedure was used to optimize the monolithic reactor with variables including control of the surface polarity of the monolith via variations in the length of the hydrocarbon chain in alkyl methacrylate monomer, time of grafting of 1-vinyl-4,4-dimethylazlactone used to activate the monolith, and time used for the immobilization of porcine lipase. Optimal conditions involved the use of a poly(stearyl methacrylate-co-ethylene dimethacrylate) monolith, grafted first with vinylazlactone, then treated with lipase for 2 h to carry out the immobilization of the enzyme. Best conditions for the transesterification of glyceryl tributyrate included a temperature of 37°C and a 10 min residence time of the substrate in the bioreactor. The reactor did not lose its activity even after pumping through it a solution of substrate equaling 1,000 reactor volumes. This enzymatic reactor was also used for the transesterification of triacylglycerides from soybean oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.