Biocatalysis of immobilized chlorophyllase in a ternary micellar system.

The immobilization of chlorophyllase was optimized by physical adsorption on various inorganic supports, including alumina, celite, Dowex-1-chloride, glass beads and silica gel. The enzyme was also immobilized in different media, including water, Tris-HCl buffer solution and a ternary micellar system containing Tris-HCl buffer solution, hexane and surfactant. The highest immobilization efficiency (84.56%) and specific activity (0.34 mumol hydrolyzed chlorophyll mg protein-1 per min) were obtained when chlorophyllase was suspended in Tris-HCl buffer solution and adsorbed onto silica gel. The effect of different ratios of chlorophyllase to the support and the optimum incubation time for the immobilization of chlorophyllase were determined to be 1-4 and 60 min, respectively. The experimental results showed that the optimum pH and temperature for the immobilized chlorophyllase were 8.0 and 35 degrees C, respectively. The use of optimized amounts of selected membrane lipids increased the specific activity of the immobilized chlorophyllase by approximately 50%. The enzyme kinetic studies indicated that the immobilized chlorophyllase showed a higher affinity towards chlorophyll than pheophytin as substrate.     

Adsorption of trypsin on commercial silica gel

The immobilization of trypsin onto various commercial silica gels was studied. Silica gels were used directly and characterized by mercuric porosimetry. Agitation rates (100–740 rpm) and particles size (35–75 to 250–500 μm) of silica gels did not affect the trypsin immobilization capacity. The pore size (3 to 15 nm) is a limiting factor of the trypsin adsorption onto the mesopores structure of silica gels. The adsorption of trypsin was determined as a function of their initial concentration and multilayer formed at high trypsin concentration.     

Immobilization of a lipoxygenase in silica gels for application in aqueous media

The encapsulation of soybean lipoxygenase-1 (LOX-1) in silica gels and its application in an aqueous medium, were studied. The main silica precursor was tetramethoxysilane (TMOS) but the introduction of hydrophobic SiCH₃ groups brought with methyltrimethoxysilane (MTMS) was evaluated. Other sol–gel synthesis parameters investigated comprised partial or complete drying by evaporation and CO₂ supercritical drying. The influence on LOX-1 activity of the various chemicals with which the enzyme was in contact during encapsulation (acetone, methanol, polyvinyl alcohol), as well as the temperature and pH, were examined. The activity of free and encapsulated LOX-1 was assayed on the oxygenation reaction of linoleic acid by dioxygen from air dissolved in aqueous medium, in a UV–vis spectrophotometer. With free LOX-1, the reaction advancement could be followed in continuous in the spectrophotometer. With the gels, in a first approach, the conversion was simply determined after 15 min reaction after filtration of the liquid, to discriminate between active and inactive gels. For the most interesting gels, the kinetics were then assessed by continuous recording in the UV spectrophotometer, after placing a small piece of gel (≈15 mg) directly in the cell. The best gels had an activity ≈30% of free LOX. The present studies, supplemented by characterization of the gels texture and structure, respectively by nitrogen adsorption and ²⁹Si MAS NMR, showed that drying a gel before use in aqueous media was detrimental to the activity. This effect is due to a contraction of the gel network which occurs when a dry aerogel sample is dipped in water after drying. Hence gels containing LOX-1 enzyme must not be dried but kept in water impregnated state, for optimum use.     

Biocatalysis of lipoxygenase in selected organic solvent media

The biocatalysis of purified soybean lipoxygenase (LOX) (EC 1.13.11.12), using linoleic acid as a substrate model, was investigated in selected organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene. The results indicated that there was a 2.6-fold increase in LOX activity in the monophasic iso-octane medium compared to that obtained in the aqueous medium. The results also showed that there was an increase of 2.2- and 1.8-fold in LOX activity in the monophasic reaction media of octane and hexane, respectively. However, an inhibitory effect on enzyme activity was observed when the monophasic reaction media of toluene, chloroform and dichloromethane were used. In addition, the results showed that the optimum concentration of octane and iso-octane in the biphasic medium containing the organic solvent and Tris–HCl buffer solution, was determined to be 3.5% and 4%, respectively, for LOX activity. Moreover, the biocatalysis of LOX in a ternary micellar system, containing either 3.5% octane or 4% iso-octane, Tris–HCl buffer solution and an emulsifier, resulted in an overall increase in enzyme activity. The K_m and V_max values, substrate specificity, optimum protein concentration, optimum reaction temperature as well as the enzymatically catalyzed end-products were investigated for LOX biocatalysis in both ternary micellar systems.     

Application of a lipase-immobilized silica monolith bioreactor to the production of fatty acid methyl esters

We developed a highly efficient bioreactor loaded with a lipase-immobilized non-shrinkable silica monolith by adopting a two-step sol–gel method, i.e., preparing a methyltrimethoxysilane (MTMS)-based silica monolith followed by coating of the latter with more hydrophobic alkyl-substituted silicates that entrapped lipase. We applied this type of bioreactor to the production of fatty acid methyl esters through methanolysis of rapeseed oil in n-hexane by Rhizopus oryzae lipase. As the result of screening alkyltrimethoxysilanes mixed with tetramethoxysilane (TMOS) during sol–gel coating, propyltrimethoxysilane (PTMS) gave the best performance, and the lipase immobilized therein exhibited ca. 10 times higher activity than non-immobilized lipase powder. The amount of the PTMS-based silicates with which the MTMS-based silica monolith was coated was optimized by adjusting the molar ratio of silanes (mixture of PTMS and TMOS at 4:1) to 100 mol of water. Conversion rate was highest at the molar ratio of 0.4 mol silanes to 100 mol of water, which was ca. 10 times higher than that with lipase deposited on the MTMS-based silica monolith. Conversion rate was approximately 1.5 times higher in the flow-through monolith bioreactor than in the batch slurry reactor.     

Application of silica aerogel encapsulated lipases in the synthesis of biodiesel by transesterification reactions

Two types of commercial lipases preparations, one from Burkholderia cepacia, the other one from Candida antartica, were encapsulated in silica aerogels reinforced with silica quartz fibre felt and dried by the CO₂ supercritical technique. These immobilized biocatalysts were applied in biodiesel synthesis by transesterification of sunflower seed oil with methyl acetate. They were found to be efficient even with mixtures of both substrates without any solvent addition. The aerogel encapsulation technique made it possible to maintain the enzymes in a dispersion state similar to the dispersion prevailing in an aqueous solution, even for further use in organic hydrophobic media. In transesterification in excess iso-octane, the two lipases encapsulated in aerogels made from 40% MTMS, were found to have activities relatively close to each other and comparable with commercial Novozyme 435. On the other in transesterification with mixture of oil and methyl acetate without any solvent, the kinetics were severely limited by substrate diffusion inside the aerogels. This was particularly true with the C. antartica, so that the corresponding aerogel encapsulated enzyme was much less active than commercial Novozyme 435, although it improved after a few tests.     

Biocatalysis in Spain: A field of success and innovation

Abstract

This is the presentation of the Special Issue “Biocatalysis in Spain”, covering the work of several Spanish groups in the field of Biocatalysis and Biotransformations. Thus, both research articles and reviews allow one to draw an accurate view of the state-of-the-art development in Spain.     

Improvement of enzymatic biodiesel production by controlled substrate feeding using silica gel in solvent free system

A silica gel-based substrate feeding system was developed to prevent methanol inhibiting the catalyst during enzymatic biodiesel synthesis. In the system, silica gel swelled upon methanol addition and subsequently released it in a controlled manner to prevent excess methanol affecting the enzyme. Biodiesel was synthesized by the enzymatic transesterification of canola oil with methanol. For this reaction, enzyme loading, methanol/oil molar ratio, silica gel dosage, glycerol content, and methanol feeding method were tested using commercial immobilized enzymes (Novozym 435 and Lipozyme RM IM from Novozymes). The results showed that conversion was highest with controlled substrate feeding rather than direct methanol addition, suggesting that the method developed here can easily prevent enzyme inhibition by limiting methanol concentration to an acceptable level.     

Immobilization of Candida cylindracea lipase on poly lactic acid,polyvinyl alcohol and chitosan based ternary blend film: Characterization,activity, stability and its application for N-acylation reactions

The ecofriendly ternary blend polymer film was prepared from the chitosan (CH), polylactic acid (PLA) and polyvinyl alcohol (PVA). Immobilization of Candida cylindracea lipase (CCL) was carried out on ternary blend polymer via entrapment methodology. The ternary blend polymer and immobilized biocatalyst were characterized by using N₂ adsorption–desorption isotherm, SEM, FTIR, DSC, and (%) water content analysis through Karl Fischer technique. Biocatalyst was then subjected for the determination of practical immobilization yield, protein loading and specific activity. Immobilized biocatalyst was further applied for the determination of biocatalytic activity for N-acylation reactions. Various reaction parameters were studied such as effect of immobilization support (ratio of PLA:PVA:CH), molar ratio (dibutylamine:vinyl acetate), solvent, biocatalyst loading, time, temperature, and orbital speed rotation. The developed protocol was then applied for the N-acylation reactions to synthesize several industrially important acetamides with excellent yields. Interestingly, immobilized lipase showed fivefold higher catalytic activity and better thermal stability than the crude extract lipase CCL. Furthermore various kinetic and thermodynamic parameters were studied and the biocatalyst was efficiently recycled for four successive reuses. It is noteworthy to mention that immobilized biocatalyst was stable for period of 300 days.     

Influence of the sol–gel chemistry on the activity of a lipase encapsulated in a silica aerogel

Silica aerogels have been shown to be efficient encapsulation media for the lipase of Burkholderia cepacia. The present study has focused on the encapsulation of this lipase in an aerogel made from 80% tetramethoxysilane (TMOS) and 20% methyltrimethoxylsilane (MTMS), dried by the supercritical CO₂ method. By varying different parameters in the synthesis chemistry of such materials, the structure and texture of the resulting gels can be significantly affected. The aim of the present study was to examine the possible existence of correlations between modifications of the gel’s synthesis procedure, and the catalytic activity of the gel-encapsulated lipase in the esterification reaction of lauric acid with 1-octanol. The synthesis parameters studied included aging of the wet gels in different solvents, variation of the solvent used during gel synthesis, variation in the molar ratio of hydrolysis water to silicon precursor and replacement of MTMS by another alkoxide. The biocatalytic activity was found to depend significantly on these different treatments. The results were analyzed in the light of the gel texture and structure characterization, respectively analyzed by nitrogen adsorption isotherms and ²⁹Si, ¹H and ¹³C NMR. These results suggest that the main role of the aerogel is to maintain the enzyme dispersed as it would be in an aqueous solution, even though it is used in an organic solvent where the lyophilized enzyme is insoluble. The nature of the gel surface groups, in particular their capability to modify the substrates concentration inside the gel, close to the active site of the enzyme, by comparison with the concentration in the solvent outside the gel, seem to have a more secondary effect.     

Evaluation of biodiesel production using lipase immobilized on magnetic silica nanocomposite particles of various structures

Nonporous and mesoporous silica-coated magnetite cluster nanocomposites particles were fabricated with various silica structures in order to develop a desired carrier for the lipase immobilization and subsequent biodiesel production. Lipase from Pseudomonas cepacia was covalently bound to the amino-functionalized particles using glutaraldehyde as a coupling agent. The hybrid systems that were obtained exhibited high stability and easy recovery regardless of the silica structure, following the application of an external magnetic field. The immobilized lipases were then used as the recoverable biocatalyst in a transesterification reaction to convert the soybean oil to biodiesel with methanol. Enzyme immobilization led to higher stabilities and conversion values as compared to what was obtained by the free enzyme. Furthermore, the silica structure had a significant effect on stability and catalytic performance of immobilized enzymes. In examining the reusability of the biocatalysts, the immobilized lipases still retained approximately 55% of their initial conversion capability following 5 times of reuse.     

Biocatalysis of tyrosinase using catechin as substrate in selected organic solvent media

The enzymatic activity of mushroom tyrosinase was investigated using catechin as substrate in selected organic solvent media. The results showed that optimal tyrosinase activity was obtained at pH 6.2, 6.6, 6.0 and 6.2 in the organic solvent media of heptane, toluene, dichloromethane, and dichloroethane, respectively, and at a temperature between 25°C and 27.5°C. In addition, the kinetic studies showed that the K_m values were 5.38, 1.03, 2.52 and 4.03 mM, for the tyrosinase-catechin biocatalysis in the reaction media of heptane, toluene, dichloromethane, and dichloroethane, respectively, while the corresponding V_max values were 1.22×10⁻³, 0.33×10⁻³, 1.47×10⁻³ and 1.20×10⁻³ δA per μg protein per second, respectively. The use of acetone as co-solvent for the tyrosinase-catechin biocatalysis showed that acetone concentrations ranging from 5% to 30% (v/v) in the heptane reaction medium produced a decrease of 4.3% to 96.7% in tyrosinase activity. The results also indicated that the presence of 12.5% acetone in the reaction medium of dichloromethane, and 22.0% in those of toluene and dichloroethane produced a maximal increase of 42.6%, 92.1% and 71.8%, respectively, in tyrosinase activity. However, the overall findings indicated that additional increases in acetone concentration resulted in an inhibition of tyrosinase activity.     

Formation of the nanostructure on a silica surface as mercury(II) ions adsorption sites

The present work investigates the adsorptive interactions of Hg(II) ions with hydroxylated silica, aminopropylsilica and silica chemically modified by β-cyclodextrin in aqueous medium. Batch adsorption studies were carried out with various agitation time and mercury(II) concentration. The maximum adsorption was observed within 15-30 min of agitation. The kinetics of the interactions, tested with model of Lagergren for pseudo-first and pseudo-second order equations, showed better agreement with first order kinetics (k₁ = 3.4 ± 0.2 to 5.9 ± 0.3 min⁻¹). The adsorption data gave good fits with Langmuir isotherms. The results have shown that β-cyclodextrin-containing adsorbent has the largest adsorption specificity to Hg(II) : K_L = 14 400 ± 700 L/mg. “β-Cyclodextrin-” inclusion complexes with ratio 1:1 and super molecules with composition С₄₂H₇₀O₃₅ · 3Hg(NO₃)₂ are formed on the surface of β-cyclodextrin-containing silica.     

Enantioselective synthesis of amines via reductive amination with a dehydrogenase mutant from Exigobacterium sibiricum: Substrate scope,co-solvent tolerance and biocatalyst immobilization

In recent years, the reductive amination of ketones in the presence of amine dehydrogenases emerged as an attractive synthetic strategy for the enantioselective preparation of amines starting from ketones, an ammonia source, a reducing reagent and a cofactor, which is recycled in situ by means of a second enzyme. Current challenges in this field consists of providing a broad synthetic platform as well as process development including enzyme immobilization. In this contribution these issues are addressed. Utilizing the amine dehydrogenase EsLeuDH-DM as a mutant of the leucine dehydrogenase from Exigobacterium sibiricum, a range of aryl-substituted ketones were tested as substrates revealing a broad substrate tolerance. Kinetics as well as inhibition effects were also studied and the suitability of this method for synthetic purpose was demonstrated with acetophenone as a model substrate. Even at an elevated substrate concentration of 50?mM, excellent conversion was achieved. In addition, the impact of water-miscible co-solvents was examined, and good activities were found when using DMSO of up to 30% (v/v). Furthermore, a successful immobilization of the EsLeuDH-DM was demonstrated utilizing a hydrophobic support and a support for covalent binding, respectively, as a carrier.     

Optimization of accessible catalase activity in polyacrylamide gel-immobilized Saccharomyces cerevisiae

The permeabilization of Saccharomyces cerevisiae (baker's yeast), either before or after immobilization in polyacrylamide gel (PAG), has been examined as a means to increase the catalase activity of PAG-immobilized yeast cells. Prior permeabilization of the cells resulted in large losses of catalase activity during immobilization, but permeabilization after immobilization produced increases in the catalase activity of yeast/PAG particles. A dependence of the accessible catalase activity on the concentration of polyacrylamide in permeabilized yeast/PAG particles, and on the method of permeabilization of the immobilized cells, was observed. Optimal levels of stable catalase activity (1000-2000 IU/g PAG particles; ca. 5%-10% of total available activity) were obtained by immobilizing yeast cells (0.5 g wet cells/mL gel) in 10% (w/v) PAG, followed by permeabilization of the entrapped cells with either cetyltrimethylammonium bromide, Triton X-100 and one freeze-thaw, or five freeze-thaw cycles. (c) 1992 John Wiley & Sons, Inc.     

Reversible adsorption of carbon dioxide on amine surface-bonded silica gel

Carbon dioxide adsorbs reversibly on a silica gel containing 3-aminopropyl groups bonded to surface atoms of silicon. These act as the active sites for the chemisorption of CO₂ at room temperature which is liberated by temperature programmed desorption at about 100 °C. The material is capable of adsorbing about 10 STP cm³ of dry CO₂ per gram and can be regenerated upon heating. It might be used as scrubber for carbon dioxide from industrial gaseous streams. Adsorption of humid CO₂ produces a small amount of formaldehyde which suggests activation of the carbon dioxide molecule.     

Biocatalysis in ionic liquids: the stereoconvergent hydrolysis of trans-β-methylstyrene oxide catalyzed by soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) was shown to catalyze hydrolysis of epoxides using the ionic liquids (ILs) [bmim][PF₆], [bmim][N(Tf)₂], and [bmim][BF₄] (where bmim=1-butyl-3-methylimidazolium, PF₆=hexafluorophosphate, N(Tf)₂=bis(trifluoromethylsulfonyl)imide, and BF₄=tetrafluoroborate) as reaction medium. Reaction rates were generally comparable with those observed in buffer solution, and when the cress enzyme was used the hydrolysis of trans-β-methylstyrene oxide gave, through a stereoconvergent process, the corresponding optically active (1S,2R)-erythro-1-phenylpropane-1,2-diol.     

五种益生菌在纤维固定化连续培养系统的共存定殖

模拟肠道的半固态生境,建立以玉米纤维为载体的固定化连续搅拌培养系统、并以非固定化连续搅拌培养系统为对照,稀释率为0.0417,分别连续培养12 d,双歧杆菌、酪酸菌、嗜酸乳杆菌、肠膜芽孢杆菌、粪肠球菌五种菌在两种系统中均可稳定共存,固定化系统固相五种菌数均高于非固定化系统,以双歧杆菌增幅最大,半固态的固定化连续培养较液态连续培养的菌相与肠道菌相较一致,能更好地模拟肠道微生态.扫描电镜观察五种菌在玉米纤维上固定形成了生物膜.     

五种益生菌在纤维固定化连续培养系统的共存定殖

模拟肠道的半固态生境, 建立以玉米纤维为载体的固定化连续搅拌培养系统、并以非固定化连续搅拌培养系统为对照, 稀释率为0.0417, 分别连续培养12 d, 双歧杆菌、酪酸菌、嗜酸乳杆菌、肠膜芽孢杆菌、粪肠球菌五种菌在两种系统中均可稳定共存, 固定化系统固相五种菌数均高于非固定化系统, 以双歧杆菌增幅最大, 半固态的固定化连续培养较液态连续培养的菌相与肠道菌相较一致, 能更好地模拟肠道微生态。扫描电镜观察五种菌在玉米纤维上固定形成了生物膜。     

Effects of relative humidity on enzyme activity immobilized in sol–gel-derived silica nanocomposites

Enzyme immobilization has attracted great interest in biotechnology processes. Herein we report the immobilization of urease from Canavalia ensiformis (jack bean) in sol–gel-derived silica nanocomposites. Urease activity, differential scanning calorimetry (DSC), nitrogen and water adsorption isotherms were used to characterize the effect of storage at various relative humidities on enzyme activity immobilized in sol–gel-derived silica nanocomposites. In this study, the nanocomposites consist of tetraethoxysilane, as inorganic silicate precursor, in combination with glycerol or trehalose as organic additives. Entrapped urease was more stable for all the formulations aged with a relative humidity of 80%. However, significant differences (p < 0.05) in enzyme activity recovered at this relative humidity were observed between samples with different formulations, reflecting the effect of additives during the immobilization process. The applications of biocompatible sol–gel-derived matrices can be further extended and utilized in the development of biosensors with immobilized biomolecules that can be used for long time periods by taking into account different factors, among which the storage relative humidity has permitted to greatly improve the stability of the immobilized urease.