Solid-phase chemical amination of a lipase from Bacillus thermocatenulatus to improve its stabilization via covalent immobilization on highly activated glyoxyl-agarose

In this paper, the stabilization of a lipase from Bacillus thermocatenulatus (BTL2) by a new strategy is described. First, the lipase is selectively adsorbed on hydrophobic supports. Second, the carboxylic residues of the enzyme are modified with ethylenediamine, generating a new enzyme having 4-fold more amino groups than the native enzyme. The chemical amination did not present a significant effect on the enzyme activity and only reduced the enzyme half-life by a 3-4-fold factor in inactivations promoted by heat or organic solvents. Next, the aminated and purified enzyme is desorbed from the support using 0.2% Triton X-100. Then, the aminated enzyme was immobilized on glyoxyl-agarose by multipoint covalent attachment. The immobilized enzyme retained 65% of the starting activity. Because of the lower p K of the new amino groups in the enzyme surface, the immobilization could be performed at pH 9 (while the native enzyme was only immobilized at pH over 10). In fact, the immobilization rate was higher at this pH value for the aminated enzyme than that of the native enzyme at pH 10. The optimal stabilization protocol was the immobilization of aminated BTL2 at pH 9 and the further incubation for 24 h at 25 degrees C and pH 10. This preparation was 5-fold more stable than the optimal BTL2 immobilized on glyoxyl agarose and around 1200-fold more stable than the enzyme immobilized on CNBr and further aminated. The catalytic properties of BTL2 could be greatly modulated by the immobilization protocol. For example, from (R/S)-2- O-butyryl-2-phenylacetic acid, one preparation of BTL2 could be used to produce the S-isomer, while other preparation produced the R-isomer.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

Improvement of the functional properties of a thermostable lipase from alcaligenes sp. via strong adsorption on hydrophobic supports

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at 55 °C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to different activated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the most interesting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135% of catalytic activity for the hydrolysis of p-nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilized enzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 °C, pH 8.5 (a 25-fold stabilizing factor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 °C (soluble enzyme) up to 70 °C (hydrophobic support enzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on the experimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highly hydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacial activation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols used to immobilize the enzyme hardly improved the properties of the lipase.     

Medium engineering on modified Geobacillus thermocatenulatus lipase to prepare highly active catalysts

The influence of additives on the activity of different covalently and site-specific chemically modified immobilized preparations of a lipase from Geobacillus thermocatenulatus (BTL2) was investigated with a view to obtain a very high active biocatalyst. Non-ionic surfactant and co-solvents at different concentration range were applied. The CNBr-BTL2 immobilized preparation, a very mild immobilized enzyme with similar properties to the soluble enzyme, exhibited an increase in activity of 3 fold in the presence of 20% (v/v) co-solvent (e.g., 1,4-dioxane) and 2.6 fold when Triton X-100 (v/v) was added in the hydrolysis of p-nitrophenylbutyrate. This immobilized preparation was hyper-activated in the presence of both additives although without a synergistic effect. The CNBr-BTL2 modified with polymers showed mild hyperactivation in the presence of each additives and even a synergy in the presence of both.In the best of cases, the HOOC-PEG1500-CNBr-BTL2 preparation showed up to 11 fold higher activity in the presence of additives combination than in absence of them.The glyoxyl-BTL2 preparation was hyper-activated in a similar way than CNBr-BTL2 in the presence of detergents but much less with co-solvents. However, the modified glyoxyl-BTL2 preparations were hyper-activated with solvent (2 fold) but not with detergent. An increase of 3 fold in activity for the modified glyoxyl-BTL2 preparations was observed in the presence of both additives.     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Different properties of the lipases contained in porcine pancreatic lipase extracts as enantioselective biocatalysts

The porcine pancreatic lipase (PPL) extracts contain a mixture of several lipases. Their fractioning was performed by sequential adsorption via interfacial activation on supports with different hydrophobicity. A protein of 25 KDa was preferentially adsorbed on octyl-Sepharose, another protein of 33 kDa was mainly adsorbed on octadecyl-Sepabeads support, and the PPL was mainly adsorbed on the support bearing phenyl groups. The different immobilized preparations showed different properties and different response due to change in the experimental conditions. Thus, in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester [(+/-)-1] to produce the corresponding acid [2], the octyl-25KDa preparation showed the best enantioselectivity (E) value (E = 7) at pH 5 and 25 degrees C, whereas the phenyl-PPL was the most enantioselective (E = 10) at pH 5, 4 degrees C, and 10% dioxane. Using different preparations at different pHs it was possible to resolve (+/-)-2-O-butyryl-2-phenylacetic acid [(+/-)-3] with a high E value (E > 100); for example, with octadecyl-33 KDa enzyme at pH 8.     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

One-step purification,covalent immobilization,and additional stabilization of a thermophilic poly-His-tagged beta-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate-epoxy Sepabeads

Using the poly-His-tagged-beta-galactosidase from Thermus sp. strain T2 overexpressed in Escherichia coli (MC1116) as a model enzyme, we have developed a strategy to purify and immobilize proteins in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification. The aforementioned enzyme could not be immobilized onto standard epoxy supports with good yields, and after purification and storage, it exhibited a strong trend to yield very large aggregates as shown by ultracentrifugation experiments. That preparation could not be immobilized in any support, very likely because the pores of the solid became clogged by the large aggregates. These novel epoxy-metal chelate heterofunctional supports contain a low concentration of Co(2+) chelated in IDA groups and a high density of epoxy groups. This enabled the selective adsorption of poly-His-tagged enzymes, and as this adsorption step is necessary for the covalent immobilization procedure, the selective covalent immobilization of the target enzyme could take place. This strategy allowed similar maximum loadings of the target enzyme using either pure or crude preparations of the enzyme. The enzyme derivative presented a very high activity at 70 degrees C (over 1000 IU in the hydrolysis of lactose) and very high stability and stabilization when compared to its soluble counterpart (activity remained unaltered after several days of incubation at 50 degrees C). In fact, this preparation was much more stable than when the same enzyme was immobilized onto standard epoxy Sepabeads.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

The effect of lead on the calcium-handling capacity of rat heart mitochondria.

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.     

Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis

A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.     

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.     

Rapid preparation of a relatively stable, partially purified lipoprotein lipase from perfused rat heart

Rat hearts, extensively washed with cold 0.15 M NaCl solution, were perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol to release endothelium-bound lipoprotein lipase. Approximately 100 mU of enzyme activity could be released from each heart (weighing about 1.7 g). Several hearts could be sequentially perfused with the same heparin solution to enrich it in lipase activity. When compared with other equally rapid and frequently used sources of rat lipoprotein lipase (such as heart acetone powder or postheparin plasma), our enzyme preparation had a much higher specific activity suggesting that a greater purification level had been already achieved in a single step. In addition, this lipoprotein lipase preparation contained only trace amounts of lipids, was stable for an hour at 37 degrees C and retained 75% of its activity after 10 days at 4 degrees C. The described procedure is a quick way to prepare a soluble, partially purified and relatively stable lipoprotein lipase that may be useful especially for the in vitro preparation of triacylglycerol-rich lipoprotein remnants.     

Covalent immobilization of lipase in organic solvents

Lipase from Rhizopus sp. has been immobilized covalently on tresyl activated silica. Three different coupling media were evaluated: aqueous buffer, n-hexane, and a microemulsion based on n-hexane, aqueous buffer, and the nonionic surfactant triethylene glycol monododecyl ether. In addition, coupling via a very long, hydrophilic spacer arm, polyethylene glycol 1500 (PEG 1500), was compared with attachment to the silica via a short silane bridge only. The enzyme preparations were tested in hydrolysis and transesterification reactions. In the hydrolysis no marked differences in activity were found between the coupling media used. In the transesterification, on the other hand, the choice of immobilization medium had a very large effect on lipase activity, the preparation from microemulsion being the most active one. The use of the hydrophilic spacer had a large effect on activity in the hydrolysis reaction. Whereas direct coupling gave an activity of immobilized lipase of 26-34% of that of free enzyme, depending on the reaction medium, lipase bound via the spacer exhibited 56-67% activity. The latter values are considerably higher than previously reported in the literature for covalently immobilized lipase. The hydrophilic spacer had no effect on enzyme activity in the transesterification, however, a fact which is attributed to the hydrophobic medium of this reaction. The spacer is incompatible with the reaction medium and will, therefore, adsorb on the particles rather than stretch out into the bulk phase. The stability of the bound lipase was extremely good, no loss in activity being observed after a period of three weeks in aqueous solution of 37 degrees C.     

Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil

Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.     

Immobilization of pectinase fromSclerotium rolfsii

Immobilization of pectinase fromS. rolfsii was studied on different matrices of which Amberlite XAD-7 showed maximum adsorption and expression of the enzyme. The most active preparation was obtained when XAD-7 was activated with 2% glutaraldehyde and 1.7 μkat of enzyme per g resin was used for immobilization at pH 5.5 and 28°C. Optimum pH and temperature of theS. rolfsii pectinase remain unaltered, 3.5 and 55°C, respectively, after immobilization. However, the apparentK _M value of the enzyme decreased from 1.75 g/L for soluble enzyme preparation to 1.4 g/L for immobilized enzyme preparation. Both soluble and immobilized enzyme preparations were most stable at pH 4.0. The immobilized enzyme preparation was more stable than the soluble enzyme.     

Novozym 435 displays very different selectivity compared to lipase from Candida antarctica B adsorbed on other hydrophobic supports

This paper shows that the properties of lipase B from Candida antarctica (CAL-B) may be easily modulated using different hydrophobic supports to immobilize it (octyl and butyl-agarose, octadecyl-Sepabeads or Lewatit). CAL-B could be fully desorbed from the supports by just incubating the biocatalyst with Triton X-100, although the concentration of detergent necessary was to fully desorb the enzyme varied with the support employed (from 1% for butyl-agarose to 4% for octadecyl-Sepabeads), suggesting that in all cases, the main reason for the enzyme immobilization was hydrophobic interactions. Lewatit VP OC 1600 yielded very different results in terms of activity, selectivity or enantioselectivity in the hydrolysis of rac-2-O-butyryl-2-phenylacetic acid (1) and 3-phenylglutaric acid dimethyl diester (3) compared to the other preparations. For example, in the hydrolysis of 1, Novozym 435 preferred the S-isomer (with an E value higher than 100) whereas all the other preparations preferred the R isomer (e.g. octyl-agarose-CAL-B with E value of 50). In the hydrolysis of 3, Novozym 435 gave S-3-phenylglutaric acid methyl ester with an ee higher than 99%, by coupling the first asymmetric hydrolysis to the enantiospecific hydrolysis of the monoester. CAL-B immobilized on Lewatit at low ionic strength not only behaved similarly to Novozym 435, but also presented some differences that should be due to the exact protocol of the enzyme immobilization in Novozym 435.     

Lipase immobilization on differently functionalized vinyl-based amphiphilic polymers: influence of phase segregation on the enzyme hydrolytic activity

Microbial lipase from Candida rugosa was immobilized by physical adsorption onto an ethylene-vinyl alcohol polymer (EVAL) functionalized with acyl chlorides. To evaluate the influence of the reagent chain-length on the amount and activity of immobilized lipase, three differently long aliphatic fatty acids were employed (C8, C12, C18), obtaining EVAL functionalization degrees ranging from 5% to 65%. The enzyme-polymer affinity increased with both the length of the alkyl chain and the matrix hydrophobicity. In particular, the esterified polymers showed a tendency to give segregated hydrophilic and hydrophobic domains. It was observed the formation of an enzyme multilayer at both low and high protein concentrations. Desorption experiments showed that Candida rugosa lipase may be adsorbed in a closed form on the polymer hydrophilic domains and in an open, active structure on the hydrophobic ones. The best results were found for the EVAL-C18 13% matrix that showed hyperactivation with both the soluble and unsoluble substrate after enzyme desorption. In addition, this supported biocatalyst retained its activity for repetitive cycles.     

A bioconjugate of Pseudomonas cepacia lipase with alginate with enhanced catalytic efficiency

A bioconjugate of Pseudomonas cepacia lipase with alginate was prepared by simple adsorption. Atomic force microscope (AFM) images showed that this bioconjugate resulted from adsorption rather than entrapment of the enzyme as enzyme molecules were visible on the gel surface. The soluble bioconjugate exhibited increased enzyme activity in terms of high effectiveness factor (effectiveness factor was 3 for the immobilized preparation) and greater Vmax/Km value (Vmax/Km increased 25 times upon immobilization). This constitutes one of the less frequently observed instances of lipase activation by lid opening as a result of binding to a predominantly hydrophilic molecule. The bioconjugate was also more stable at 55 degrees C as compared to the free enzyme and could be reused for oil hydrolysis up to 4 cycles without any loss in activity. Fluorescence emission spectroscopy showed that the immobilized enzyme had undergone definite conformational changes.