Reactivation of a thermostable lipase by solid phase unfolding/refolding effect of cysteine residues on refolding efficiency

Lipase from Geobacillus thermocatenulatus (BTL2) was immobilized in two different matrixes. In one derivative, the enzyme was immobilized on agarose activated with cyanogen bromide (CNBr-BTL2) via its most reactive superficial amino group, whereas the other derivative was covalently immobilized on glyoxyl agarose supports (Gx-BTL2). The latter immobilization protocol leads to intense multipoint covalent attachment between the lysine richest region of enzyme and the glyoxyl groups on the support surface. The resulted solid derivatives were unfolded by incubation under high concentrations of guanidine and then resuspended in aqueous media under different experimental conditions. In both CNBr-BTL2 and Gx-BTL2 derivatives, the oxidation of Cys residues during the unfolding/refolding processes led to inefficient folding for the enzyme because only 25-30% of its initial activity was recovered after 3 h in refolding conditions. Dithiothreitol (DTT), a very mild reducing agent, prevented Cys oxidation during the unfolding/refolding process, greatly improving activity recovery in the refolded forms. In parallel, other variables such as pH, buffer composition and the presence of polymers and other additives, had different effects on refolding efficiencies and refolding rates for both derivatives. In the case of solid derivatives of BTL2 immobilized on CNBr-agarose, the surface's chemistry was crucial to guarantee an optimal protein refolding. In this way, uncharged protein vicinities resulted in better refolding efficiencies than those charged ones.     

Effect of solid-phase chemical modification on the features of the lipase from Thermomyces lanuginosus

The lipase from Thermomyces lanuginosus (TLL) was immobilized on octyl Sepharose and further modified with ethylenediamine (EDA) after activation of the carboxylic groups with carbodiimide. Different degrees of modification of the carboxyl groups were carried out by controlling the concentration of carbodiimide (10%, 50% or 100%). Subsequently, the effect of incubation of the modified preparations on hydroxylamine to recover the modified tyrosine was also studied. The modified enzymes exhibited a mobility in native electrophoresis quite different from that of the unmodified lipase (as expected by the changes in charge), and required higher concentrations of cationic detergent to become desorbed from the support. Interestingly, the chemical modification of the immobilized TLL produced an improvement in its activity, proportional to the amination degree. This increase in activity was much more significant at pH 10, where the fully modified preparation increased the activity by a factor of 10 as compared to the unmodified preparation. Moreover, the incubation of the chemically aminated preparations in a hydroxylamine solution improved the activity by an additional factor of 1.2. The fully aminated and incubated in hydroxylamine preparation exhibited a thermostability higher than that of the unmodified preparation, mainly at pH 5 (almost a 30 fold factor). In the presence of tetrahydrofurane, some stabilization was observed at pH 7, while at pH 9 the stability of the modified enzyme decreased (under all the assayed amination degrees) when compared to that of the unmodified enzyme. Thus, this simple protocol may be a rapid and efficient way of preparing a TLL biocatalyst with higher activity and stability, although this will depend on the inactivation conditions.     

Immobilization of laccase from Cerrena unicolor on controlled porosity glass

White-rot basidiomycete Cerrena unicolor grown in non-induced and induced conditions was tested for production of laccase, lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP). A typical correlation between the concentration of phenolic compounds in the culture fluid and the extracellular laccase activity was observed. The heterogeneous crude laccase preparation obtained after the non-induced fermentor cultivation was immobilized both on controlled porosity glass (CPG) activated by γ-aminopropyltriethoxysilane (APTES) and on CPG with its surface covered by dextran layers. The laccase activities were tested in the aqueous solution for the native and immobilized preparations using different pH and temperature conditions. Laccase activities were additionally examined for native and immobilized forms of laccase preparations in the aqueous solution containing organic solvents. The greatest activity toward the substrate used in the presence of organic solvents was shown by the laccase preparation coupled with the CPG covered by a dextran layer. Potential inhibitors such as thioglycolic acid, thiourea and EDTA used in 1-mM concentration did not show inhibiting properties towards the laccase preparations.     

Positive effects of the multipoint covalent immobilization in the reactivation of partially inactivated derivatives of lipase from Thermomyces lanuginosus

Different immobilized preparations of lipase from Thermomyces lanuginosus (TLL) have been inactivated by exposure to high temperatures, guanidine or 95% of dioxane. The studied preparations were: non-stabilized cyanogen bromide (CNBr-TLL), aminated CNBr-TLL (CNBr-TLL-A), and two stabilized preparations of aminated TLL by immobilization on glyoxyl support, Gx(9/10)-TLL-A (TLL-A immobilized at pH 9 and later incubated at pH 10) or Gx(10)-TLL-A (directly immobilized at pH 10). The reactivation of the partially inactivated immobilized enzymes under mild conditions by incubation in aqueous buffer, allowed recovery of some of the original activity, which was improved when it was pre-incubated in guanidine. Amination produced a fairly negative effect on the reactivation of the enzyme, but the multipoint covalent attachment of this aminated enzyme reversed the effect (e.g., recovered activity increased from 20% for CNBr-TLL to 80% for Gx(9/10)-TLL-A). The negative effect of the amination was clearer when the inactivation was caused by exposure to high temperatures, although the multipoint attachment of aminated enzyme was able to improve the recovered activity. The determination of enzyme activity in the presence of hexadecyltrimethylammonium bromide slowed the inactivation rates of all preparations and improved the recovery of activity after incubation under mild conditions, suggesting that the opening mechanism of the lipase could be a critical step in the TLL inactivation/reactivation. The use of multipoint attached TLL preparations did not only improve enzyme stability, but it also increased activity recovery when the preparation was incubated under mild conditions.     

Immobilization of lipases on hydrophobic supports involves the open form of the enzyme

The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1 M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength.The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.     

Immobilization of a soluble chemically thermostabilized enzyme

Cellobiase was coupled to a dialdehyde dextran by reductive alkylation in the presence of sodium cyanoborohydride. The resulting conjugate, obtained without loss of enzymic activity, presents properties of thermoresistance largely superior to those of native enzyme: the rate of inactivation is reduced compared to that of native enzyme and its optimal temperature of activity is 70-75 degrees C instead of 65 degrees C. Finally the conjugate presents increased longevity when subjected to experiments of operational stability; its hydrolytic activity is maintained at 60 degrees C in a 10% (w/v) cellobiose solution for more than 100 h whereas the native enzyme is inactivated after 45 h. The cellobiase-dextran conjugate was immobilized by covalent coupling on aminated silica by reductive alkylation in the presence of NaBH(3)CN. The characteristics of thermoresistance of this stabilized and immobilized conjugate were studied and compared to those of a preparation of native cellobiase immobilized on a silica support activated with glutaraldehyde. Analysis of the thermoresistance of these two cellobiase preparations clearly shows that immobilization has maintained and even enhanced their properties. In particular, the operational stability, measured at 68 degrees C on 10% (w/v) cellobiose shows an increased longevity of the stabilized and immobilized enzyme for 120 h compared to 60 h for the native immobilized enzyme. Two successive incubations of these cellobiase derivatives show that it is possible to obtain 2.5 times more glucose with the stabilized-immobilized enzyme than with the immobilized preparation. The procedure described above enables us to prepare a thermostabilized immobilized cellobiase.     

Modulation of the properties of immobilized CALB by chemical modification with 2,3,4-trinitrobenzenesulfonate or ethylendiamine. Advantages of using adsorbed lipases on hydrophobic supports

Lipase B from Candida antarctica (CALB) has been adsorbed on octyl-agarose or covalently immobilized on cyanogen bromide agarose. Then, both biocatalysts have been modified with ethylenediamine (EDA) or 2,4,6-trinitrobenzensulfonic acid (TNBS) just using one reactive or using several modifications in a sequential way (the most complex preparation was CALB–TNBS–EDA–TNBS). Covalently immobilized enzyme decreased the activity by 40–60% after chemical modifications, while the adsorbed enzyme improved the activity on p-nitrophenylbutyrate (pNPB) by EDA modification (even by a 2-fold factor). These biocatalysts were further characterized. The results showed that the effects of the chemical modification on the enzyme features were strongly dependent on the immobilization protocol utilized, the experimental conditions where the catalyst will be utilized, and the substrate. Significant changes in the activity/pH profile were observed after the chemical modifications. The effect of the modifications on the enzyme activity depends on the substrate and the reaction conditions: enzyme specificity is strongly altered by the chemical modification. Moreover, enzyme activity versus pNPB (using octyl-CALB–EDA) or versus R methyl mandelate (using octyl-CALB–TNBS) increased by almost a 2-fold factor at pH 5. The stability of the modified enzymes at different pH and in the presence of organic solvents generally decreased after the modifications, usually by no more than a 2-fold factor. However, under some conditions, some stabilization was found. CALB enantioselectivity in the hydrolysis of R/S methyl mandelate could be also improved by these chemical modifications (e.g., E-value went from 11 to 16 using octyl-CALB–TNBS at pH 5). Therefore, solid phase chemical modification of immobilized lipases may become a powerful tool in the design of lipase libraries with very different properties, each immobilized preparation may be used to produce a variety of forms with altered properties.     

Microwave-assisted synthesis of galacto-oligosaccharides from lactose with immobilized beta-galactosidase from Kluyveromyces lactis

Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free -galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating. Immobilization of the -galactosidase on to Duolite A-568 increased the synthesis of GOS. GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media. The advantage of microwave heating on GOS formation was also examined. Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS. The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized -glucosidase and with added co-solvents such as hexanol.     

Enzymic interesterification of fats: The effect of non-lipase material on immobilized enzyme activity

An immobilized lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) suitable for fat interesterification has been prepared by precipitation onto diatomaceous earth (Celite) with acetone of a crude lipase preparation from an Aspergillus. Non-lipase material present in the preparation which precipitated at high acetone concentrations or ovalbumin added prior to the immobilization reduced the measured interesterification activity without affecting lipolytic activity. The non-lipase material reduced the interesterification activity by as much as 50%. The interesterification activity of immobilized preparations was enhanced by the use of higher concentrations of the crude lipase or, more substantially, by admixture of purified lipase.     

Oriented immobilization of stem bromelain via the lone histidine on a metal affinity support

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from the stem of the pineapple plant (Ananas comosus) and is unique for it contains a single histidine residue (His-158) in the polypeptide. Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of bromelain was selected. Bromelain was successfully immobilized on iminodiacetic acid carrier Sepharose 6B. Cu²⁺ complexed with iminodiacetate (IDA) was used as the chelating ligand to bind the lone histidine on bromelain. Simultaneously, preparation of a high affinity immobilized preparation was attempted using a soluble cross-linked preparation of bromelain on Cu-IDA-Sepharose. However this second method proved unsuccessful, possibly due to poor histidine accessibility in the cross-linked preparation. The immobilized preparation obtained using uncrosslinked bromelain was more resistant to thermal inactivation, as evidenced by retention of over enzyme 50% activity after incubation at 60 °C, as compared to 20% retained by the native enzyme. The immobilized preparation also exhibited a broader pH-activity profile in acidic range. The native, immobilized and soluble cross-linked bromelain showed apparent Michaelis constant (K_m) values of 1.08, 0.42, 1.56 mg/ml, respectively, using casein as the substrate. While the maximum velocity (V_max) values of the soluble and immobilized preparations were comparable, cross-linked preparation showed a 20% decrease, suggesting inactivation. The mild conditions used for predominantly oriented immobilization exploiting the unique property of single histidine, the high recovery of immobilized preparations, the stability, reusability and the regenerability of the matrix are the main features of the method reported here.     

Enhanced activity of an immobilized lipase promoted by site-directed chemical modification with polymers

The activity of a lipase from Geobacillus thermocatenulatus (BTL2) can be greatly improved by site-directed chemical modification of a single external Cys64. This residue is placed in the proximity of the region where the lid is allocated when the lipase exhibits its open and active form. Thiol group of Cys64 was modified by thiol-disulfide exchange with pyridyldisulfide poly-aminated-dextrans or mono-carboxylated-polyethyleneglycol. The modification was performed on the covalently immobilized lipase on CNBr-agarose or glyoxyl-agarose. The activity of modified derivatives was strongly dependent on the immobilized preparation, the polymer used and the substrate assayed. For example, the modification with PEG-COOH of BTL2 immobilized on glyoxyl-agarose increased 5-fold the enzyme activity towards the hydrolysis of 2-O-butyryl-2-phenylacetic acid. However, the modification with 3-(2-pyridyldithio)-propionyl-dextran-NH₂ reduced the activity to 40%.The fact that the modified enzymes can be inhibited by an irreversible inhibitor much more rapidly than the unmodified ones suggested that the main effect of the modification is to somehow stabilize the open form of the lipase.     

Laccase immobilization on copolymer of butyl acrylate and ethylene glycol dimethacrylate

Extracellular laccase produced by the wood-rotting fungus Cerrena unicolor was immobilized by covalent bonds formation on the copolymer of butyl acrylate and ethylene glycol dimethacrylate. The carrier had a fixed superstructure and three kinds of anchor groups: –NH₂, –OH, and –COOH. Three procedures were used for the activation of the carrier: (i) glutaraldehyde, (ii) divinyl sulfone, and (iii) carbodiimide. It was found that laccase coupling to the carrier via glutaraldehyde yielded an enzyme-carrier preparation of very high activity and storage stability. Consideration was also given to the problem of how the pH, ionic strength, protein concentration and the presence of additives (syringaldazine, guaiacol, Cu²⁺) affect the coupling procedure via glutaraldehyde. Thermal- and pH-stability, as well as the activity profiles of the best enzyme-carrier preparation, was evaluated. The very high operational stability investigated in a packed bed reactor at 30 °C shows the potential of the preparation for practical use.     

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.     

Enzymatic transglycosylation of natural and modified nucleosides by immobilized thermostable nucleoside phosphorylases from Geobacillus stearothermophilus

Natural and modified purine nucleosides have been synthesized using the recombinant thermostable enzymes purine nucleoside phosphorylase II (E. C. 2.4.2.1) and pyrimidine nucleoside phosphorylase (E. C. 2.4.2.2) from Geobacillus stearothermophilus B-2194. The enzymes were produced in recombinant E. coli strains and covalently immobilized on aminopropylsilochrom AP-CPG-170 after heating the cell lysates and the removal of coagulated thermolabile proteins. The resulting preparations of thermostable nucleoside phosphorylases retained a high activity after 20 reuses in nucleoside transglycosylation reactions at 70–75°C with a yield of the target products as high as 96%. Owing to the high catalytic activity, thermal stability, the ease of application, and the possibility of repeated use, the immobilized preparations of thermostable nucleoside phosphorylases are suitable for the production of pharmacologically important natural and modified nucleosides.     

Purification, immobilization, and stabilization of a lipase from Bacillus thermocatenulatus by interfacial adsorption on hydrophobic supports

A lipase from Bacillus thermocatenulatus (BTL2) cloned in E. coli has been purified using a very simple method: interfacial activation on a hydrophobic support followed by desorption with Triton. Only one band was detected by SDS-PAGE. The pure enzyme was immobilized using different methodologies. BTL2 adsorbed on a hydrophobic support (octadecyl-Sepabeads) exhibited a hyperactivation with respect to the soluble enzyme, whereas the other immobilized preparations suffered a slight decrease in the expressed activity. The soluble enzyme was very stable, but all immobilized preparations were much more stable than the soluble enzyme, the octadecyl-Sepabeads-BTL2 preparation being the most stable one in all conditions (high temperature or in the presence of organic cosolvents), maintaining 100% of the activity at 65 degrees C or 30% of dioxane and 45 degrees C after several days of incubation. The glyoxyl preparation, the second more stable, retained 80% of the initial activity after 2 days, respectively. The adsorption of this thermophilic lipase on octadecyl-Sepabeads permitted an increase in the optimal temperature of the enzyme of 10 degrees C.     

Additive Effect of Dextrans on the Stability of Horseradish Peroxidase

The influence of various concentration (10, 20, and 30% w/v) of different molar weighted dextrans as additives on the stability of HRP has been studied in aqueous medium. Native HRP preparations were formulated with different additives for storage stabilization and better performance at high temperature and pH. The results obtained show a stabilizing effect in the presence of an additive (75 kDa dextran). The enzyme with 75 kDa dextran (in concentration 10% w/v) showed the highest thermal resistance and the best performance for long-term storage at pH 5.0. In the presence of the 75 kDa dextran, the enzyme activity was increased threefold at 25 °C and lost only 15% activity in 2 h at 50 °C in comparison to the native enzyme which lost all its activity. In addition, dextran protected HRP against inactivation by air bubbles.     

Studies of pyridine nucleotide oxidizing enzymes from human neutrophils

An NADH cytochrome c reductase has been identified in plasma membrane fractions from neutrophils in addition to the superoxide producing NADPH oxidase which has been extensively studied by other investigators. Activation of neutrophils resulted in increased enzyme activities but to different degrees; the NADH cytochrome c reductase increased 2 fold in specific activity and the NADPH oxidase 30 fold. Treatment of the plasma membrane fraction with sonication and differential centrifugation yielded a particulate fraction (R2) with a 2 fold increase in specific activities of both enzymes and concentrations of cytochrome b and FAD. The cytochrome b in the preparation was not reduced under anaerobic conditions by either NADH or NADPH. Treatment of preparations of R2 with deoxycholate or potassium thiocyanate separated the two enzymes yielding particulate preparations with only NADPH oxidase or NADH cytochrome c reductase activity, respectively.     

Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers

Lecitase Ultra has been covalently immobilized on cyanogen bromide cross-linked 4% agarose (CNBr) beads, maintaining 70% of the initial activity. The activity of the immobilized enzyme was improved in the presence of Triton X-100, sodium dodecyl sulfate (SDS), and cetyltrimethyl ammonium bromide (CTAB) (e.g., up to 800% when using CTAB). However, CTAB and Triton X-100 presented a negative effect on enzyme stability even at low concentrations, and SDS cannot be used for a long time at 1% concentration. To maintain the hyperactivated conformation of the enzyme in the absence of detergent, ionic polymers were added during incubation of the immobilized enzyme in the presence of detergents. Coating the immobilized enzyme with polyethylenimine in aqueous buffer (PEI) produced a 3-fold increase in enzyme activity. However, in the presence of 0.1% SDS (v/v), this coating produced a 50-fold increase in enzyme activity. Using PEI and 0.01% (v/v) CTAB, the Lecitase activity decreased to 10%. Using irreversible inhibitors, it could be shown that the PEI/SDS-CNBr-Lecitase preparation allowed its catalytic Ser to be more accessible to the reaction medium than the unmodified CNBr-Lecitase.     

Bioremediation of polycyclic aromatic hydrocarbons by fungal laccases engineered by directed evolution

Fungal laccases are useful for several remarkable transformations, such as bioremediation of polycyclic aromatic hydrocarbons (PAHs), synthesis of phenolic-based resins, oxidation of lignin derivatives and others. Most of these substrates are barely water-soluble, and although polar organic co-solvents may be added to enhance their solubility, transformation rates dramatically decrease due to the negative effect of organic solvents on the protein structure. Laccase from Myceliophthora thermophila variant T2 (MtLT2) has been submitted to laboratory evolution in Saccharomyces cerevisiae with the aim of improving activity and stability in organic co-solvents. Some 4500 clones created by random mutagenesis were screened in two rounds of directed evolution. Libraries were explored under increasing concentrations of acetonitrile and ethanol, and several mutants with improved features were purified and further characterised. Turnover rates of MtLT2 in 30% (v/v) acetonitrile and 50% (v/v) ethanol were increased up to 6.5- and 7.5-fold, respectively. The best variants showed similar rates in 20% (v/v) acetonitrile or 30% (v/v) ethanol as the parent type in aqueous media. Mutant laccases were also tested for the oxidation of anthracene in the presence of 20% (v/v) acetonitrile.     

Chaperone function in organic co-solvents: experimental characterization and modeling of a hyperthermophilic chaperone subunit from Methanocaldococcus jannaschii

Molecular chaperones play a central role in maintaining protein structure within a cell. Previously, we determined that the gene encoding a molecular chaperone, a thermosome, from the hyperthermophilic archaeon Methanocaldococcus jannaschii is upregulated upon lethal heat shock. We have recombinantly expressed this thermosome (rTHS) and show here that it is both stable and fully functional in aqueous solutions containing water-miscible organic co-solvents. Based on circular dichroism the secondary structure of rTHS was not affected by one-hour exposures to a variety of co-solvents including 30% v/v acetonitrile (ACN) and 50% methanol (MeOH). By contrast, the secondary structure of a mesophilic homologue, GroEL/GroES (GroE), was substantially disrupted. rTHS reduced the aggregation of ovalbumin and citrate synthase in 30% ACN, assisted refolding of citrate synthase upon solvent-inactivation, and stabilized citrate synthase and glutamate dehydrogenase in the direct presence of co-solvents. Apparent total turnover numbers of these enzymes in denaturing solutions increased by up to 2.5-fold in the presence of rTHS. Mechanistic models are proposed to help ascertain specific conditions that could enhance or limit organic solvent-induced chaperone activity. These models suggest that thermodynamic stability and the reversibility of enzyme unfolding play key roles in the effectiveness of enzyme recovery by rTHS.