Stabilization of a highly active but unstable alcohol dehydrogenase from yeast using immobilization and post-immobilization techniques

The alcohol dehydrogenase (ADH) from Baker's yeast is very active but extremely unstable under several different conditions. Mild immobilization methods such as one-point attachment to agarose activated with cyanogen bromide groups or ionic adsorption to agarose activated with charged groups allow high activity recoveries (80–100%) but do not promote protein stabilization. In contrast, immobilization methods that force the enzyme to be covalently attached at multiple points on the support fully inactivate the enzyme. Herein, we propose an interesting solution to address the dichotomy between activity and stability. We have developed a protocol in which the enzyme is immobilized on agarose activated with glyoxyl groups in the presence of acetyl cysteine, which results in the recovery of 25% of the enzyme activity but increases the thermal stability of the soluble enzyme 50-fold. However, this immobilization technique does not stabilize the enzyme quaternary structure. Hence, a post-immobilization technique using functionalized polymers has been used to cross-link all enzyme subunits. In this method, polycationic polymers (polyethylenimine) cross-link the quaternary structure with a negligible effect on catalytic activity, which results in a derivative that is 5-fold more stable than non-cross-linked derivatives under very dilute and acidic conditions that highly favor subunit dissociation. Therefore, the stability was increased 500-fold for this optimal derivative compared to diluted soluble enzyme, although the relative expressed activity was low (25%). However, the low expressed activity may be overcome by designing immobilized biocatalysts with high volumetric activities.     

Immobilization and stabilization of d-hydantoinase from Vigna angularis and its use in the production of N-carbamoyl-d-phenylglycine. Improvement of the reaction yield by allowing chemical racemization of the substrate

This work reports the immobilization of a multimeric d-hydantoinase (DHTase) from Vigna angularis (E.C. 3.5.2.2.) on agarose beads activated with glyoxyl groups aiming to improve its stability via multipoint covalent attachment. The final reduction with sodium borohydride resulted in a drop in enzyme activity that could be decreased by adding Zn²⁺ or Mg²⁺. The optimal preparation with high activity (58 % recovered activity) and stability (around 86-fold more stable than the free enzyme) was obtained by DHTase immobilization on glyoxyl agarose for 24 h at 25 °C and pH 10.05, and a borohydride reduction step in the presence of 10 mM Zn²⁺ (DHTase-Glx). The enzyme was almost fully immobilized on glyoxyl agarose (19.8 mg/g of support) when offering 20 mg/g. This immobilized biocatalyst was used to catalyze the hydrolysis of d,l-phenylhydantoin under substrate racemization conditions, which produced 99 % of N-carbamoyl-d-phenylglycine after 9 h reaction.     

Immobilization of Bacillus circulans β-galactosidase and its application in the synthesis of galacto-oligosaccharides under repeated-batch operation

A highly active and stable derivate of immobilized Bacillus circulans β-galactosidase was prepared for the synthesis of galacto-oligosaccharides (GOS) under repeated-batch operation. B. circulans β-galactosidase was immobilized on monofunctional glyoxyl agarose and three heterofunctional supports: amino-, carboxy-, and chelate-glyoxyl agarose. Glyoxyl agarose was the support with highest immobilization yield and stability being selected for the optimization of immobilization conditions and application in GOS synthesis. A central composite rotatable design was conducted to optimize contacted protein and immobilization time, using maximum catalytic potential as the objective function. Optimal conditions of immobilization were 28.9 mg/g and 36.4 h of contact, resulting in a biocatalyst with 595 IU/g and a half-life 89-fold higher than soluble enzyme. Immobilization process did not alter the synthetic capacity of β-galactosidase, obtaining the same GOS yield and product profile than the free enzyme. GOS yield and productivity remained unchanged along 10 repeated batches, with values of 39% (w/w) and 5.7 g GOS/g of biocatalyst·batch. Total product obtained after 10 batches of reaction was 56.5 g GOS/g of biocatalyst (1956 g GOS/g protein). Cumulative productivity in terms of mass of contacted protein was higher for the immobilized enzyme than for its soluble counterpart from the second batch of synthesis onwards.     

Use of physicochemical tools to determine the choice of optimal enzyme: stabilization of D-amino acid oxidase

An evaluation of the stability of several forms (including soluble and two immobilized preparations) of d-amino acid oxidases from Trigonopsis variabilis (TvDAAO) and Rhodotorula gracilis (RgDAAO) is presented here. Initially, both soluble enzymes become inactivated via subunit dissociation, and the most thermostable enzyme seemed to be TvDAAO, which was 3-4 times more stable than RgDAAO at a protein concentration of 30 microg/mL. Immobilization on poorly activated supports was unable to stabilize the enzyme, while highly activated supports improved the enzyme stability. Better results were obtained when using highly activated glyoxyl agarose supports than when glutaraldehyde was used. Thus, multisubunit immobilization on highly activated glyoxyl agarose dramatically improved the stability of RgDAAO (by ca. 15,000-fold) while only marginally improving the stability of TvDAAO (by 15-20-fold), at a protein concentration of 6.7 microg/mL. Therefore, the optimal immobilized RgDAAO was much more stable than the optimal immobilized TvDAAO at this enzyme concentration. The lower stabilization effect on TvDAAO was associated with the inactivation of this enzyme by FAD dissociation that was not prevented by immobilization. Finally, nonstabilized RgDAAO was marginally more stable in the presence of H(2)O(2) than TvDAAO, but after stabilization by multisubunit immobilization, its stability became 10 times higher than that of TvDAAO. Therefore, the most stable DAAO preparation and the optimal choice for an industrial application seems to be RgDAAO immobilized on glyoxyl agarose.     

Investigating and optimizing the immobilization of levansucrase for increased transfructosylation activity and thermal stability

Levansucrase (LS) represents a key enzyme in glycoside synthesis of novel prebiotics and β-2,6-levan. The study of the effects of immobilization parameters of LS, produced from Bacillus amyloliquefaciens, onto glyoxyl agarose-iminodiacetic acid/Cu (glyoxyl agarose-IDA/Cu) by response surface methodology revealed the significance of their interactive effects. Retention of activity was altered by interactive effects from buffer molarity/time and buffer pH/buffer molarity. The optimized immobilization conditions were identified to be a protein loading of 9.09 mg protein/g support, a buffer concentration of 608 mM at pH 6.8 and an incubation time of 49 h. Normally a reducing agent is applied to the immobilized enzyme in order to promote the formation of covalent bonds. This step was replaced with the addition of the ionic polymer polyethylenimine (PEI), which provided a better compromise between retained activity and thermal stability of the immobilized LS. Indeed, LS immobilized onto glyoxyl agarose-IDA/Cu/PEI had a retention of activity of 70.91% with a protein yield of 44.73% and an activity yield of 54.69%, while exhibiting a half-life 4.7 times higher than that of the free LS at 50 °C.     

Improvement of fungal arabinofuranosidase thermal stability by reversible immobilization

A gene encoding α-l-arabinofuranosidase (abfA) from Aspergillus niveus was identified, cloned, and successfully expressed in Aspergillus nidulans. Based on amino acid sequence comparison, the 88.6 kDa enzyme could be assigned to the GH family 51. The characterization of the purified recombinant AbfA revealed that the enzyme was active at a limited pH range (pH 4.0–5.0) and an optimum temperature of 70 °C. The AbfA was able to hydrolyze arabinoxylan, xylan from birchwood, debranched arabinan, and 4-nitrophenyl arabinofuranoside. Synergistic reactions using both AbfA and endoxylanase were also assessed. The highest degree of synergy was obtained after the sequential treatment of the substrate with endoxylanase, followed by AbfA, which was observed to release noticeably more reducing sugars than that of either enzyme acting individually. The immobilization of AbfA was performed via ionic adsorption onto various supports: agarose activated by polyethyleneimine polymers, cyanogen bromide activated Sepharose, DEAE-Sepharose, and Sepharose-Q. The Sepharose-Q derivative remained fully active at pH 5 after 360 min at 60 °C, whereas the free AbfA was inactivated after 60 min. A synergistic effect of arabinoxylan hydrolysis by AbfA immobilized in Sepharose-Q and endoxylanase immobilized in glyoxyl agarose was also observed. The stabilization of arabinofuranosidases using immobilization tools is a novel and interesting topic.     

Preparation of a very stable immobilized biocatalyst of glucose oxidase from Aspergillus niger

Glucose oxidase (GOX) has been immobilized on different activated supports, including glyoxyl agarose, epoxy sepabeads and glutaraldehyde-activated supports. Immobilization onto supports pre-activated with glutaraldehyde rendered the most thermo-stable preparation of GOX. Therefore, as the glutaraldehyde chemistry gave a high stabilization of the enzyme, we proposed another technique for improving the multipoint attachment through glutaraldehyde: the enzyme was ionically adsorbed on cationic supports with primary amino groups and then the immobilized preparation was treated with a glutaraldehyde solution. The decrease on enzyme activity was <20%. Following this methodology, we achieved the highest stability of all the immobilization systems analyzed, showing a half-life 100 times higher than the soluble enzyme. Moreover, this derivative showed a higher stability in the presence of organic solvents (for instance methanol) or hydrogen epoxide than the ionically adsorbed enzyme or the soluble one. Therefore, the adsorption of GOX on aminated cationic support and subsequent treatment with glutaraldehyde was presented as a very successful methodology for achieving a very stable biocatalyst.     

Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques

Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi-subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross-linking of previously immobilized enzyme with tailor-made dextran-aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl-agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl-agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross-linked with dextran-aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.     

Modulation of the distribution of small proteins within porous matrixes by smart-control of the immobilization rate

The distribution of enzymes attached to porous solid supports is a major concern in multienzymatic bioreactors. Herein, as proof of the concept that protein localization on porous surfaces can be controlled by tuning the protein immobilization rate. We study the distribution of two poly-histidine-tagged fluorescent proteins (His-GFP and His-mCherryFP) immobilized on different 4% crosslinked agarose-type carriers by confocal laser scanning microscopy. In this context, immobilization rate is easily modulated by controlling the (i) nature of physico-chemical interaction between protein and surface (reactive groups on surface), (ii) by controlling the reactive group density and (iii) by adding competitors to the immobilization process. His-GFP is 350-fold more rapid immobilized on agarose surfaces activated with either glyoxyl groups or chelates than the same matrix activated with primary amine groups instead. A similar effect is seen with agarose matrixes activated with lower glyoxyl densities that immobilize His-GFP roughly 350-fold slower than the corresponding highly activated matrix. When His-GFP is immobilized on agarose activated with chelates groups in presence of imidazol which competes with the protein for the reactive groups on the support, the immobilization rate is again 400-fold slower than when the same protein was immobilized on the same support but with no imidazol during the immobilization process. In all cases, it was observed that rapid immobilizations (quantitative immobilization in less than 10 min) located 100% of the loaded protein at the crown of the carrier beads, meaning that only the 10% of the bead radius was colonized by the protein. On the contrary, when immobilization is much slower, a homogeneous distribution is obtained, resulting in beads whose whole radius is occupied by the protein. Therefore, we set that the more rapid immobilization, the more heterogeneous distribution. All the knowledge gained in protein distribution by immobilization rate alteration of a single protein is applied to the co-immobilization of the two fluorescent proteins in order to develop four different co-immobilization patterns with an enormous applied potential to other multi-protein systems.     

Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose

Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD(+). In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD(+)-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.     

Design of β‐galactosidase/silica biocatalysts: Impact of the enzyme properties and immobilization pathways on their catalytic performance

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β‐galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro‐mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120‐fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β‐galactosidase immobilized on glyoxyl‐activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl‐activated silica was similar to that observed in macroporous agarose‐glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml^?1, 290 UI g^?1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.     

Preparation of immobilized/stabilized biocatalysts of β-glucosidases from different sources: Importance of the support active groups and the immobilization protocol

β-Glucosidases from two different commercial preparations, Pectinex Ultra SP-L and Celluclast® 1.5L, were immobilized on divinylsulfone (DVS) supports at pH 5.0, 7.0, 9.0, and 10. In addition, the biocatalysts were also immobilized in agarose beads activated by glyoxyl, and epoxide as reagent groups. The best immobilization results were observed using higher pH values on DVS-agarose, and for Celluclast® 1.5L, good results were also obtained using the glyoxil-agarose immobilization. The biocatalyst obtained using Pectinex Ultra SP-L showed the highest thermal stability, at 65°C, and an operational stability of 67% of activity after 10 reuses cycles when immobilized on DVS-agarose immobilized at pH 10 and blocked with ethylenediamine. The β-glucosidase from Celluclast® 1.5L produced best results when immobilized on DVS-agarose immobilized at pH 9 and blocked with glycine, reaching 7.76-fold higher thermal stability compared to its free form and maintaining 76% of its activity after 10 successive cycles. The new biocatalysts obtained by these protocols showed reduction of glucose inhibition of enzymes, demonstrating the influence of immobilization protocols, pH, and blocking agent.     

Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization

The immobilization of a glutamate dehydrogenase from Thermus thermophilus (GDH) on glyoxyl agarose beads at pH 7 has permitted to perform the immobilization, purification and stabilization of this interesting enzyme. It was cloned in Escherichia coli and a first thermal shock of the crude preparation destroyed most mesophilic multimeric proteins. Glyoxyl agarose can only immobilize enzymes via a multipoint and simultaneous attachment. Therefore, only proteins having several terminal amino groups in a position that permits their interaction with a flat surface can be immobilized. GDH became rapidly immobilized at pH 7 and its multimeric structure became stabilized as evidenced by SDS-PAGE. This derivative was stable at acidic pH value while the non-stabilized enzyme was very unstable under these conditions due to subunit dissociation. After immobilization, a further incubation at pH 10 improved enzyme stability under any inactivating conditions by increasing the enzyme–support bonds. In fact, GDH immobilized at pH 7 and incubated at pH 10 preserved more activity than GDH directly immobilized at pH 10 (50% versus 15% after 24 h of incubation) and was also more stable (1.5- to 3-fold, depending on the conditions).This method could be extended to any other multimeric enzyme expressed in mesophilic hosts.     

Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus

First, the enzyme immobilized on cyanide bromide agarose beads (CNBr) (that did not involve all enzyme subunits in the immobilization) has been crosslinked with aldehyde-dextran. This preparation did not any longer release enzyme subunits and become fully stable at pH 4 and 25 °C.Then, the stabilities of many different enzyme preparations (enzyme immobilized on CNBr, that derivative further crosslinked with aldehyde-dextran, enzyme immobilized on highly activated amino-epoxy supports, GDH immobilized on supports having a few animo groups and many epoxy groups, GDH immobilized on glyoxyl-agarose beads at pH 7, and that preparation further incubated at pH 10, and finally the enzyme immobilized on this support directly at pH 10) were compared at pH 4 and high temperatures, conditions where both dissociation and distortion play a relevant role in the enzyme inactivation. The most stable preparation was that prepared at pH 7 and incubated at pH 10, followed by GDH immobilized on amino and epoxy supports and the third one was the enzyme immobilized on glyoxyl-agarose at pH 10.The incubation of all enzyme preparations in saturated guanidine solutions produced the full inactivation of all enzyme preparations. When not all enzyme subunits were immobilized, activity was not recovered at all. Among the other derivatives, only glyoxyl preparations (the most inert supports and those where a more intense multipoint covalent attachment were expected) gave significant reactivation when re-incubated in aqueous medium. After optimization of the reactivation conditions, the enzyme immobilized at pH 7 and later incubated at pH 10 recovered 100% of the enzyme activity.     

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.     

Stabilization of a formate dehydrogenase by covalent immobilization on highly activated glyoxyl-agarose supports

Formate dehydrogenase (FDH) is a stable enzyme that may be readily inactivated by the interaction with hydrophobic interfaces (e.g., due to strong stirring). This may be avoided by immobilizing the enzyme on a porous support by any technique. Thus, even if the enzyme is going to be used in an ultra-membrane reactor, the immobilization presents some advantages. Immobilization on supports activated with bromocianogen, polyethylenimine, glutaraldehyde, etc., did not promote any stabilization of the enzyme under thermal inactivation. However, the immobilization of FDH on highly activated glyoxyl agarose has permitted increasing the enzyme stability against any distorting agent: pH, T, organic solvent, etc. The time of support-enzyme reaction, the temperature of immobilization, and the activation of the support need to be optimized to get the optimal stability-activity properties. Optimized biocatalyst retained 50% of the offered activity and became 50 times more stable at high temperature and neutral pH. Moreover, the quaternary structure of this dimeric enzyme becomes stabilized by immobilization under optimized conditions. Thus, at acidic pH (conditions where the subunit dissociation is the first step in the enzyme inactivation), the immobilization of both subunits of the enzyme on glyoxyl-agarose has allowed the enzyme to be stabilized by hundreds of times. Moreover, the optimal temperature of the enzyme has been increased (even by 10 degrees C at pH 4.5). Very interestingly, the activity with NAD(+)-dextran was around 60% of that observed with free cofactor.     

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.     

Optimization of an industrial biocatalyst of glutaryl acylase: stabilization of the enzyme by multipoint covalent attachment onto new amino-epoxy Sepabeads

Glutaryl-7-aminocephalosporanic acid acylase (GA), an industrially relevant enzyme, has been immobilized onto very different supports, including glyoxyl agarose, heterofunctional epoxy Sepabeads, glutaraldehyde and cyanogen bromide (CNBr) activated supports. Immobilization onto amino-epoxy Sepabeads rendered the most thermo stable preparation of GA, with a half-life time eight times higher than the soluble enzyme, keeping 80% of the enzyme activity. Several parameters that affect the enzyme-support interaction (pH and incubation time) were studied. It was found that after immobilization onto amino-epoxy Sepabeads, incubation at alkaline pH and low temperature exerted dramatic stabilizing effects, increasing the half-life time of the derivative 130 times with respect to the soluble enzyme, while keeping unaltered its intrinsic activity. The loading capacity of the amino-epoxy Sepabeads proved to be very good with a maximum load of 62 mg of protein per g of support with 85 IU/g at 25 degrees C and 200 IU/g at 37 degrees C which makes it a biocatalyst of possible industrial application.     

Immobilization–stabilization of a new recombinant glutamate dehydrogenase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.     

Immobilization of functionally unstable catechol-2,3-dioxygenase greatly improves operational stability

Thermophilic catechol 2,3-dioxygenase (EC 1.13.11.2) from Bacillus stearothermophilus has been immobilized on highly activated glyoxyl agarose beads. The enzyme could be fully immobilized at 4 degrees C and pH 10.05 with a high retention of activity (around 80%). Enzyme immobilized under these conditions showed little increase in thermostability compared with the soluble enzyme, but further incubation of immobilized enzyme at 25 degrees C and pH 10.05 for 3 h before borohydride reduction resulted in conjugates exhibiting a 100-fold increase in stability (c.f. the free enzyme). The stability of catechol 2,3-dioxygenase immobilized under these conditions was essentially independent of protein concentration whereas free enzyme was rapidly inactivated at low protein concentrations. An apparent stabilization factor of over 700-fold was recorded in the comparison of free and immobilized catechol 2,3-dioxygenases at protein concentrations of 10 μg/ml. Immobilization increased the 'optimum temperature' for activity by 20 degrees C, retained activity at substrate concentrations where the soluble enzyme was fully inactivated and enhanced the resistance to inactivation during catalysis. These results suggest that the immobilization of the enzyme under controlled conditions with the generation of multiple covalent links between the enzyme and matrix both stabilized the quaternary structure of the protein and increased the rigidity of the subunit structures.