Some factors affecting the stability of glucoamylase immobilized on hornblende and on other inorganic supports

Glucoamylase from four different companies was studied: three had similar stability (half-life at 50°C about 140 hr); the fourth was less stable (half-life at 50°C about 20 hr). The immobilized enzymes were all less stable than their soluble counterparts: immobilized enzyme stability depended on the soluble enzyme used, the support, and method of immobilization. Thus enzyme bound to Enzacryl-TIO was less stable than enzyme bound to hornblende (metal-link method); this, in turn, was less stable than enzyme bound to hornblende by a silane–glutaraldehyde process. Bound enzyme stability was also improved by the presence of substrate or product (starch maltose or glucose). After 110 hr at 50°C in the presence of maltose (10% (w/v)) one preparation (a more stable soluble enzyme boul1d to hornblende by a silane–glutaraldehyde process) retained over 95% of its activity: activity loss was too low to permit the estimation of a half-life.     

Compositions and compositional-behavioural relationships of enzymes immobilized on porous inorganic supports via titanium(IV) species

The compositions and compositional-behavioural relationships of glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) immobilized on titanium(IV)-activated porous inorganic supports have been investigated for several transition metal activation techniques based on the metal-link/chelation method developed by our group. The highest activity (239 Ug^?1 matrix) of immobilized glucoamylase was obtained with the hydrous titanium(IV) oxide derivative of the support when this and a 15% w/v TiCl₄ solution were dried at 45°C in vacuum for 30 h. However, the immobilized enzyme preparation displayed a very unstable behaviour, as did also the preparation which was obtained by drying the mixture of support and transition metal solution at atmospheric pressure. This was mainly due to an enzyme deactivation by titanium inhibition instead of enzyme loss in substrate solution. When amination and carbonylation steps were included in the immobilization technique much more stable preparations were obtained, mainly when the support was activated by drying at 45°C with a 15% w/v TiCl₄ solution ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1495}\text{h}$) although with a lower initial activity (35.6 Ug^?1 matrix). The pure TiCl₄ support activation rather than TiCl₄/HCl solution support activation led to less stable immobilized enzyme preparations (washing and amination solvent chloroform, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 365}\text{h}$; washing and amination solvent water, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 276}\text{h}$) than the preparation obtained with the dried titanium(IV)-activated support. This was due to loss of enzyme-titanium(IV) complex in solution, as the interactions between the titanium(IV) and the silanol groups of the porous silica are weak. However, the amination (with 1,6-diaminohexane) and carbonylation (with glutaraldehyde) steps always led to immobilized enzyme preparations with constant specific activities and protein/titanium(IV) ratio. This suggests that the spacing effect introduced by these reactions removes the titanium(IV) inhibition of glucoamylase.     

Enzyme immobilization via monoclonal antibodies I. Preparation of a highly active immobilized carboxypeptidase A

A novel method for the preparation of highly active immobilized enzymes is described. It is based on the binding of enzymes to suitable carriers via monoclonal antibodies, which bind to the enzyme with high affinity without affecting its catalytic activity. The applicability of the method forwarded has been illustrated by the preparation of two samples of highly active immobilized carboxypeptidase A (CPA) preparations as follows: A mouse monoclonal antibody (mAb 100)to CPA that binds to the enzyme with a high-affinity constant without affecting its catalytic activity was prepared, purified, and characterized. Covalent binding of this monoclonal antibody to Eupergit C (EC) or noncovalent binding to Sepharose-protein A (SPA)yielded the conjugated carriers EC-mAb and SPA.mAb, respectively, which reacted specifically with CPA to give the immobilized enzyme preparations EC-mAb.CPA and SPA.mAb.CPA displaying full catalytic activity and improved stability. At pH 7.5 and a temperature range of 4-37 degrees C an apparent binding constant of approximately 10(8)M(-1) characterizing the interaction of CPA with EC-mAb and SPA.mAb, was obtained. To compare the properties of EC-mAb.CPA and SPA.mAb.CPA with those of immobilized CPA preparations obtained by some representative techniques of covalent binding of the enzyme with a corresponding carrier, the following immobilized CPA preparations were obtained and their properties investigated: EC-CPA (I), a preparation obtained by direct binding of EC with CPA; EC-NH-GA-CPA (II), a derivative obtained by covalent binding of CPA to aminated EC via glutaraldehyde; EC-NH-Su-CPA (III), a CPA derivative obtained by binding the enzyme to aminated EC via a succinyl residue; and EC-HMD-GA-CPA (IV), obtained by binding the enzyme via glutaraldehyde to a hexamethylene diamine derivative of EC. Full enzymic activity for all of the bound enzyme, such as that recorded for the immobilized CPA preparations EC-mAb.CPA and SPA.mAb.CPA, was not detected in any of the insoluble covalently bound enzyme preparations.     

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.     

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.     

Immobilization of yeasts on titanium activated inorganic supports

Summary A study of the immobilization of yeast cells with invertase activity by the metal link method was performed. Baker's yeast cells were immobilized on titanium activated porous silica support and on its alkylamine and aldehyde derivatives, their initial activities being 19.6, 39.9 and 10.6 U/ml of reactor respectively. When crosslinking of the immobilized cells was performed, an initial activity of 48.2 U/ml was achieved on the titanium activated support. Batch long-term stability tests were car ried out for 400 hours and the crosslinked preparations showed an unsta ble behaviour compared with the very stable preparations obtained with the simple metal-link method.A higher activity (56.2 U/ml) was obtained when a titanium activated macroporous support, pumice stone, was used as cell carrier, which compared favourably with calcium alginate entrapped cells (17.7 – 31.3 U/ml)     

Immobilization of a proteinase from the extremely thermophilic organism Thermus Rt41A

An extracellular proteinase from Thermus strain Rt41A was immobilized to controlled pore glass (CPG) beads. The properties of the free and CPG-immobilized enzymes were compared using both a large (azocasein) and a small (peptidase) substrate. The specific activity of the immobilized proteinase was 5284 azoU/mg with azocasein and 144 sucU/mg for SucAAPFpNA. The percentage recovery of enzyme activity was unaffected by pore size when it was immobilized at a fixed level of activity/g of beads, whereas it increased with increasing pore size when added at a fixed level/m(2) of support. Saturation of the CPG beads was observed at 540 azoU/m(2) of 105-nm beads. Lower levels (50 azoU/m(2) of 50-nm beads) were used in characterization experiments. The pH optimum of the immobilized Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA, compared with the free proteinase which was 10.5 for both substrates. The immobilized enzyme retained 65% of its maximum activity against azocasein at pH 12, whereas the free proteinase retained less than 10% under the same conditions. Stability at 80 degrees C increased on immobilization at all pH values between 5 and 11, the greatest increase in half-life being approximately 12-fold at pH 7.0. Temperature-activity profiles for both the free and immobilized enzymes were similar for both substrates. The stability of the immobilized proteinase, however, was higher than that of the free enzyme in the absence and presence of CaCl(2). Overall, the results show that low levels of calcium (10 muM) protect against thermal denaturation, but that high calcium or immobilization are required to protect against autolysis. (c) 1994 John Wiley & Sons, Inc.     

Spherical vs. granular immobilization support selection and performance on an optical flow cell immunosensor based on the fluorescence of Cyanine-5

A spherical porous glass support Trisoperl (TRISO) with four pore diameters (? 47.8; 55.9; 102.6, and 108.8 nm) was characterized and selected for application in an optical flow cell immunosensor, in comparison with controlled pore glass (CPG). The TRISO support was functionalized with aldehyde and isothiocyanate (-NCS) groups to attach bovine serum albumin and alkaline phosphatase (AP). The TRISO isothiocyanate pore diameter 47.8 nm (TRISO(-NCS) 47.8 nm) showed the better potential to be used in the immunosensor. It immobilized more protein (19.3 mg AP per g support) while presenting an optical performance comparable to the CPG. CPG(-NCS) and TRISO(-NCS) 47.8 nm were tested in the immunosensor model where the saturation of the Goat IgG immobilized in the supports with Monoclonal Anti-Goat IgG conjugated with Cyanine-5 was reached, followed by regeneration with the elution buffer modified PBS pH 2.0. The TRISO(-NCS) 47.8 nm presented lower fluorescence intensity at saturation (around 39 AU) than CPG(-NCS) (150 to 104 AU), but revealed a major advantage related to the uniform arrangement of the spherical particles in the flow cell, generating no significant fluorescence differences between gravity and flow package.     

Investigation of the binding mechanism of glucoamylase to alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) has been coupled to several porous silica matrices by a new covalent process using alkylamine derivatives of titanium(IV)-activated supports. In order to investigate the interaction of the titanium element with the silanol groups of the inorganic matrices, activation was performed at different times, using titanium(IV) chloride, either pure or as a 15% w/v solution, in 15% w/v hydrochloric acid at 25, 45 and 80°C, followed by washing with sodium acetate buffer (0.02m, pH 4.5) or chloroform. Using pure TiCl₄, the highest activities of all preparations were obtained at 80°C and with acetate buffer washing, resulting from a higher content of titanium coating of the carrier. When activation was performed in aqueous TiCl₄ solution, followed by a drying step, the highest activity was obtained with preparations washed with chloroform, with or without amination. When reacting pure TiCl₄ with controlled pore glass (CPG) and with porous silica (Spherosil), colour formation was observed after reaction of glutaraldehyde with the aminated support. This did not happen when Celite was used as the support. As a criterion for comparison of the different immobilized enzyme preparations, the concept of an ‘instability factor’, which measures the percentage of immobilized enzyme activity due to release of enzyme into solution, is introduced. Instability factors of immobilized enzyme preparations on Celite were always higher than those obtained with the other matrices, confirming that there was no covalent coupling of the enzyme to Celite. However, when the activation was performed with aqueous TiCl₄ solution with drying, Schiff's base formation was observed in all preparations and very stable immobilized enzyme preparations were obtained. The results of the activation of controlled pore glass and porous silica with pure titanium(IV) chloride suggest the existence of a true reaction between the titanium element and the silanol groups of these carriers by formation of a bridge, Si-O-Ti, while with the titanium(IV) chloride solution in hydrochloric acid, a coating of hydrous titanium(IV) oxide is obtained.     

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.     

Post-immobilization of modified macromolecular reagents using assembled penicillin acylase for microenvironmental regulation of nanopores and enhancement of enzyme stability

Penicillin acylase (PA) is known to regulate the microenvironment of nanospores. In this study, nanopores containing chemically-modified macromolecules co-assembled with immobilized PA were constructed. We also investigated the various types of functionalized mesocellular siliceous foams (MCFs) commonly used for the immobilization of PA by measuring the catalytic performance and stability of each PA preparation. Amino-MCF activated by p-benzoquinone was chosen as the optimum support for PA immobilization. Successful modification of macromolecules was verified by FT-IR and ultraviolet (UV) spectroscopy. The specific activity of PA co-assembled with dextran 10 k was 99.1 U/mg, which was 1.5-fold that of pristine immobilized PA, while the optimum pH was shifted to neutral. Compared to pristine immobilized and free PA, the optimum temperatures for the modified PA were 5 and 10°C higher, respectively. The residual activity of the ficoll derivative of PA after treatment at 50°C for 6 h was 70%, and this was later increased to 214.5% compared to that of pristine immobilized PA. The dextran 10 k derivative of PA exhibited 90.2% residual activity after 25 times of continuous use. The results show that chemically-modified macromolecules co-assembled with PA in amino-MCF provided a suitable microenvironment for enzyme stability.     

Bioaffinity Based Oriented Immobilization of Stem Bromelain

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K _m values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V _max values remained unaffected on immobilization.     

Immobilization studies of whole microbial cells on transition metal activated inorganic supports

Summary Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial -d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.     

Characterization of Nitrosomonas europaea immobilized in calcium alginate

Nitrosomonas europaea cells have been immobilized in calcium alginate and the resulting preparation was used as a biocatalyst for the oxidation of NH⁺₄ to NO^?₂. Characterization of this immobilized biocatalyst was done according to the guidelines recommended by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The most important indications obtained from the results are: (a) at low concentrations of substrate, either ammonium ions or oxygen, diffusion limitation will play a role; (b) inhibition by nitrite ions accumulating in the support is not rapidly controlling the efficiency of the immobilized cells; (c) accumulation of hydrogen ions is a rate-limiting factor, especially in unbuffered solutions; (d) the activity of immobilized N. europaea can increase as a result of growth in the support under conditions which would cause washout of free cells. This last result shows the potential of immobilized N. europaea for nitrification of wastewater. The development of a system applying a cheaper and more stable support is, however, a prerequisite for this application.     

Immobilization of trypsin on chitosan gels: use of different activation protocols and comparison with other supports

Trypsin was immobilized on chitosan gels coagulated with 0.1 or 1 M NaOH and activated with glutaraldehyde or glycidol. The derivatives were characterized by their recovered activity, thermal (40, 55 and 70 degrees C) and alkaline (pH 11) stabilities, amount of enzyme immobilized on gels for several enzyme loads (8-14 mg(protein)/g(Gel)) and compared to agarose derivatives. Enzyme loads higher than 14 mg(protein)/g(Gel) can be immobilized on glutaraldehyde derivatives, which showed 100% immobilization yield and, for loads up to 8 mg(protein)/g(Gel), 100% recovered activity. Activation with glycidol led to lower immobilization yields than the ones obtained with glutaraldehyde, 61% for agarose-glyoxyl (AgGly) with low grade of activation and 16% for the chitosan-glyoxyl (ChGly), but allowed obtaining the most stable derivative (ChGly), that was 660-fold more stable than the soluble enzyme at 55 and 70 degrees C-approximately threefold more stable than AgGly. The ChGly derivative presented also the highest stability during incubation at pH 11. Analyses of lysine residue contents in soluble and immobilized trypsin indicated formation of multipoint bonds between enzyme and support, for glyoxyl derivatives.     

An improved CPG support for the synthesis of 3'-amine-tailed oligonucleotides.

A new controlled-pore glass (CPG) support is described that allows for the direct synthesis of oligonucleotides bearing a 3'-aminohexyl tail. This solid support (AH-CPG) exhibits superior performance as compared to a commercially available 3'-amine CPG. The AH-CPG is prepared from 6-aminohexan-1-ol with a unique protecting group for the amine that also functions as the site of attachment to the CPG. A 3'-amine-tailed oligodeoxynucleotide (ODN) was prepared from this support using standard phosphoramidite coupling and deprotection conditions. The 3'-amine-tailed ODN was subsequently modified with an acridinylpropionic acid tetrafluorophenyl ester. Facile synthesis of the AH-CPG and the stability of the deprotected product makes this functionalized solid support especially useful for preparation of oligonucleotides bearing 3'-amine tails and other modifications.     

Production of xylo-oligosaccharides by immobilized-stabilized derivatives of endo-xylanase from Streptomyces halstedii

An endoxylanase from Streptomyces halstedii was stabilized by multipoint covalent immobilization on glyoxyl-agarose supports. The immobilized enzyme derivatives preserved 65% of the catalytic activity corresponding to the one of soluble enzyme that had been immobilized. These immobilized derivatives were 200 times more stable 200 times more stable than the one-point covalently immobilized derivative in experiments involving thermal inactivation at 60 °C. The activity and stability of the immobilized enzyme was higher at pH 5.0 than at pH 7.0. The optimal temperature for xylan hydrolysis was 10 °C higher for the stabilized derivative than for the non-stabilized derivative. On the other hand, the highest loading capacity of activated 10% agarose gels was 75 mg of enzyme per mL of support. To prevent diffusional limitations, low loaded derivatives (containing 0.2 mg of enzyme per mL of support) were used to study the hydrolysis of xylan at high concentration (close to 1% (w/v)). 80% of the reducing sugars were released after 3 h at 55 °C. After 80% of enzymatic hydrolysis, a mixture of small xylo-oligosaccharides was obtained (from xylobiose to xylohexose) with a high percentage of xylobiose and minimal amounts of xylose. The immobilized-stabilized derivatives were used for 10 reaction cycles with no loss of catalytic activity.     

Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K_m value of the immobilized α-amylase was decreased by immobilization while V_max was unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.     

Optimal pyoverdin-CPG composites for development of an optical biosensor to detect iron

Optical fluorescence-quenching-based biosensing cell is described and optimization of covalent binding of highly selective natural iron-chelating peptide secreted by bacteria is suggested. Pyoverdin biosynthesized by Pseudomonas monteilii and having 70% iron chelating activity was immobilized on amino alkylated controlled pore glass (CPG) and cross-linked with glutaraldehyde (2.5%, 28°C, 30 min). The pyoverdin-CPG immobilization was confirmed using fluorescence microscopic images (excitation range, 465–495 nm) for bright green fluorescence and by FTIR spectrum stretching at 3406.4 cm⁻¹ for amino group. The pyoverdin loading capacity of activated CPG matrix was 25 mg g⁻¹ of CPG and its rinsing analysis (leaking profile of the immobilized peptide vs. washing) detected negligible (2–3 μg) pyoverdin in the second wash.     

Silica carriers for immobilization of thermostable α-amylase

The possibilities for immobilization of thermostable α-amylase from Bacillus licheniformis 44MB82 on silica carriers activated by different methods have been studied. Immobilization on Ti(IV)-activated CPG, Chromosorb P, quartz powder and pumice stone was sufficiently effective. The preparation immobilized on CPG showed a shift in pH and temperature-profiles of enzyme action. They were found to be changed from pH 6,0–6,5 to pH 8,5 and from 90°C to 80°C, respectively, when compared to these parameters of the soluble enzyme. Immobilization leads to enhancement of thermostability. Possibilities for batchwise use of the immobilized preparation were established.