Influence of the immobilization chemistry on the properties of immobilized β-galactosidases (original) (raw)
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Influence of the immobilization chemistry on the properties of immobilized �-galactosidases
J Mol Catal B Enzym, 2001
Uruguaý´´Á bstract Ž) Ž. Neutral b-galactosidases from E. coli and K. lactis were bound to glutaraldehyde-agarose Glut-agarose through Ž. amino groups, and to thiolsulfinate-agarose TSI-agarose through thiol groups. In general, TSI-gels exhibited higher yields Ž. Ž. after immobilization 60-85% than Glut-gels 36-40%. The kinetic parameters of the enzymes bound to TSI-gels Ž. particularly those with lower concentration of active groups were less affected than those of the Glut-gels. This might indicate that the binding to TSI-agarose is more conservative of the protein conformation. However, the Glut-derivatives exhibited in general better thermal and solvent stabilities than TSI-derivatives. The stability of the derivatives was studied in Ž. the presence of ethanol, dioxane and acetone 18% vrv. The stabilization of the immobilized enzymes, for some of the solvents assayed, was evidenced by the existence of final very stable enzyme states with high residual activities, thus allowing the utilization of the derivatives in the presence of organic cosolvents.
Influence of the immobilization chemistry on the properties of immobilized [beta]-galactosidases
Journal of Molecular Catalysis B Enzymatic
Neutral b-galactosidases from E. coli and K. lactis were bound to glutaraldehyde-agarose Glut-agarose through Ž . amino groups, and to thiolsulfinate-agarose TSI-agarose through thiol groups. In general, TSI-gels exhibited higher yields Ž . Ž . after immobilization 60–85% than Glut-gels 36–40% . The kinetic parameters of the enzymes bound to TSI-gels Ž . particularly those with lower concentration of active groups were less affected than those of the Glut-gels. This might indicate that the binding to TSI-agarose is more conservative of the protein conformation. However, the Glut-derivatives exhibited in general better thermal and solvent stabilities than TSI-derivatives. The stability of the derivatives was studied in Ž . the presence of ethanol, dioxane and acetone 18% vrv . The stabilization of the immobilized enzymes, for some of the solvents assayed, was evidenced by the existence of final very stable enzyme states with high residual activities, thus allowing the utilization of ...
β-?-Galactosidases immobilized on soluble matrices: Kinetics and stability
Enzyme and Microbial Technology, 1985
Three ~-o-galactosidases {13-D-galactoside galactohydrolase, I';C 3.2.1.23) from different origins have been immobilized on sucrose-polyacrolein and sucrose sulphate-polyacrolein. This gave enzyme conjugates insoluble in the immobilization medium 6ut which could be made soluble by reduction with sodium borohydride before use. The optimum conditions fbr both copolymer synthesis and the immobilization reaction were investigated. Lr. and 13C n.m.r, spectroscopy were used to jbllow the sulphation and the copoO'merization reaction. The characteristics of the enzyme conjugates were compared with those of the free enzymes: the Vma x values of the enzyme conjugates were lower t/tan those of the corresponding free enzymes, whilst the K m values were similar. The thermal stability of the enzyme conjugates depended on the enzyme origin, while their ptl stability was in all cases higher than that of the free enzymes. These data suggest some ad~,antages in using enzyme immobilization supports which can be made soluble after separation of the immobilized enzyme without altering the enzyme characteristics.
Journal of Molecular Catalysis B: Enzymatic, 1998
. The covalent immobilization of b-galactosidase from KluyÕeromyces lactis b-gal on to two different porous carriers, CPC-silica and agarose, is reported. CPC-silica was silanizated and activated with glutaraldehyde. The activation of agarose Ž . via a cyanylating agent CDAP was optimized. Gel-bound protein and gel-bound activity were both measured directly, Ž . allowing the determination of apparent specific activities S.A. . Higher amounts of b-gal were immobilized on the activated Ž y1 . CPC-silica maximum capacity, 23 mg ml of packed support than on the CDAP-activated agarose. For the lower enzyme Ž y1 . loading assayed 12.6 mg ml packed support , 100% of the enzyme was immobilized but only 34% of its activity was Ž y1 expressed. This inactivation during immobilization was confirmed by the S.A. values 22-29 EU mg for the CPC-deriva-y1 . Ž . tives and 80 EU mg for soluble b-gal . The K 3.4 mM for the CDAP-derivative with ONPG as substrate was higher app Ž . than the K value for soluble b-gal 2 mM . When the enzyme loading was increased five-fold, the K increased M app four-fold, to 13 mM. The V values for the CPC-derivatives were remarkably lower than the V for soluble app max b-galactosidase. CDAP-derivatives showed better thermal stabilities than CPC-derivatives but neither of them enhanced the stability of the soluble enzyme. When stored at 48C, the activity of both derivatives remained stable for at least 2 months. Ž . Both derivatives displayed high percentages of lactose conversion 90% in packed bed mini-reactors. Glucose production was 3.3-fold higher for the CPC-derivative than for the CDAP-derivative, as a consequence of the higher flow rates achieved. q 1998 Elsevier Science B.V. All rights reserved.
Immobilization and stabilization of α-galactosidase on Sepabeads EC-EA and EC-HA
International Journal of Biological Macromolecules, 2011
␣-Galactosidase from tomato has been immobilized on Sepabead EC-EA and Sepabead EC-HA, which were activated with ethylendiamino and hexamethylenediamino groups, respectively. Two strategy was used for the covalent immobilization of ␣-galactosidase on the aminated Sepabeads: covalent immobilization of enzyme on glutaraldehyde activated support and cross-linking of the adsorbed enzymes on to the support with glutaraldehyde. By using these two methods, all the immobilized enzymes retained very high activity and the stability of the enzyme was also improved. The obtained results showed that, the most stable immobilized ␣-galactosidase was obtained with the second strategy. The immobilized enzymes were characterized with respect to free counterpart. Some parameters effecting to the enzyme activity and stability were also analyzed. The optimum temperature and pH were found as 60 • C and pH 5.5 for all immobilized enzymes, respectively. All the immobilized ␣-galactosidases were more thermostable than the free enzyme at 50 • C. The stabilities of the Sepabead EC-EA and EC-HA adsorbed enzymes treated with glutaraldehyde compared to the stability of the free enzyme were a factor of 6 for Sepabead EC-EA and 5.3 for Sepabead EC-HA. Both the free and immobilized enzymes were very stable between pH 3.0 and 6.0 and more than 85% of the initial activities were recovered. Under the identical storage conditions the free enzyme lost its initial activity more quickly than the immobilized enzymes at the same period of time. The immobilized ␣-galactosidase seems to fulfill the requirements for different industrial applications.
International Journal of Biological Macromolecules, 2018
In the present work, we aimed to explore the molecular binding between alginate and β-galactosidase, as well as the effect of this interaction on the activity retention, thermal stability, and kinetic properties of the enzyme. The impact of pH and enzyme/alginate ratio on physicochemical properties (turbidity, morphology, particle size distribution, ζ-potential, FTIR, and isothermal titration calorimetry) was also evaluated. The ratio of biopolymers and pH of the system directly affected the critical pH of complex formation; however, a low alginate concentration (0.1 wt%) could achieve an electrical charge equivalence at pH 3.4 with 93.72% of yield. The binding between β-galactosidase and alginate was an equilibrium between enthalpic and entropic contributions, which promoted changes in the structure of the enzyme. Nevertheless, this conformational modification was reversible after the dissociation of the complex, which allowed the enzyme to regain its activity. These findings will likely broaden functional applications of enzyme immobilization.
J Biosciences, 1982
β-D-Galactosidase (EC 3.2.1.23) from Lactobacillus bulgaricus (1373) was immobilized in a Polyacrylamide gel lattice in the presence of dithiothreitol, glutathione, cysteine, bovine serum albumin, casein, lactose and glucono-δ-lactone. Cysteine, bovine serum albumin, and lactose were found very effective in preserving the activity. With cysteine, bovine serum albumin and lactose, the activity yields were 61, 60 and 66% respectively, as compared to 31% without protective agents. The yield improved upto 85% when all the three protective agents, cysteine, bovine serum albumin and lactose were added during immobilization. The addition of protective agents did not have any effect on optimum pH, optimum temperature, kinetic constants and pH stability when compared with β-galactosidase immobilized without the use of protective agents; however the heat and storage stabilities were found to increase.
Enzyme and Microbial Technology, 2008
A very stable ␣-galactosidase from Thermus sp. T2 has been immobilized on different supports activated with glyoxyl, epoxy or glutaraldehyde groups. Although all preparations retained very high activity (usually over 90%) and all immobilization protocols improved the enzyme stability, the best stability was obtained by immobilization on glutaraldehyde activated supports. Using glutaraldehyde, we compared the immobilization of the enzyme on pre-activated supports or the modification with glutaraldehyde of the enzyme previously adsorbed on amino-supports. The last strategy gave even more stable preparations, retaining over 90% of initial activity. Optimal conditions for the preparation of the immobilized preparations were 1% (v/v) glutaraldehyde and support activated with 40 mol/mL of support. This preparation retained 90% initial activity after 48 h at pH 7 and 75 • C while the soluble enzyme was fully inactivated after only 8 h. Moreover, this immobilization protocol improved the optimal temperature from 65 • C (soluble enzyme) to 70 • C.
Optimal Immobilization of β-Galactosidase onto κ
2014
Galactosidase (-gal) was immobilized by covalent binding on novel-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 2 2 full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55 ∘ C compared to the free enzyme. The apparent after immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocity max was 131.2 mol⋅min −1 while it was 177.1 mol⋅min −1 for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of the-carrageenan beads loaded with-galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.