Effect of immobilization on the stability and substrate specificity of α-d-galactosidase isolated from the invertebrate Turbo cornutus (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.
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 of ?-galactosidases from Thermus aquaticus YT-1 for oligosaccharides synthesis
Biotechnology Techniques, 1995
B-galactosidases of Thermus aquaticus YT-1, exhibiting a galactosyl transferase activity, were immobilized using different techniques. Entrapment in agarose or gellan gum beads was unsuitable for enzyme immobilization due to enzyme leakage. A technique that efficiently immobilized the enzymes was developed using glutaraldehyde co-crosslinking of B-galactosidases with bovine serum albumin, followed by entrapment in agarose beads.
Influence of the immobilization chemistry on the properties of immobilized β-galactosidases
Journal of Molecular Catalysis B: Enzymatic, 2001
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. q
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.
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.
Applied Biochemistry and Biotechnology, 2009
Partially purified α-Galactosidase from Penicillium griseoroseum was immobilized onto modified silica using glutaraldehyde linkages. The effective activity of immobilized enzyme was 33%. Free and immobilized α-galactosidase showed optimal activity at 45 °C and pH values of 5 and 4, respectively. Immobilized α-galactosidase was more stable at higher temperatures and pH values. Immobilized α-galactosidase from P. griseoroseum maintained 100% activity after 24 h of incubation at 40 °C, while free enzyme showed only 32% activity under the same incubation conditions. Defatted soybean flour was treated with free and immobilized α-galactosidase in batch reactors. After 8 h of incubation, stachyose was completely hydrolyzed in both treatments. After 8 h of incubation, 39% and 70% of raffinose was hydrolyzed with free and immobilized α-galactosidase respectively. Immobilized α-galactosidase was reutilized eight times without any decrease in its activity.