Co-immobilization of pectinase and glucoamylase onto sodium aliginate/graphene oxide composite beads and its application in the preparation of pumpkin-hawthorn juice

Abstract

Co‐immobilization of pectinase and glucoamylase onto sodium alginate/graphene oxide beads was achieved by N,N′‐dicyclohexylcarbodiimide/N‐hydroxysuccinimide as activating agent. The co‐immobilized pectinase‐glucoamylase (I‐PG) prepared under optimal conditions (pH 4.0, 40°C and 35 min) possessed pectinase activity of 1,227.5 ± 36.5U/g and glucoamylase activity of 1,027.2 ± 29.2U/g, with activity recovery of 73.8% and 85.2%, respectively. Both pectinase and glucoamylase in I‐PG possessed wider pH tolerance and superior thermal stability to those of their free counterparts. Reusability studies indicated that both enzymes in I‐PG retained over 60% of initial activity after six times of reuse. Conditions for the hydrolysis of the pumpkin–hawthorn compound juice by I‐PG were optimized using orthogonal experiments. After treatment with I‐PG, light transmittance, soluble solids, and reducing sugar content in the resulting juice increased significantly, whereas soluble protein and pectin content decreased appreciably. Therefore, the use of I‐PG provided an effective and feasible method for improving quality of the pumpkin–hawthorn juice. Practical applications In order to overcome the drawbacks of using free pectinase and glucoamylase, an effective method for the co‐immobilization of these two enzymes onto sodium alginate/graphene oxide beads was developed. The co‐immobilized pectinase/glucoamylase developed in this study could be applied in the clarification of juice rich in pectin and starch.

Enhanced Performance of Immobilized Xylanase/Filter Paper-ase on a Magnetic Chitosan Support

“…Catalysts 2019, 9, x FOR PEER REVIEW 6 of 10 stability could be to immobilize the labile and hard-to-stabilize enzyme by ion exchange on the already-immobilized resistant enzyme to desorb the least-stable enzyme when its activity decreases. Other authors have reported outstanding reusability when using immobilized hydrolase systems, attributed to high retention of biocatalysts in the nanoparticles [11,17]. However, previous studies using a complex of hydrolytic enzymes immobilized on magnetic particles have reported activity drops to 20% after the second reuse cycle [16,28].…”

“…Many reports have shown a wide variety of results for immobilization yield and efficiency that are attributed to the nature of the immobilization protocol, supporting material, cross-linker agent, and the particular sizes of enzymes and their substrates [6,7,10,19]. A synergistic effect was demonstrated when using immobilized enzymes degrading complex substrates rather than using a mixture of individual immobilized enzymes, which was attributed to the reduction of product inhibition, a limit to the accumulation of intermediates, and more bound enzymes able to attack large substrates, minimizing steric hindrance [11,13,17]. Cross-linking alters the microstructure of chitosan by the formation of covalent bonds between enzymes and Fe 3 O 4 @chitosan, as was previously discussed [22], thus impacting in biochemical behavior of the biocatalyst.…”

“…Then, the immobilized enzymes (Fe3O4@Chitosan@Enzymes) were collected from the reaction using an external neodymium magnet (1.2 T), rinsed with ethanol 70% v/v and then with Milli-Q water, and speed vac dried (DyNA vap centrifugal evaporator, Labnet, Edison, NJ, USA) at 60 °C. The immobilization yield (Equation (1)), immobilization Other authors have reported outstanding reusability when using immobilized hydrolase systems, attributed to high retention of biocatalysts in the nanoparticles [11,17]. However, previous studies using a complex of hydrolytic enzymes immobilized on magnetic particles have reported activity drops to 20% after the second reuse cycle [16,28].…”

“…Moreover, in recent years, the development of immobilized multienzymatic systems is increasingly guided by economic and environmental drawbacks, leading to the development of new multistep synthetic methods [10]. Further, it has been reported that multienzyme immobilization could confer superior activity, stability, and performance compared to individual immobilized enzymes, especially when using complex substrates [11][12][13]. However, among the disadvantages of multienzyme immobilization are the different stabilities of each biocatalyst involved, protocol design, or the occurrence of undesired interactions [14].…”

Biocatalysis in the winemaking industry: Challenges and opportunities for immobilized enzymes

“…Enzyme co-immobilization requires adequate immobilization methods considering a judicious selection of the support and mechanism of immobilization, and a thorough characterization of the enzymes and the kinetics of the reactions involved (Arana-Peña et al, 2019;El-Zahab, Meza, Cutright, & Wang, 2004;Hwang & Lee, 2019;Lopez-Gallego & Schmidt-Dannert, 2010;Ren et al, 2019;Sun et al, 2014;Torres & Batista-Viera, 2019;Yang, Dai, Wei, Zhu, & Zhou, 2019). It is expected that the activity of the enzymes can be preserved during co-immobilization while increasing their stability, hopefully attaining similar operational half-life values of each enzyme partner (Betancor & Luckarift, 2010).…”