Improvement of covalent immobilization procedure of β‐galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study

Abstract

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by β-galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane-polyvinyl alcohol (POS-PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL initial lactose concentration and 6 U mL enzyme concentration, obtaining 25.46 ± 0.01 gL yield of trisaccharides. Although below the HPLC-IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL of trisaccharides and 13.79 ± 0.21 gL of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady-state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568-1578, 2017.

Kinetic Model for Galacto-Oligosaccharide Synthesis

“…GOS synthesis depends on different reaction parameters, except for the type of enzyme. In the literature, optimization of various parameters, such as initial lactose and enzyme concentration, reaction time, pH, and temperature, has been reported as crucial for improving the GOS synthesis. ,,− According to research, the effect of temperature and pH, in the enzyme’s working range, was proven to be insignificant compared to the influence of the initial substrate and enzyme concentration on GOS production. ,,, Thus, a detailed understanding of the reaction kinetics and the development of mechanistic models that would fit experimental data, obtained within a wide range of conditions, is required for the design and process optimization of biochemical reactors.…”

“…Micro-kinetic modeling is widely used to describe various enzymatic conversions of various biologically based resources. − For example, there were numerous attempts to describe the reaction mechanism of GOS synthesis, all of which emphasized its complexity due to the existence of several parallel reactions and the simultaneousness of processes of transgalactosylation and lactose hydrolysis. Upon these findings, a few kinetic models, based on Michaelis–Menten-type equations or the micro-kinetic approach, ,,,− have been proposed. All reported models are based on a reaction mechanism describing the simultaneous synthesis of GOS and lactose hydrolysis with the central role of the galactose–enzyme (EGAL) complex, which can then decompose to form galactose in hydrolysis or react with different sugars to form GOS of different degree of polymerization and composition.…”

“…The proposed models differ in level of complexity with regard to GOS composition, number of reaction steps and constants, and occurrence of product inhibition. Hence, several reports include only modeling of cumulative GOS concentration, ,,,, while others include models for each detected GOS species. , Also, in two reports, , inhibition of enzyme with products (glucose and galactose) was observed and related reactions were included in the kinetic model. Consequently, the complexity of estimation differs, since the number of rate constants for estimation varies significantly in the reported studies, between 9 and 16.…”

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β‐galactosidase

“…Several compounds have been used for modification of epoxy groups in commercial supports aiming to evaluate different methods of immobilization. Each compound provides its own characteristics to the material, such as the crosslinking of glutaraldehyde; oxidation through acid solution action to produce carboxyl groups; activation of carboxyl groups with 1,1‐(3‐dimethylamino‐propyl)‐ethyl‐carbodiimide hydrochloride by the reaction of nucleophilic agents on protonated carbodiimide, under slightly acid conditions, with amino groups of proteins, and addition of amine groups using ethylenediamine . However, few manuscripts reported the modification of epoxy groups on Immobead 150, and to our knowledge, the use of acid solution has not yet been described.…”

Sodium bicarbonate‐gelled chitosan beads as mechanically stable carriers for the covalent immobilization of enzymes

“…β‐Gal was immobilized via entrapment, adsorption, and covalent techniques. β‐Gal was also affinity immobilized where the β‐gal antibodies were first attached to the immobilization carrier, and then the β‐gal was immobilized onto this affinity carrier . The immobilization of enzymes is a highly beneficial process.…”