Immobilization of glucose oxidase in poly(2-hydroxyethyl methacrylate) membranes

doi:10.1016/0142-9612(87)90087-1

Biomaterials

Volume 8, Issue 6, November 1987, Pages 489-495

https://doi.org/10.1016/0142-9612(87)90087-1 Get rights and content

Abstract

Glucose oxidase (GOD) was immobilized in a poly(2-hydroxyethyl methacrylate) (HEMA) membrane through matrix entrapment in order to investigate the effect of various parameters (e.g. concentration of ingredients, temperature, repeated interaction with glucose and shelf storage) on the activity of the enzyme. Permeability of the membrane to a model permeant was tested and SEMs were obtained. It was observed that upon immobilization the affinity of GOD towards glucose was substantially decreased, and increasing the GOD content of the membrane adversely affected the activity. Membranes with the highest enzyme activity were also found to be the most permeable. Changes were detected in the pH and temperature where GOD is most active. Membrane permeability was observed to increase when crosslinker, and/or HEMA concentrations were low. The same parameters were also found to alter the morphology of the membrane as observed under SEM.

References (29)

C. Bertrand et al.
Multipurpose electrode with different enzyme systems bound to collagen films
Anal. Chim. Acta.
(1981)
T. Yao
A chemically modified enzyme membrane electrode as an amperometric glucose sensor
Aial. Chim. Acta
(1983)
E. Staude et al.
Reactions with enzymes covalently bonded to heterogeneous ultrafiltration membranes
J. Membrane Sci.
(1982)
O.H. Lowry et al.
Protein measurement with the folin phenol reagent
J. Biol. Chem.
(1951)
V.M. Hasirci
Synthesis and characterization of PVNO-PVP hydrogels
Biomaterials
(1981)
R. Axen
Methods for preparing immobilized enzymes
E. Katzir-Katchalski
Enzyme membrane
O.O. Zaborsky
K.F. O'Driscoll
Technique of enzyme entrapment in gels
J. Kost et al.
Glucose sensitive membranes containing glucose oxidase activity. Swelling and permeability studies
J. Biomed. Mater. Res.
(1985)

F. Tsuchida et al.

Application of l-( + )-lactate electrode for clinical analysis

Biotechnol. Bioeng.

(1985)

N.A. Peppas et al.

The structure of highly crosslinked poly(2-hydroxyethyl methacrylate) hydrogels

J. Biomed. Mater. Res.

(1985)

R.Y.M. Huang et al.

lonically crosslinked hydrophilic polymer membranes: synthesis and measurement of transport properties

J. Polymer Sci.

(1973)

Cited by (98)

Modulation of polymer-based immobilized enzymes for industrial scale applications
2021, Polymeric Supports for Enzyme Immobilization: Opportunities and Applications
Enzymes have become crucial part of various industries like development and production of nutritious food and beverage products, processing of fruit and vegetable, cheese, protein, grains, and fats and oils, as well as in several other industries like dairy, baking, brewing, and cereal extraction. It has been seen since last decade that enzymes have been sharing major part in global market with its increasing usage in various sectors. R&D centers of several companies have been now focusing on evaluation, development, upscaling (usually by expression and cloning of enzymes), validation of technologies, and innovative enzyme formulation for various commercial processes. Enzyme technology has been known for several hundreds of years; however, 21st century has revolutionized it due to its increased applications in almost every sector. According to MarketsandMarkets, the enzymes have market of USD 5.9 billion (estimated for 2020), while it has been expected to reach USD 8.7 billion by 2026 according to CAGR. The industrial applications of enzymes have prerequisite of enzyme to be in its insoluble state as soluble state is fragile requiring extensive protocols for its stability during long storage as well as cost expensive due to its single use. The enzymes in its immobilized state can be used for several times with easier recovery from reaction mixture and maintained for large time periods. The enzyme immobilization is actually a platform that involves the combination of enzyme selectivity and kinetics with the physical and chemical properties of the immobilizing matrix in a specialized formulation to maximize both its stability and catalysis and make cost-effective industrial process. Most importantly, scaling up of immobilized process from lab to industries requires lesser optimization in a cost-effective manner than that based on soluble enzymes. Further, immobilized enzyme–based industrial processes have been enticing due to least time consumption and cost efficacious downstream processing. The present chapter is based on modulating enzyme immobilization so that lab scale experimentation can be easily scaled up to industrial scale processes with maximum product-yield and minimal downstream processing.
Phospholipid bilayer functionalized membrane pores for enhanced efficiency of immobilized glucose oxidase enzyme
2017, Journal of Membrane Science
Citation Excerpt :
On the other hand, maximum activity of immobilized GOx was observed at pH 6.5. Other studies have also reported a shift in optimum pH for maximum activity of immobilized enzyme [45–47]. The reason for this behavior was the change in the microenvironment (H+ and OH− concentrations) of the enzyme due to the functionalization within membranes [48].
This study integrates the advantages of biomimetic characteristics of phospholipid bilayer and convective flow through microporous membrane within the same configuration and reports the development of novel biofunctionalized membranes by incorporating phospholipid bilayer within membrane pores for efficient enzymatic catalysis. Two different approaches were used for membrane functionalization – (i) direct deposition of lipid bilayer within bare membrane pores, and (ii) immobilization of a polymer cushion prior to lipid bilayer within membrane pores. As a model enzyme, glucose oxidase (GOx) was immobilized into the phospholipid bilayer network within the functionalized membrane pores. In-depth activity study was conducted with different GOx-functionalized membranes while monitoring the production of H₂O₂ by oxidation of glucose. Biofunctionalized membrane, containing polymer cushion supported phospholipid bilayer and operating under convective mode of flow, exhibited important advancements in the field of immobilized enzymatic catalysis in terms of activity, stability and operating range of pH. Comparison of the results obtained in this study with other literature reported results establish the benefits of the biofunctionalized membrane discussed here. To the best of our knowledge this is the first study that investigates the advantages of incorporation of phospholipid bilayer within membrane pores for immobilized enzymatic catalysis.
Maltase entrapment approach as an efficient alternative to increase the stability and recycling efficiency of free enzyme within agarose matrix
2016, Journal of the Taiwan Institute of Chemical Engineers
Citation Excerpt :
Entrapment is one of the widely investigated immobilization methods where enzymes are enclosed or confined within polymer matrix without altering their native structure and used to develop bioreactors for commercial applications [9–11]. This method is based on the simple and single step procedure which induces no conformation alteration on the active site of enzyme and makes the process easy for the diffusion of substrate and product [12,13]. A variety of matrices such as alginate, agar-agar, carrageenan, agarose, polyacrylamide, pectin, chitosan and gelatin have been evaluated as a polymer matrix support for the immobilization of enzymes.
Maltase catalyses the hydrolysis of maltose and is widely used in the synthesis of various food and pharmaceutical products. In the current study, matrix entrapment technique was applied to immobilize maltase within agarose beads. Maximum immobilization yield (76.11%) was achieved at 3% agarose concentration and 5.0 mm beads size was found to be optimum combination for maximum catalytic activity of entrapped maltase. It was noticed that entrapment increased the optimum reaction temperature and pH of maltase from 45°C to 60°C and 6.5 to 7.0, respectively with reference to its free enzyme whereas no effect was observed on reaction time. Entrapped maltase showed increase in K_m value from 1.717 to 1.912 mM ml⁻¹ and decrease in V_max value from 8411.0 to 6214.0 U ml⁻¹ min⁻¹ as compared to free enzyme. Entrapped maltase displayed broad thermal stability up to 80°C whereas; free enzyme completely lost it activity when temperature reached up to 60°C. Scanning electron microscopy of agarose beads before and after maltase entrapment revealed significant morphological change on the matrix surface. Considering the economic feasibility, the entrapped maltase indicated imperative recycling efficiency up to ten reaction cycles.
Photo-irradiation for preparation, modification and stimulation of polymeric membranes
2009, Progress in Polymer Science (Oxford)
Citation Excerpt :
Hydrogel membranes for biomedical and biosensor applications are common examples for membrane preparation using photo-initiated polymerization technique [73]. A multi-step strategy to prepare membranes for enzyme immobilization was proposed by Arica et al. [74,75]. PolyHEMA membranes were prepared by UV-initiated polymerization of HEMA using azobisisobutyronitrile (AIBN) as “type I” photo-initiator (cf. Fig. 2).
The recent developments in combining photo-irradiation-based and membrane technologies are analyzed in this review. It is emphasized that the effects of photo-initiated reactions onto properties of polymeric membranes will largely depend on the nature of the membrane's barrier (i.e., porous vs. non-porous, uncharged vs. charged, or involving affinity interactions). For de novo preparation of membranes from low molar mass or soluble precursors, photo-initiated polymerization and photo-cross-linking are the main pathways, while photo-degradation for pore formation is only rarely applied. Membrane functionalization (modification) is described with many examples, organized into photo-cross-linking of membranes and photo-grafting of membrane surfaces, either via “grafting-to” or via “grafting-from” routes. Photo-stimulation of barrier properties is an attractive concept to create stimuli-responsive membranes, and various ways to use photo-chromic moieties for that purpose are discussed. Overall, photo-irradiation-based methods can be very versatile enabling technologies to improve the performance of polymeric membranes in technical separations (e.g., in gas separation, pervaporation, ultra- and microfiltration or as membrane adsorbers) and other processes (e.g., for controlled release or in sensor systems), and they will definitely also contribute to the development of entirely novel membrane-based materials.
Reversible immobilization of catalase by using a novel bentonite-cysteine (Bent-Cys) microcomposite affinity sorbents
2008, Colloids and Surfaces A: Physicochemical and Engineering Aspects
Citation Excerpt :
Single amino acid molecules could hold certain advantages as pseudo-affinity ligands for industrial application. These molecules are resistant to harsh chemicals and high temperatures as well as to their low cost [10,11]. Many supports have been employed to immobilize enzymes, the most important being polymers.
This study focused on developing bentonite–cysteine (Bent–Cys) microcomposite affinity sorbents (38–105 μm) for catalase adsorption from aqueous solutions. Pseudo-biospecific affinity ligand l-cysteine was covalently binded onto the bentonite structures. X-ray diffraction and FTIR analysis of Bent–Cys composite affinity sorbents were performed. According to X-Ray diffraction data, cysteine molecule is parallel to the tetrahedral–octahedral–tetrahedral layer (TOT silicate layer) and is a monolayer in the inner layer space of the structure. The surface areas of the Bentonite and Bent–Cys microcomposite structures were found as 33.00 ± 0.30 and 22.80 ± 0.30, respectively. An elemental analysis of immobilized l-cysteine for nitrogen was estimated to be 541.3 μmol g⁻¹ bentonite. Catalase adsorption onto the microcomposite affinity sorbents from aqueous solutions containing different amounts of catalase at different pH was investigated in a batch system. The maximum catalase adsorption capacity of the Bent–Cys microcomposite affinity sorbents was 175 mg g⁻¹. The non-specific catalase adsorption onto the bentonite was very low (about 2.7 mg g⁻¹). The activity yield decreased with the increase of the enzyme loading. It was observed that there was a significant change between V_max value of the free catalase and V_max value of the adsorbed catalase on the Bent–Cys microcomposite affinity sorbents. The K_m value of the immobilized enzyme was higher 1.1 times than that of the free enzyme. Optimum operational temperature was 10 °C higher than that of the free enzyme and was significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed without loss of adsorption capacity or enzyme activity.
The application of methacrylate-based polymers to enzyme biosensors
2006, Biomolecular Engineering
Enzyme electrodes based on methacrylates have received significant attention in the development of biosensors. This article reviews the use and application of methacrylate and its derivatives as an immobilization system for the preparation of enzyme electrodes. Resent examples, extracted from the literature, illustrate the superior performance of such materials in the fabrication of biosensors and bioreactors.

View all citing articles on Scopus

View full text

Immobilization of glucose oxidase in poly(2-hydroxyethyl methacrylate) membranes

Abstract

Anal. Chim. Acta.

Aial. Chim. Acta

J. Membrane Sci.

J. Biol. Chem.

Biomaterials

Methods for preparing immobilized enzymes

Enzyme membrane

Technique of enzyme entrapment in gels

Glucose sensitive membranes containing glucose oxidase activity. Swelling and permeability studies

J. Biomed. Mater. Res.

Application of l-( + )-lactate electrode for clinical analysis

Biotechnol. Bioeng.

The structure of highly crosslinked poly(2-hydroxyethyl methacrylate) hydrogels

J. Biomed. Mater. Res.

lonically crosslinked hydrophilic polymer membranes: synthesis and measurement of transport properties

J. Polymer Sci.