Immobilization Of Glucoamylase Research Articles

Research progress of glucoamylase with industrial potential.

Glucoamylase is one of the most widely used enzymes in industry, but the development background and existing circumstances of industrial glucoamylase were not described by published articles. CiteSpace, a powerful tool for bibliometric, was used to analyze the past, existing circumstances, and trends of a professional field. In this study, 1820 Web-of-Science-indexed articles from 1991 to 2021 were collected and analyzed by CiteSpace. The research hotspots of industrial glucoamylase, like glucoamylase strain directional improvement, Aspergillus niger glucoamylase, glucoamylase immobilization, application of glucoamylase in ethanol production, and "customized production" of porous starch, were found by analyzing countries, institutions, authors, keywords, and references of articles. PRACTICAL APPLICATIONS: The research progress of glucoamylase with industrial potential was analyzed by CiteSpace, and a significant research direction of glucoamylase with industrial potential was found. This is helpful for academic and corporate audiences to understand the current situation of glucoamylase with industrial potential and carry out follow-up works.

Glucoamylase Immobilization in Corncob Powder: Assessment of Enzymatic Hydrolysis of Starch in the Production of Glucose

Amylases are environmentally attractive enzymes and are employed in food processing technology; for example, the starch hydrolysis process is used for glucose syrup production. The use of biocatalysts attached to a support promotes greater stabilization of the enzyme and allows its reuse, this being interesting for industrial applications. The choice of the support for enzyme immobilization must be made according to the process costs, therefore, the employment of agro-industrial byproducts is an alternative. We evaluated the immobilization of commercial glucoamylase in corncob powder (CCP) and the application of the immobilized enzyme (CCP-glucoamylase) in response to starch hydrolysis process The yield of glucoamylase immobilization in CCP was 95%. Soluble glucoamylase and CCP-glucoamylase presented maximum activity temperatures at 50 °C and 60 °C, respectively. Both biocatalysts showed a maximum activity at pH 5. Soluble glucoamylase and CCP-glucoamylase exhibited a good thermal stability at 50 °C, resulting in half-lives of 33 h and 46 h, respectively. Soluble glucoamylase and CCP-glucoamylase reached starch conversion into glucose of 75% and 16%, respectively, after 12 h of reaction, with an enzymatic load of 30 U per gram of starch. However, after an increase in the enzymatic load of CCP-glucoamylase, starch conversion was equivalent CCP-glucoamylase has the potential to hydrolyze starch into glucose and can be reused, thus becoming an enhanced value-added bioproduct and an alternative for industrial applications. A strategy for future industrial application would be the hydrolysis of starch using CCP-glucoamylase in a fixed bed reactor operated continuously.

Development of a novel bi-enzymatic silver dendritic hierarchical nanostructure cascade catalytic system for efficient conversion of starch into gluconic acid

The natural cascade processes lead to inspire researchers to bring diverse biocatalysts together in an artificial way. In this work, we are going to introduce a brilliant double enzyme microsystem prepared from co–immobilization of glucose oxidase (GOD) and glucoamylase (GA) on silver dendrites hierarchical (Ag–DH) nanostructure through Ugi four-component reaction (Ugi–4CR) in water, as the green solvent at ambient temperature. The success preparation of the unique biocatalyst system was confirmed by FT–IR, UV–Vis, TGA, XRD, TEM, FE–SEM, AFM and elemental analysis. The properties of free and immobilized enzymes were investigated and compared. The immobilized GA and GOD had higher Km (Michaelis constant) and lower Vmax (maximum reaction velocity) than their native forms. The values of activation energy (Ea) for both the immobilized enzymes were smaller than those with native enzymes, implying that the immobilized enzymes are more temperature insensitive. The obtained double enzyme microsystem was employed as a biocatalyst for one pot transformation of starch to gluconic acid as an exclusive cascade reaction under mild conditions and in aqueous medium, and the final product obtained in high yield. Moreover, the artificial biomimetic microsystem exhibited high stability and reusability after eight cycles. These results demonstrated the feasibility of this approach for co-immobilization of enzyme on hierarchical structures may be extended to other biocatalytic cascades, thereby opening a new window for the other artificial biotransformations in chemistry.

Mixture design of starchy substrates hydrolysis by an immobilized glucoamylase from Aspergillus brasiliensis

Starch has great importance in human diet, since it is a heteropolymer of plants, mainly found in roots, as potato, cassava and arrowroots. This carbohydrate is composed by a highly-branched chain: amylopectin; and a linear chain: amylose. The proportion between the chains varies according to the botanical source. Starch hydrolysis is catalyzed by enzymes of the amilolytic system, named amylases. Among the various enzymes of this system, the glucoamylases (EC 3.2.1.3 glucan 1,4-alpha-glucosidases) are the majority because they hydrolyze the glycosidic linkages at the end of starch chains releasing glucose monomers. In this work, a glucoamylase secreted in the culture medium, by the ascomycete Aspergillus brasiliensis, was immobilized in Dietilaminoetil Sepharose-Polyethylene Glycol (DEAE-PEG), since immobilized biocatalysts are more stable in long periods of hydrolysis, and can be recovered from the final product and reused for several cycles. Glucoamylase immobilization has shown great thermal stability improvement over the soluble enzyme, reaching 66% more activity after 6 h at 60 °C, and 68% of the activity after 10 hydrolysis cycles. A simplex centroid experimental mixture design was applied as a tool to characterize the affinity of the immobilized enzyme for different starchy substrates. In assays containing several proportions of amylose, amylopectin and starch, the glucoamylase from A. brasiliensis mainly hydrolyzed the amylopectin chains, showing to have preference by branched substrates.

Triazine-functionalized chitosan-encapsulated superparamagnetic nanoparticles as reusable and robust nanocarrier for glucoamylase immobilization

Aspergillus niger glucoamylase (GLA) was covalently immobilized on 1,3,5-triazine-functionalized chitosan coated superparamagnetic nanoparticles (MNPCh-CC). The morphology, structure and properties of functionalized nanocomposite were investigated, as well as GLA immobilization process, through different analytical tools. Different experimental parameters such as optimum temperature, pH, reaction time and enzyme concentration were studied for free and immobilized enzyme. The GLA immobilized on nanocarrier exhibited excellent catalytic activity at pH 4.5 and 60°C. Notably, the immobilized GLA showed quite impressive stability, even after 10 reaction cycles, it could still retain about 70% of the initial activity. The results showed that immobilization process couldn’t significantly inhibit enzyme-substrate interaction and subsequently retained its effective catalytic activity. The substantial improvement of reactivity, reusability, and stability of this biocatalyst system may confer it a wider range of applications in industrial processes.

Immobilization of Glucoamylase onto Electrospun PAA/PVA Microfibrous Membrane by Active Ester Method

Immobilization of Glucoamylase onto Electrospun PAA/PVA Microfibrous Membrane by Active Ester Method

Immobilization of glucoamylase on triazine-functionalized Fe3O4/graphene oxide nanocomposite: Improved stability and reusability

Immobilization of an enzyme can enhance its catalytic activity, depending on the properties of the enzyme and the matrix. Graphene oxide is a nontoxic material and selective modulator for enzyme activity and is also a thermostable molecule that is important in large-scale nanostructure sheet applications. Herein, we have successfully developed a strategy for preparing a nanocomposite for enzyme immobilization model with high loading capacity. Nanostructures of hybrid graphene oxide-Fe3O4-cyanuric chloride (GO/MNP-CC) have adjustable surface chemistry that is an excellent candidate for covalent immobilization of enzymes. The morphology, structure and properties of GO/MNP-CC nanocomposite were investigated through different analytical tools. Glucoamylase, an important enzyme in industrial food products, was immobilized on GO/MNP-CC and exhibited excellent catalytic activity at pH 6.5 and 60°C. The results of this study indicated that the catalytic activity, reusability and stability of immobilized enzyme have been obviously improved compared to the free enzyme. The apparent Km and νmax for free and immobilized glucoamylase were also determined. These properties make them a good candidate to improve the practicality and further the development of the capacity enzyme attachment. Thus, the synthesized matrix has the potential for practical applications in other and binary enzyme immobilization and would have a wide prospect for their applications in bio-industry and biosensing.

Preparation and characterization of a green nano-support for the covalent immobilization of glucoamylase from Neurospora sitophila

The preparation of green nano supports for the covalent immobilization of enzymes is of special interest both from the economic and environmental point of view. In this contribution, we report on the synthesis of phytochemicals coated silver nanoparticles, which were used as a novel green support for the covalent immobilization of glucoamylase isolated from Neurospora sitophila. The aqueous extract of Fagonia indica was used as a source of reducing and capping agents for the reduction of silver ions into silver nanoparticles. The prepared nanoparticles were characterized by various analytical techniques. UV–visible spectroscopy was used to detect the characteristic surface plasmon resonance bands (426, 438nm) of the silver nanoparticles. The biosynthesized silver nanoparticles were mostly spherical in shapes with an average particle size of 30–40nm (TEM and DLS measurements). X-ray diffraction and energy dispersive X-ray studies confirmed the face centered cubic crystalline form and elemental composition of the biogenic silver nanoparticles respectively. FTIR study revealed that plant polyphenolics and protein were mainly involved in the reduction and capping of silver ions. Glucoamylase from Neurospora sitophila was covalently immobilized to these nanoparticles via EDC (1-(3-(dimethylamino) propyl) 3-ethylcarbodiimidehydrochloride) coupling reaction. The immobilized enzyme exhibited higher pH and thermal stabilities as compared to the free enzyme. The kinetic constant (KM) value for the immobilized glucoamylase was higher (0.73mg/mL) than its free counterpart (0.44mg/mL), whereas the Vmax value was slightly higher for the immobilized glucoamylase. The findings of this study conclude that the newly developed green method for the synthesis of green nano-support is simple, cost effective and could be successfully used for the immobilization of various enzymes and other macromolecules.

Reversible immobilization of glucoamylase onto magnetic polystyrene beads with multifunctional groups

A novel and simple process for the surface functionalization of micron-sized monodisperse magnetic polystyrene (PS) microbeads was reported. The polystyrene seed particles were prepared prior to the dispersion polymerization method. Afterwards, series of surface chemical modifications on polystyrene microspheres were conducted, and three end-functional microspheres with carboxyl, imidazolyl and sulphydryl groups were obtained. The functional magnetic polystyrene microspheres were prepared by impregnation and subsequent precipitation of ferric and ferrous ions into the polystyrene particles. Finally, the functional magnetic polystyrene was used for the reversible immobilization of glucoamylase via metal-affinity adsorption. The results indicated that the obtained immobilized glucoamylase presented excellent reusability, applicability, magnetic response and regeneration of supports. The magnetic PS microspheres retained >65% of its initial activity at 65°C over 6h; and the lowest residual activity of immobilized glucoamylase prepared by regenerated supports still remained about 50% of the initial activity after the 10th cycles.

Improved Stabilities of Immobilized Glucoamylase on Functionalized Mesoporous Silica Synthesised using Decane as Swelling Agent

Ordered mesoporous silica, with high porosity was used to immobilize glucoamylase via adsorption and covalent binding. Immobilization of glucoamylase within mesoporous silica was successfully achieved, resulting in catalytically high efficiency during starch hydrolysis. In this study, mesoporous silica was functionalized by co-condensation of tetraethoxysilane (TEOS) with organosilane (3-aminopropyl) triethoxysilane (APTES) in a wide range of molar ratios of APTES: TEOS in the presence of triblock copolymer P123 under acidic hydrothermal conditions. The prepared materials were characterized by Small angle XRD, Nitrogen adsorption – desorption and 29Si MAS solid state NMR. N2 desorption studies showed that pore size distribution decreases due to pore blockage after functionalization and enzyme immobilization. Small angle XRD and 29Si MAS NMR study reveals mesophase formation and Si environment of the materials. The main aim of our work was to study the catalytical activity, effect of pH, temperature storage stability and reusability of covalently bound glucoamylase on mesoporous silica support. The result shows that the stability of enzyme can be enhanced by immobilization. © 2013 by Authors, Published by BCREC Group. This is an open access article under the CC BY-SA License (https://creativecommons.org/licenses/by-sa/4.0)

Enzymatic processes for the purification of trehalose

A dual-enzyme process aiming at facilitating the purification of trehalose from maltose is reported in this study. Enzymatic conversion of maltose to trehalose usually leads to the presence of significant amount of glucose, by-product of the reaction, and unreacted maltose. To facilitate the separation of trehalose from glucose and unreacted maltose, sequential conversion of maltose to glucose and glucose to gluconic acid under the catalysis of glucoamylase and glucose oxidase, respectively, is studied. This study focuses on the hydrolysis of maltose with immobilized glucoamylase on Eupergit® C and CM Sepharose. CM Sepharose exhibited a higher protein adsorption capacity, 49.35 ± 1.43 mg/g, and was thus selected as carrier for the immobilization of glucoamylase. The optimal reaction temperature and reaction pH of the immobilized glucoamylase for maltose hydrolysis were identified as 40°C and 4.0, respectively. Under such conditions, the unreacted maltose in the product stream of trehalose synthase-catalyzed reaction was completely converted to glucose within 35 min, without detectable trehalose degradation. The conversion of maltose to glucose could be maintained at 0.92 even after 80 cycles in repeated-batch operations. It was also demonstrated that glucose thus generated could be readily oxidized into gluconic acid, which can be easily separated from trehalose. We thus believe the proposed process of maltose hydrolysis with immobilized glucoamylase, in conjunction with trehalose synthase-catalyzed isomerization and glucose oxidase-catalyzed oxidation, is promising for the production and purification of trehalose on industrial scales.

Temperature-Triggered Enzyme Immobilization and Release Based on Cross-Linked Gelatin Nanoparticles

A glucoamylase-immobilized system based on cross-linked gelatin nanoparticles (CLGNs) was prepared by coacervation method. This system exhibited characteristics of temperature-triggered phase transition, which could be used for enzyme immobilization and release. Their morphology and size distribution were examined by transmission electron microscopy and dynamic light scattering particle size analyzer. Their temperature-triggered glucoamylase immobilization and release features were also further investigated under different temperatures. Results showed that the CLGNs were regularly spherical with diameters of 155±5 nm. The loading efficiencies of glucoamylase immobilized by entrapment and adsorption methods were 59.9% and 24.7%, respectively. The immobilized enzyme was released when the system temperature was above 40°C and performed high activity similar to free enzyme due to the optimum temperature range for glucoamylase. On the other hand, there was no enzyme release that could be found when the system temperature was below 40°C. The efficiency of temperature-triggered release was as high as 99.3% for adsorption method, while the release of enzyme from the entrapment method was not detected. These results indicate that CLGNs are promising matrix for temperature-triggered glucoamylase immobilization and release by adsorption immobilization method.

Direct binding glucoamylase onto carboxyl-functioned magnetic nanoparticles

A novel and efficient immobilization of glucoamylase from Aspergillus niger has been developed by using carboxyl functioned magnetic Fe3O4 nanoparticles (CMNPs) as support. The CMNPs were prepared by co-precipitation of Fe2+/Fe3+ with oleic acid as surfactant and consequent oxidation of CC into COOH by KMnO4 solution in situ. And then glucoamylase was directly bonded onto the magnetic nanoparticles. The bonding was verified by Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis. The immobilization process was investigated by examining immobilization time, enzyme concentration and pH. As a result, it could gain an enzyme loading with 120mg glucoamylase/g magnetic nanoparticles and specific activity of 90U/mg protein while 1mg/mL glucoamylase and 10mg/mL ferrofluid were mixed at pH 6.0 and 25°C for 16h. The immobilized enzyme presented a same or better storage, pH and thermal stability than the free enzyme and could be reused. And the residual activity was about 40% after 8 cycles. The Michaelis constant (Km) showed that affinity of immobilized enzyme for binding substrate was lower than free enzyme.

Immobilization of glucoamylase onto polyaniline-grafted magnetic hydrogel via adsorption and adsorption/cross-linking

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of "starch" dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7mg/g from 2.0mg/mL enzyme solution at pH4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55°C and pH4.5 compared to free enzyme (50°C and pH5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase.

Preparation and characterization of graphite nanosheets decorated with Fe3O4 nanoparticles used in the immobilization of glucoamylase

Graphite nanosheets (GNSs) decorated with Fe3O4 nanoparticles were synthesized by an inverse coprecipitation method. The Fe3O4–GNS hybrid material was characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, nitrogen adsorption/desorption isotherms, thermogravimetry and magnetic hysteresis studies. We demonstrated that the Fe3O4–GNS hybrid material had a flexible interleaved structure, a large specific surface area, high magnetic sensitivity, and good thermal stability. The amino functionalization of the material with 3-aminopropyltriethoxysilane produced a magnetic material that was used in the immobilization of glucoamylase by using glutaraldehyde as a coupling reagent. The results showed that the immobilized glucoamylase exhibited excellent activity and reusability.

Reversible immobilization of glucoamylase onto magnetic carbon nanotubes functionalized with dendrimer

Magnetic carbon nanotubes (MCNTs) with necklace-like nanostructures was prepared via hydrothermal method, and hyperbranched poly(amidoamine) (PAMAM) was grafted on the surface of MCNTs on the basis of the Michael addition of methyl acrylate and the amidation of the resulting ester with a large excess of ethylenediamine (EDA), which could achieve generational growth under such uniform stepwise reactions. The terminal -NH(2) groups from the dendritic PAMAM were reacted with differently functionalized groups to form functionalized MCNTs. Subsequently, enzyme was immobilized on the functionalized MCNTs through adsorption, covalent bond, and metal-ion affinity interactions. The immobilization of glucoamylase, hereby chosen as model enzyme, onto the differently functionalized MCNTs is further demonstrated and assessed based on its activity, thermal stability, as well as reusability. Besides ease in recovery by magnetic separation, the immobilized glucoamylase on functionalized MCNTs offers superior stability and reusability, without compromising the substrate specificity of free glucoamylase. Furthermore, the results indicate that the metal-chelate dendrimer offers an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase.

Immobilization of Glucoamylase onto Lewis Metal Ion Chelated Magnetic Affinity Sorbent: Kinetic, Isotherm and Thermodynamic Studies

In this study, magnetic metal-chelate beads, m-poly(ethylene glycol dimethacrylate-vinyl imidazole) [m-poly(EGDMA–VIM)] with an average diameter 150–200 μm was synthesized by copolymerizing ethylene glycol dimethacrylate (EGDMA) with vinyl imidazole (VIM). The spesific surface area of the m-poly(EGDMA–VIM) beads was found 63.1 m2/g. Cu2+ ions were chelated on the m-poly(EGDMA–VIM) beads and used in immobilization of Aspergillus niger glucoamylase in a batch system. The maximum glucoamylase adsorption capacity of the m-poly(EGDMA–VIM)–Cu2+ beads was observed as 120 mg/g at pH 6.5. The optimum pH for free and m-poly(EGDMA–VIM)–Cu2+ immobilized glucoamylase were found 4.0 and 4.5, respectively. The optimum temperature of glucoamylase was not changed after immobilization and determined as 60 o C for free and immobilized enzyme preparations. The glucoamylase adsorption capacity and adsorbed enzyme activity slightly decreased after 10 batch successive reactions, demonstrating the usefulness of the enzyme-loaded beads in biocatalytic applications. Storage stability was found to increase with immobilization. The effect of various experimental parameters such as pH, glucoamylase concentration, contact time and temperature in aqueous solution were also investigated. Adsorption isotherm obtained for m-poly(EGDMA–VIM)–Cu2+ was consistent with Langmuir model. Kinetic studies showed that the adsorption process agreed with both the pseudo-second-order kinetic model and the modified Ritchie's-second-order kinetic model. Various thermodynamic parameters, free energy (ΔG 0), enthalpy (ΔH 0) and entropy (ΔS 0), were also calculated and the results showed that the adsorption process strongly depended on temperature of medium.

Vinyl triazole carrying metal‐chelated beads for the reversible immobilization of glucoamylase

AbstractPoly(ethylene glycol dimethacrylate–1‐vinyl‐1,2,4‐triazole) [poly(EGDMA–VTAZ)] beads with an average diameter of 100–200 μm were obtained by the copolymerization of ethylene glycol dimethacrylate (EGDMA) with 1‐vinyl‐1,2,4‐triazole (VTAZ). The copolymer hydrogel bead composition was determined by elemental analysis and was found to contain 5 EGDMA monomer units for each VTAZ monomer unit. The poly(EGDMA–VTAZ) beads were characterized by swelling studies and scanning electron microscopy (SEM). The specific surface area of the poly(EGDMA–VTAZ) beads was found 65.8 m2/g. Cu2+ ions were chelated on the poly(EGDMA–VTAZ) beads. The Cu2+ loading was 82.6 μmol/g of support. Cu2+‐chelated poly(EGDMA–VTAZ) beads with a swelling ratio of 84% were used in the immobilization of Aspergillus niger glucoamylase in a batch system. The maximum glucoamylase adsorption capacity of the poly(EGDMA–VTAZ)–Cu2+ beads was 104 mg/g at pH 6.5. The adsorption isotherm of the poly(EGDMA–VTAZ)–Cu2+ beads fitted well with the Langmuir model. Adsorption kinetics data were tested with pseudo‐first‐ and second‐order models. The kinetic studies showed that the adsorption followed a pseudo‐second‐order reaction model. The Michaelis constant value for the immobilized glucoamylase (1.15 mg/mL) was higher than that for free glucoamylase (1.00 mg/mL). The maximum initial rate of the reaction values were 42.9 U/mg for the free enzyme and 33.3 U/mg for the immobilized enzyme. The optimum temperature for the immobilized preparation of poly(EGDMA–VTAZ)–Cu2+–glucoamylase was 65°C; this was 5°C higher than that of the free enzyme at 60°C. The glucoamylase adsorption capacity and adsorbed enzyme activity slightly decreased after 10 batch successive reactions; this demonstrated the usefulness of the enzyme‐loaded beads in biocatalytic applications. The storage stability was found to increase with immobilization. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone-Activated Alginate Beads: II. Enzyme Immobilization and Characterization

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K (m) values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V (m) value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone Activated Alginate Beads: I. Beads Preparation and Characterization

ρ-Benzoquinone-activated alginate beads were presented as a new carrier for affinity covalent immobilization of glucoamylase enzyme. Evidences of alginate modification were extracted from FT-IR and thermal gravimetric analysis and supported by morphological changes recognized through SEM examination. Factors affecting the modification process such as ρ-benzoquinone (PBQ) concentration, reaction time, reaction temperature, reaction pH and finally alginate concentration, have been studied. Its influence on the amount of coupled PBQ was consequently correlated to the changes of the catalytic activity and the retained activity of immobilized enzyme, the main parameters judging the success of the immobilization process. The immobilized glucoamylase was found kept almost 80% of its native activity giving proof of non-significant substrate, starch, diffusion limitation. The proposed affinity covalent immobilizing technique would rank among the potential strategies for efficient immobilization of glucoamylase enzyme.