The industrial preparation of glucose involves a preliminary starch saccharification to maltodextrin that uses α-amylase, and a second hydrolysis to glucose that uses glucoamylase [1
]. Glucoamylase (1,4-α-glucanglucohydrolase, EC 18.104.22.168) is an enzyme preparation that decomposes starch into glucose by tearing-off glucose units from the non-reduced end of the polysaccharide chain. As an extra-cellular enzyme, it catalyzes the hydrolysis of α-1,4glucosidic bonds in starch by progressively chopping off single glucose molecules from the ends of amylose chains. Some glucoamylases can also attack the branching α-1,6 bonds of amylopectin, but at a much slower rate than α-1,4 bonds [3
]. Immobilized enzyme preparations are useful catalysts for industrial biotransformations [4
]. Immobilization of enzymes through covalent attachment has also been demonstrated to induce higher resistance to temperature, denaturants, and organic solvents in several cases [6
]; therefore it has become a hot topic studied by scholars. The carrier bonding of enzymes may produce alterations in their observed activity, specificity, or selectivity [9
]. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Otherwise, a stabilizing hydrophilic microenvironment can be created by the introduction of hydrophilic macromolecules into the proximity of the enzyme. The stability of an immobilized enzyme is dictated by many factors such as the number of bonds formed between the enzyme and carrier [10
]. The development of oxide-coated glass has increased its durability and half-life, which are important economic advantages, but the cost of the carrier still exceeds the value of the final product [11
Currently, a large variety of matrixes have been used in immobilized glucoamylase [7
]. The enzymes are linked to an insoluble matrix by chemical bonds, which generally produce very stable derivatives in which enzyme leakage is prevented. The result shows that the stability and recovery times of immobilized enzyme were not ideal. The binding capacity and catalytic ability of the enzyme are reduced when the carrier is present, but the ability to adapt well to the microenvironment was enhanced [15
]. In recent years, the advent of self-immobilization [16
] has intrigued researchers. It has two forms, direct cross-linking and indirect cross-linking. There are disadvantages to the use of direct cross-linking of untreated enzyme molecules such as poor mechanical performance, small granules, and low enzymatic activity By contrast, pre-treatment of enzymes by physical or chemical precipitation or adding protectants makes them more robust for the same cost. Enzymes of cross-linked enzyme crystals (CLECs) and cross-linked enzyme aggregates (CLEAs) [19
] were precipitated from an aqueous solution by adding a salt or a water-miscible organic solvent or polymer, and then the physical aggregates of enzyme molecules were cross-linked with a bifunctional agent. Higher stability and suitability of immobilized enzymes were achieved by using two types of unconventional methods. CLEA have been applied extensively in many kinds of enzymes [8
] and represent one of the best potential methods of immobilizing enzymes.
Xanthan gum is a nature polysaccharide and an important industrial biopolymer [22
]. It has been used in a wide variety of enzyme modifications for a number of important reasons, such as emulsion stability, temperature stability, and pseudoplastic rheological properties [23
]. The storage stability and tolerance of immobilized enzymes were enhanced significantly with xanthan gum in previous studies. The conformation stability of enzymes is necessary for their bioactivity. In the case of CLEA with dextrin co-aggregates, the amino group of the enzyme might react with glutaraldehyde to establish enzyme–enzyme, starch–grain, and enzyme–starch linkages. In this study, dextrin and xanthan gum as protecting agents were added in the process of prepared CLEA of glucoamylase, and we focus on the characteristics of modified CLEA and free glucoamylase. The morphology and tolerance were analyzed by the comparison of immobilized and unmodified enzyme. Furthermore, the thermal stability and the optimum concentration of the protective agent and glutaraldehyde solution were studied systematically in this paper. We also examined immobilized and native glucoamylase with respect to kinetic parameters, recoverability, and performance in different conditions.
3. Experimental Section
Glucoamylase from Aspergillus niger was kindly donated by Enzyme Preparation of Luliang (Yunnan, China). Glutataldehyde solution (50%) and 3.5-Dinitrosalicylic acid (>98%, w/w) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dextrin and ammonium sulfate were also supplied by Sinopharm Chemical Reagent Co., Ltd. and were of analytical reagent grade. All other reagents were of analytical grade. All the solutions were prepared with distilled deionized water.
Glucoamylase (2 g crude enzyme power) was dissolved in 100 mL sodium acetic buffer (200 mM, pH 5.6) in a 200-mL beaker and stirred gently at room temperature for 15 min, which produced a supernatant after centrifugation. To the enzyme solution, the precipitant of (NH4)2SO4 solid powder was added, and the mixture was stirred for 20 min. Precipitate was isolated by centrifugation at 5000 rpm at 10 °C for 15 min, then 100 mL sodium acetic buffer (200 mM, pH 5.6) was added and stirred for 15 min at room temperature. Calcium chloride was added to the enzyme solution to remove ammonium sulfate until the final concentration of calcium ions in the solution reached 0.1 M. The enzyme solution was used without any further purification.
3.1.2. Glucoamylase Cross-Linked Enzyme Aggregates (CLEA)
CLEA of glucoamylase was prepared by using a modified procedure from the literature [31
]. A mixture of 400 μL glucoamylase solution, 600 μL sodium acetic buffer (200 mM, pH 5.6) and 70% (w
) powdered (NH4
(created by adding dextrin power or xanthan gum to prepared CLEA-D or CLEA-XG before precipitation and stirring for 20 min at room temperature) was prepared and aged for 15 min at room temperature with occasional gentle stirring. Next, 50% (v
) glutaraldehyde was added to reach a final concentration of 0.5% (v
) and aged for 2 h at room temperature with stirring at 300 rpm. The mixture was then diluted with the addition of 1 mL of 200 mM pH 5.6 sodium acetic buffer, vortexed for 15 s, and microfuged for 10 min at 4 °C. The observed dissolving of the enzyme aggregates upon vortexing and microfuging was indicative of incomplete cross-linking of the protein. The supernatant was decanted and the residue was washed three times with sodium acetic buffer (200 mM, pH 5.6), centrifuged, and decanted. The final enzyme preparation was kept in the same buffer at 4 °C. Prior to use, CLEA kept in the buffer was centrifuged and supernatant was decanted.
3.2. Determination and Analysis
3.2.1. Activity Assay
Standard conditions commonly used for the measurement of the activity of soluble and immobilized glucoamylase were as follows. Four hundred microliters of free or equal amounts of immobilized enzyme (the volume used in the process of immobilization) were added to a buffer solution to 600 μL of 2 wt % dextrin gelatinized in water in 0.2 M sodium acetic buffer, pH 5.6. After 10 min of incubation at 60 °C, 3 mL of 3,5-Dinitrosalicylic acid (DNS) was added in order to stop the catalytic reaction. The released glucose was measured by absorbance after 30 min at room temperature and recalculated on the base of glucose standard absorbance. The amount of glucose (in 1 μg) generated for 1 min was used as the unit of enzyme activity (U).
3.2.2. Optimum Temperature, pH, and Kinetic Parameters Assay
Determination of optimum temperature and pH was achieved by individually changing the conditions of the glucoamylase activity assay: temperature ranged from 30 to 80 °C; optimum pH was determined in the citrate (pH 3.0–5.0), phosphate (pH 5.0–7.0), and Tris-HCl (pH 7.0–8.0) buffer. Kinetic parameters (Km and Vmax values) were calculated by measuring the initial velocities of the reaction at various substrate concentrations (0.5%–5% (w/v)); the values were substituted into the Lineweaver–Burk plots to obtain Km and Vmax.
3.2.3. Determination of Stability
The thermostability of immobilized and native glucoamylase was measured by incubation at 60 and 70 °C in 0.2 M sodium acetic buffer of pH 5.6 without substrate. They will be taken out in order at times from 0 to 390 min and immediately cooled down in an ice bath. Then the activity of free and immobilized enzyme was determined under the above conditions.
The storage stability of immobilized preparations was estimated by residual enzymatic activity after storage at ambient temperature in the corresponding buffers. The residual activity was periodically measured and compared with the initial activity of the freshly prepared immobilized enzyme (assuming the initial activity of the freshly prepared biocatalyst was 100%).
To evaluate the reusability of immobilized glucoamylase, a series of experiments was carried out under standard assay conditions. The interval of determination was every 3 h and we were sure to rinse the precipitate before each use. The activity of the free enzyme was taken to be 100%.
We used a modified CLEA procedure to prepare immobilized glucoamylase with high activity recovery, with added dextrin or xanthan gum as protectors during the cross-linking process. Maltose and maltohexaose were used as active site conformational templates during the harsh cross-linking process [33
]. Compared with conventional methods (i.e.
, covalent or non-covalent combination, embedding, CLEC), the immobilized enzyme and carrier-bound CLEA of glucoamylase with tailor-made properties (e.g., enhanced activity, thermostability, and storage stability) have been designed by a simple and effective technology [31
]. Two types of immobilized enzyme exhibited the expected increase in stability and tolerance compared to the native enzyme. Moreover, the results of storability and recyclability showed that the enzyme has superior performance after immobilization. The recycling experiments suggest that an immobilized enzyme has desirable characteristics and avoids loss of a large amount of activity after 30 uses. Hence, this work has great potential in teaching about immobilization, a relatively new idea for improving the activity of enzymes.
Unfortunately, CLEA exhibited the drawback of inactivation by the generation of steric limitation compared to the free enzyme during the catalysis of large substrates, which perhaps led to fatal contact with the active site of immobilized enzymes [37
]. In addition, the enzyme may be inactivated by chemical modification in the process of CLEA preparation; this is due to the fact that the glucoamylase active site often contains lysine and arginine residues, which are potential sites for cross-linking. On cross-linking, the enzyme will be “locked” into this less favorable conformation. For these reasons, to avoid modifying failure, optimization of the CLEA procedure also involves optimization of the cross-linkers/enzyme ratio, precipitants, reaction conditions, etc.
]. The optimum conditions for precipitation and cross-linking were different for glucoamylase from different sources. We could not draw any definite conclusions regarding the influence of the numbers of surface lysine and arginine residues on the activity recovery in CLEA formation and the storage and operational stability of the resulting CLEA. Further studies are going to research the effects of crosslinking on bull serum albumin (BSA), lysine, and the linker regions of the glucoamylase enzyme. Furthermore, to accelerate the industrial application, a feasible way to promote the application was to find a suitable carrier that is combined with CLEA.