A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin

A novel method was developed for the immobilization of glucoamylase from Aspergillus niger. The enzyme was immobilized onto polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin, resulting in 85% immobilization yield. The immobilized enzyme has been fully active for 30 days. In addition, the immobilized enzyme retained 90 and 75% of its activity in 60 and 90 days, respectively. The enzyme optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4 and 65 °C, respectively. The kinetic parameters for the hydrolysis of maltodextrin by free and immobilized glucoamylase were also determined. The K_m values for free and immobilized enzyme were 7.5 and 10.1 g maltodextrin/l, respectively. The V_max values for free and immobilized enzyme were estimated as 20 and 16 μmol glucose/(min μl enzyme), respectively. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes.     

Comparative studies on soluble and immobilized yeast hexokinase

Comparative studies have been carried out on soluble and immobilized yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The enzyme was immobilized by covalent attachment to a polyacrylamide type support containing carboxylic functional groups. The effects of immobilization on the catalytic properties and stability of hexokinase were studied. As a result of immobilization, the pH optimum for catalytic activity was shifted in the alkaline direction to ～pH 9.7. The apparent optimum temperature of the immobilized enzyme was higher than that of the soluble enzyme. The apparent K_m value with D-glucose as substrate increased, while that with ATP as substrate decreased, compared with the data for the soluble enzyme. Differences were found in the thermal inactivation processes and stabilities of the soluble and immobilized enzymes. The resistance to urea of the soluble enzyme was higher at alkaline pH values, while that for the immobilized enzyme was greatest at ～pH 6.0.     

Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe₃O₄, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring.     

β-d-Galactosidases immobilized on soluble matrices: Kinetics and stability

Three β-d-galactosidases (β-d-galactoside galactohydrolase, EC 3.2.1.23) from different origins have been immobilized on sucrose-polyacrolein and sucrose sulphate-polyacrolein. This gave enzyme conjugates insoluble in the immobilization medium but which could be made soluble by reduction with sodium borohydride before use. The optimum conditions for both copolymer synthesis and the immobilization reaction were investigated. I.r. and ¹³C n.m.r. spectroscopy were used to follow the sulphation and the copolymerization reaction. The characteristics of the enzyme conjugates were compared with those of the free enzymes: the V_max values of the enzyme conjugates were lower than those of the corresponding free enzymes, whilst the K_m values were similar. The thermal stability of the enzyme conjugates depended on the enzyme origin, while their pH stability was in all cases higher than that of the free enzymes. These data suggest some advantages in using enzyme immobilization supports which can be made soluble after separation of the immobilized enzyme without altering the enzyme characteristics.     

Immobilization of pectinase fromSclerotium rolfsii

Immobilization of pectinase fromS. rolfsii was studied on different matrices of which Amberlite XAD-7 showed maximum adsorption and expression of the enzyme. The most active preparation was obtained when XAD-7 was activated with 2% glutaraldehyde and 1.7 μkat of enzyme per g resin was used for immobilization at pH 5.5 and 28°C. Optimum pH and temperature of theS. rolfsii pectinase remain unaltered, 3.5 and 55°C, respectively, after immobilization. However, the apparentK _M value of the enzyme decreased from 1.75 g/L for soluble enzyme preparation to 1.4 g/L for immobilized enzyme preparation. Both soluble and immobilized enzyme preparations were most stable at pH 4.0. The immobilized enzyme preparation was more stable than the soluble enzyme.     

Purification, Immobilization, and Some Properties of Glucose Isomerase from Streptomyces flavogriseus

Glucose isomerase (EC 5.3.1.5) produced from Streptomyces flavogriseus was purified by fractionation with (NH₄)₂SO₄ and chromatography on diethylaminoethyl (DEAE)-cellulose and DEAE-Sephadex A-50 columns. The purified enzyme was homogeneous as shown by ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Benzyl DEAE-cellulose, triethylaminoethyl-cellulose, and DEAE-cellulose were effective in the immobilization of partially purified glucose isomerase. Several differences in properties were found between purified soluble enzyme, immobilized enzyme (DEAE-cellulose-glucose isomerase), and heat-treated whole cells. Glucose and xylose served as substrate for the enzyme. Whole cells had the highest K_m values for glucose and xylose; the soluble enzyme had the lowest values. The optimum temperature for activity of the soluble and immobilized enzymes was 70°C; that for whole cells was 75°C. The pH optimum for the three enzyme preparations was 7.5. Magnesium ion or Co²⁺ was required for enzyme activity; an addition effect resulted from the presence of both Mg²⁺ and Co²⁺. The enzyme activity was inhibited by Hg²⁺, Ag⁺, or Cu²⁺. The conversion ratio of the enzyme for isomerization was about 50%. The soluble and immobilized enzymes showed a greater heat stability than whole cells. The soluble enzyme was stable over a slightly wider pH (5.0 to 9.0) range than the immobilized enzyme and whole cells (pH 5.5 to 9.0). The molecular weight of the enzyme determined by the sedimentation equilibrium method was 171,000. A tetrameric structure for the enzyme was also indicated. After operating at 70°C for 5 days, the remaining enzyme activity of the immobilized enzyme and whole cells, which were used for the continuous isomerization of glucose in a plug-flow type of column in the presence of Mg²⁺ and Co²⁺, was 75 and 55%, respectively. Elimination of Co²⁺ decreased operational stability.     

Preparation and properties of an immobilized cellulase on the reversibly soluble matrix Eudragit L-100

Abstract

To prepare a smart biocatalyst, cellulase was immobilized on the reversibly soluble matrix Eudragit L-100 by non-covalent and covalent methods. Covalent immobilization using carbodiimide coupling exhibited superior enzyme loading and reusability compared with non-covalent immobilization, and the covalent loading was increased by almost 20% through the addition of N-hydroxysuccinimide. The temperature optimum of the cellulase was not improved apparently by immobilization but the pH optimum increased from 4.75 to 5.25. Immobilized cellulase was more active than free cellulase above pH 5.0. Immobilized cellulase was more stable than free cellulase during storage at 4°C, room temperature and 50°C. K_m values of immobilized and free cellulase were 85.55 and 73.84 g L^?1, respectively. About 50% productivity was retained after five cycles for hydrolysis of steam-exploded straw.     

Immobilization of Kluyveromyces marxianus cells containing inulinase activity in open pore gelatin matrix: 1. Preparation and enzymatic properties

Kluyveromyces marxianus cells with inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7) activity have been immobilized in open pore gelatin pellets with retention of > 90% of the original activity. The open pore gelatin pellets with entrapped yeast cells were obtained by selective leaching out of calcium alginate from the composite matrix, followed by crosslinking with glutaraldehyde. Enzymatic properties of the gelatin-entrapped cells were studied and compared with those of the free cells. The immobilization procedure did not alter the optimum pH of the enzymatic preparation; the optimum for both free and immobilized cells was pH 6.0. The optimum temperature of inulin hydrolysis was 10°C higher for immobilized cells. Activation energies for the reaction with the free and immobilized cells were calculated to be 6.35 and 2.26 kcal mol^?1, respectively. K_m values were 8 mM inulin for the free cells and 9.52 mM for the immobilized cells. The thermal stability of the enzyme was improved by immobilization. Free and immobilized cells showed fairly stable activities between pH 4 and 7, but free cell inulinase was more labile at pH values below 4 and above 7 compared to the immobilized form. There was no loss of enzyme activity of the immobilized cells on storage at 4°C for 30 days. Over the same period at room temperature only 6% of the original activity was lost.     

An investigation of the properties of glucoamylase immobilized on glass beads involving 5-diazosalicylic acid bonded to a titanium (IV) oxide film

Optimum conditions have been determined for the immobilization of glucoamylase on glass involving diazotized 5-aminosalicylic acid bonded to a deposited imperfectly crystallized film of TiO₂. The changes in the kinetic and thermodynamic characteristics of the enzyme on immobilization have been determined. There are significant differences in the behaviour of the immobilized enzyme towards its substrates, maltose and starch. The apparent K_m for starch increased on immobilization whereas that for maltose decreased. The pH optimum for the immobilized preparation showed a shift to acid pH relative to that of the free enzyme.     

Immobilization of glucoamylase on DEAE-cellulose activated with chloride compounds

Partially purified glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger NRRL 330 has been immobilized on DEAE-cellulose activated with cyanuric chloride in 0.2 m acetate buffer, pH 4.2. In the matrix-bound glucoamylase, enzyme yield was 20 mg g^?1 of support, corresponding to 40 200 units g^?1 of DEAE support. Binding of the enzyme narrows the pH optimum from 3.8–5.2 to 3.6. Thermal stability of the bound glucoamylase enzyme was decreased although it showed a higher temperature optimum (70°C) than the free form (55°C). The rate of reaction of glucoamylase was also changed after immobilization. V_max values for free and bound enzyme were 36.6 and 22.6 μmol d-glucose ml^?1 min^?1 and corresponding K_m values were 3.73 and 4.8 g l^?1 respectively. Free and immobilized enzyme when used in the saccharification process gave 84 and 56% conversion of starch to d-glucose, respectively. The bound enzyme was quite stable and in the batch process it was able to operate for about five cycles without any loss of activity.     

Immobilization of Escherichia coli cells containing aspartase activity with kappa-carrageenan. Enzymic properties and application for L-aspartic acid production.

Whole cells of Escherichia coli having high aspartase (L-asparate ammonialyase, EC 4.3.1.1) activity were immobilized by entrapping into a kappa-carrageenan gel. The obtained immobilized cells were treated with glutaraldehyde or with glutaraldehyde and hexamethylenediamine. The enzymic properties of three immobilized cell preparations were investigated, and compared with those of the soluble aspartate. The optimum pH of the aspartase reaction was 9.0 for the three immobilized cell preparations and 9.5 for the soluble enzyme. The optimum temperature for three immobilized cell preparations was 5--10 degrees C higher than that for the soluble enzyme. The apparent Km values of immobilized cell preparations were about five times higher than that of the soluble enzyme. The heat stability of intact cells was increased by immobilization. The operational stability of the immobilized cell columns was higher at pH 8.5 than at optimum pH of the aspartase reaction. From the column effluents, L-aspartic acid was obtained in a good yield.     

Preparation and application of poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan hydrogels for immobilization of lipase

In the present of this study, two novel polymeric matrixes that are poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan was synthesized and applied for immobilization of lipase. For the immobilization of enzyme, two different immobilization procedures have been carried out via covalently binding and entrapment methods. On the free and immobilized enzymes activities, optimum pH, temperature, storage and thermal stability was investigated. The optimum temperature for free, covalently immobilized and entrapped enzymes was found to be 30, 35 and 30 degrees C, respectively. Optimum pH for both free and immobilized enzymes was also observed at pH 8. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for free and immobilized lipases. Furthermore, the reuse numbers of immobilized enzymes also studied. It was observed that after 40th use in 5 days, the retained activities for covalently immobilized and entrapped lipases were found as 39% and 22%, respectively. Storage and thermal stability of enzyme was also increased by as a result of immobilization procedures.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

IMMOBILIZATION OF OXALATE DECARBOXYLASE TO EUPERGIT AND PROPERTIES OF THE IMMOBILIZED ENZYME

Oxalate decarboxylase, an oxalate degradation enzyme used for medical diagnosis and decreasing the oxalate level in the food or paper industry, was covalently immobilized to Eupergit C. Different immobilization parameters, including ratio of enzyme to support, ammonia sulfate concentration, pH, and incubation time, were optimized. Under the condition of enzyme/support ratio at 1:20, pH 9, with 1.5 mol/L (NH₄)₂SO₄, room temperature, and shaking at 30 rpm for 24 hr, activity recovery of immobilized Oxdc reached 90% with an apparent specific activity of 0.44 U/mg support. The enzymatic properties of immobilized Oxdc were investigated and compared with those of the soluble enzyme. Both shared a similar profile of optimum conditions; the optimum pH and temperature for soluble and immobilized Oxdc were 3.5 and 50°C, respectively. The immobilized enzyme was more stable at lower pH and higher temperatures. The kinetic parameters for soluble and immobilized enzyme were also determined.     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

Sucrose hydrolysis by thermostable immobilized inulinases from aspergillus ficuum

The possibility of using thermostable inulinases from Aspergillus ficuum in place of invertase for sucrose hydrolysis was explored. The commercial inulinases preparation was immobilized onto porous glass beads by covalent coupling using activation by a silane reagent and glutaraldehyde before adding the enzyme. The immobilization steps were optimized resulting in a support with 5,440 IU/g of support (sucrose hydrolysis) that is 77% of the activity of the free enzyme. Enzymatic properties of the immobilized inulinases were similar to those of the free enzymes with optimum pH near pH 5.0. However, temperature where the activity was maximal was shifted of 10 degrees C due to better thermal stability after immobilization with similar activation energies. The curve of the effect of sucrose concentration on activity was bi-phasic. The first part, for sucrose concentrations lower than 0.3 M, followed Michaelis-Menten kinetics with apparent K(M) and Vm only slightly affected by immobilization. Substrate inhibition was observed at values from 0.3 to 2 M sucrose. Complete sucrose hydrolysis was obtained for batch reactors with 0.3 and 1 M sucrose solutions. In continuous packed-bed reactor 100% (for 0.3 M sucrose), 90% (1 M sucrose) or 80% sucrose conversion were observed at space velocities of 0.06-0.25 h(-1). The operational half-life of the immobilized inulinases at 50 degrees C with 2 M sucrose was 350 days.     

Characterization of molecular-sieve-bound inulinase

The enzyme inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7), prepared from Kluyveromyces marxianus has been immobilized using an inorganic solid support, molecular sieve 4A via the metal link method. The immobilized enzyme had around 22 units of inulinase activity per g of the support with retention of 72% of the original activity. The optimum protein to molecular sieve ratio for the maximum retention of inulinase activity was 9 mg/g molecular sieve. The properties of soluble and immobilized enzyme differed in many respects. The optimum pH of the enzyme shifted from 6 to 5 and the optimum temperature of enzyme activity changed from 50 to 55°C. K_m values were 6.7 mM for soluble enzyme and 10 mM for immobilized enzyme. The heat stability of the enzyme was improved by immobilization. Immobilized enzyme retained about 76% of the original activity after 40 days of storage at room temperature (30±2°C).     

Development of Bed Reactor Using Brick Dust Immobilized CIVI-Cellulase from Seeds of Cowpea (Vigna sinensis L)

Cellulase extracted from seeds of Cowpea (Vigna sinensis L var VITA-4) was partially purified and immobilized on brick dust as solid support via glutaraldehyde. The percentage retention of the enzyme activity on brick dust was nearly 85%. After immobilization specific activity of the enzyme increased from 0.275 to 0.557 U mg^?1 protein with about 2 fold enrichment. The optimum pH and temperature of soluble enzyme were determined as pH 4.6 and WC, respectively whereas immobilized enzyme showed at pH 5.0 and 37°C, respectively. The V_max values for soluble and immobilized enzyme were determined as 6.67 and 1.25 mg min^?1, respectively whereas K_m values were 4.35 and 4.76 mg ml^?1, respectively. The immobilized enzyme displayed higher thermal stability than soluble enzyme and retained about 50% of its initial activity after 12 reuses. Immobilized enzyme was packed in an indigenously designed double walled glass bed reactor for continuous production of reducing sugars.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Characterization of functionally active immobilized carbonic anhydrase purified from sheep blood lysates

Carbonic anhydrase (CA) catalyzes the reversible reaction of hydration of CO₂ to bicarbonate and the dehydration of bicarbonate back to CO₂. Sequestration of CO₂ from industrial processes or breathing air may require a large amount of highly active and stable CA. Therefore, the objectives of the present study were to purify large amounts of CA from a cheap and easily accessible source of the enzyme and to characterize the enzymatic and kinetic properties of soluble and immobilized enzyme. We recovered 80% of pure enzyme with a specific activity of 4870 EU/mg protein in a single step using sheep blood lysates from slaughter house waste products and CA specific inhibitor affinity chromatography. Since affinity pure CA showed both anhydrase and esterase activities, we measured the esterase activities for enzymology. The Michaelis–Menten constant, K_M, pH optimum, activation energy, and thermal stability of soluble enzymes were 8 × 10^?2 M, 7.3 pH, 7.3 kcal/mol and 70 °C, respectively.The immobilization of the enzyme to Affigel-10 was very efficient and 83% of purified enzyme was immobilized. The immobilized enzyme showed a K_M of 5 × 10^?2 M and activation energy of 8.9 kcal/mol, suggesting a better preference of substrate for immobilized enzyme in comparison to soluble enzyme. In contrast to soluble enzyme, immobilized enzyme showed relatively higher activity at pH 6–8. From these results, we concluded that a shift in pH profile toward acidic pH is due to modification of lysine residues involved in the immobilization process. The immobilized enzyme was stable at higher temperatures and showed highest activity at 80 °C. The activity of immobilized enzyme in a flow reactor at 0.5–2.2 ml/min flow rate was unaffected. Collectively, results from the present study suggested the application of blood lysate waste from animal slaughterhouses for purification of homogeneous enzyme for CO₂ capture in a flow reactor.