Some factors affecting the stability of glucoamylase immobilized on hornblende and on other inorganic supports

Glucoamylase from four different companies was studied: three had similar stability (half-life at 50°C about 140 hr); the fourth was less stable (half-life at 50°C about 20 hr). The immobilized enzymes were all less stable than their soluble counterparts: immobilized enzyme stability depended on the soluble enzyme used, the support, and method of immobilization. Thus enzyme bound to Enzacryl-TIO was less stable than enzyme bound to hornblende (metal-link method); this, in turn, was less stable than enzyme bound to hornblende by a silane–glutaraldehyde process. Bound enzyme stability was also improved by the presence of substrate or product (starch maltose or glucose). After 110 hr at 50°C in the presence of maltose (10% (w/v)) one preparation (a more stable soluble enzyme boul1d to hornblende by a silane–glutaraldehyde process) retained over 95% of its activity: activity loss was too low to permit the estimation of a half-life.     

Studies on the stability of immobilized xanthine oxidase and urate oxidase.

The stability of immobilized preparations of xanthine oxidase and urate oxidase was studied, and optimized, because of the potential joint use of both enzymes in clinical analysis. Xanthine oxidase was immobilized on cellulose, Sepharose, hornblende, Enzacryl-TIO, and porous glass. Thehalf-lives of these preparations at 30 degree C ranged from 40 min to 5.0 hr. In this respect immobilized enzyme resembled soluble enzyme in dilute solution (0.11 mg/ml), when the half-live was about 3.5 hr. More concentrated enzyme solution (1 mg/ml) had a half-life of 64 hr, and was, therefore, considerably more stable than the untreated immobilized xanthine oxidase preparations. Inclusion of albumen in storage and assay buffer increased the half-life of bound xanthine oxidase. So also did treatment with glutaraldehyde: in the case of xanthine oxidase bound to Enzarcyl-TIO such treatment increased the half-life at 30 degree C from 3 hr to about 100 hr. Immobilized xanthine dehydrogenase was more stable than immobilized xanthine oxidase: the dehydrogenase lost no activity during continuous assay for 5 hr at 30 degree C. The stability of immobilized urate oxidase depended on the quantity of enzyme used and on the time of stirring during immobilization: thus a preparation was made (by stirring urate oxidase (48 mg/g support) with Enzacryl-TIO for 24 hr) which lost no activity during 350 hr at 30 degree C.     

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.     

The effect of lead on the calcium-handling capacity of rat heart mitochondria.

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.     

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.     

Immobilization of lactate dehydrogenase on polyacrylamide beads

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.     

Immobilization of pig muscle 3-phosphoglycerate kinase

3-Phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 88 units g^?1 xerogel. The activity versus pH profile showed a sharper maximum at pH 6.5 in the case of the immobilized enzyme. The immobilized enzyme had a broad apparent optimum temperature range between 40 and 50°C. The apparent K_m values of the immobilized 3-phosphoglycerate kinase were lower for both 3-phosphoglycerate and ATP than those of the soluble enzyme. In the case of the immobilized enzyme stabilities were enhanced.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

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.     

Physico-Chemical Characterization of Watermelon Urease upon Immobilization in Alginate Beads

Watermelon (Citrullus vulgaris) urease was immobilized in 3.5% alginate leading to 72% immobilization. There was no leaching of the enzyme over a period of 15 days at 4°C. It continued to hydrolyse urea at a faster rate upto 90 min of incubation. The immobilized urease exhibited a shift of apparent pH optimum by one unit towards acidic side (from pH 8.0 to 7.0). The K_m was found to be 13.3 mM; 1.17 times higher than the soluble enzyme (11.4 mM). The beads were fairly stable upto 50°C and exhibited activity even at ?10°C. The enzyme was significantly activated by ME and it exhibited two peaks of activation; one at lower concentration and another at higher concentration. Time-dependent ureolysis in presence of ME progressed at a much elevated rate. Unlike soluble enzyme, which was inhibited at 200 mM urea, the immobilized enzyme was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2000 mM urea. Moreover, the inhibition caused by high urea concentration was partially abolished by ME. The significance of the observations is discussed.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde

High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA-1 (mesh 20–50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, pH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60°C, pH 10 for alkaline protease and 50°C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the K_m values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60°C. Other peptide hydrolases, β-galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose-6-phosphate dehydrogenase, and xanthine oxidase became inactive.     

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).     

Preparation and properties of immobilized amyloglucosidase

Amyloglucosidase was immobilized on a copolymer of methyl methacrylate and 2-dimethylaminoethyl methacrylate. The resulting immobilized amyloglucosidase has 19% of the soluble enzyme specific activity. The pH optimum of immobilized amyloglucosidase is shifted towards acidity by 1.9 units. The temperature optimum of immobilized enzyme is shifted upward by 5°C. The immobilized amyloglucosidase has the maximum stability at pH 4.6, whereas the soluble enzyme has maximum stability at pH 5.5. While soluble amyloglucosidase has a maximum thermal stability at 50°C, the stability of the immobilized amyloglucosidase steadily decreases with the increase in temperature.     

Immobilization of glucansucrase for the production of gluco-oligosaccharides from Leuconostoc mesenteroides

Glucansucrase from Leuconostoc mesenteroides was immobilized in 1?% (w/v) with sodium alginate to produce oligosaccharides. Glucansucrase gave three activity bands of approx. 240, 178, and 165?kDa after periodic acid-Schiff staining with sucrose. The immobilized enzyme had 40?% activity after ten batch reactions at 30?°C and 75?% activity after a month of storage at 4?°C, which is six times more stable than the free enzyme. Immobilized enzyme was more stable at lower (3.5?4.5) and higher (6.5?7.0) pH ranges and higher temperatures (35?40?°C) compared with the free enzyme. Immobilized and free glucansucrase were employed in the acceptor reaction with maltose and each produced gluco-oligosaccharide ranging from trisaccharides to homologous pentasaccharides.     

Hydrolysis of starchy materials by repeated use of an amylase immobilized on a novel thermo-responsive polymer

A copolymer of methacrylic acid (MAA) and N-isopropyl acrylamide (NIPAM) was used as a novel, reversibly soluble-insoluble support whose solubility changes depending on the temperature of the solution. Amylase (Dabiase K-27) immobilized covalently on the thermo-responsive polymer showed good solubility response: the immobilized enzyme (D-MN) was in a soluble state below 32°C, but insoluble above 42°C. D-MN in a soluble state has a high specific activity for the hydrolysis of soluble or uncooked starch. The solubility response of D-NM to changes in the temperature of the solution was more sensitive when 0.5% NaCl was added to a buffer solution (pH 4.5) with D-MN than in the buffer solution without NaCl. D-MN was used successively for repeated hydrolysis reactions of soluble and uncooked starches, in which D-MN was insolubilized either by changing the temperature of the reaction mixture from 30°C to 36°C with 0.5% NaCl or by adjusting the NaCl concentration of the reaction mixture from 0% to 1% at 30°C. In the repeated hydrolysis, glucose was produced successively from the soluble and uncooked starches, and D-MN could be repeatedly used after being recovered from the reaction product by centrifugation at the end of each batchwise hydrolysis.     

Continuous production of fructooligosaccharides using fructosyltransferase immobilized on ion exchange resin

A continuous production of fructooligosaccharides from sucrose was investigated by fructosyltransferase immobilized on a high porous resin, Diaion HPA 25. The optimum pH (5.5) and temperature (55°C) of the enzyme for activity was unaltered by immobilization, and the immobilized enzyme became less sensitive to the pH change. The optimal operation conditions of the immobilized enzyme column for maximizing the productivity were as follows: 600 g/L of sucrose feed concentration, flow rate of superficial space velocity 2.7 h^?1. When the enzyme column was run at 50°C, about 8% loss of the initial activity of immobilized enzyme was observed after 30 days of continuous operation, during which high productivity of 1174 g/L·h was achieved. The kinds of products obtained using the immobilized enzyme were almost the same as those using soluble enzymes or free cells.     

Conformational changes of soluble mitochondrial ATPase as controlled by hydrophobic interactions within the enzyme.

At 30° C soluble mitochondrial ATPase from baker's yeast shows non-linear kinetics with respect to Mg-ATP; the apparent Km values for Mg-ATP are 0.6 and 2.0 mM. At lower temperatures, 5° C and 12° C, the kinetics of the enzyme are linear with a Km for Mg-ATP of approximately 0.6 mM. Octylguanidine induces non-linear kinetics at 12° C. As octylguanidine and increases in temperature augment hydrophobic interactions within the enzyme, it is concluded that the strength of hydrophobic bonding within the protein regulates its conformational changes. Methanol activates the enzyme only at relatively high temperature which further indicates that the protein may exist in two active conformations.     

Immobilization of horseradish peroxidase on activated wool

This work is aimed to immobilize partially purified horseradish peroxidase (HRP) on wool activated by multifunctional reactive center, namely cyanuric chloride. The effect of cyanuric chloride concentration, pH and enzyme concentration on immobilization of HRP was studied. FT-IR and SEM analyses were detected for wool, activated wool and immobilized wool-HRP. The wool-HRP, prepared at 2% (w/v) cyanuric chloride and pH 5.0, retained 50% of initial activity after seven reuses. The wool-HRP showed broad optimum pH at 7.0 and 8.0, which was higher than that of the soluble HRP (pH 6.0). The soluble HRP had an optimum temperature of 30 °C, which was shifted to 40 °C for immobilized enzyme. The soluble and wool-HRP were stable up to 30 and 40 °C after incubation for 1 h, respectively. The apparent kinetic constant values (K_ms) of wool-HRP were 10 mM for guiacol and 2.5 mM for H₂O₂, which were higher than that of soluble HRP. The wool-HRP was remarkably more stable against proteolysis mediated by trypsin. The wool-HRP exhibited more resistance to heavy metal induced inhibition. The wool-HRP was more stable to the denaturation induced by urea, Triton X-100, isopropanol, butanol and dioxan. The wool-HRP was found to be the most stable under storage. In conclusion, the wool-HRP could be more suitable for several industrial and environmental purposes.     

Comparative study of stability of soluble and cell wall invertase from Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is the most significant source of enzyme invertase. It is mainly used in the food industry as a soluble or immobilized enzyme. The greatest amount of invertase is located in the periplasmic space in yeast. In this work, it was isolated into two forms of enzyme from yeast S. cerevisiae cell, soluble and cell wall invertase (CWI). Both forms of enzyme showed same temperature optimum (60°C), similar pH optimum, and kinetic parameters. The significant difference between these biocatalysts was observed in their thermal stability, stability in urea and methanol solution. At 60°C, CWI had 1.7 times longer half-life than soluble enzyme, while at 70°C CWI showed 8.7 times longer half-life than soluble enzyme. After 2-hr of incubation in 8?M urea solution, soluble invertase and CWI retained 10 and 60% of its initial activity, respectively. During 22?hr of incubation of both enzymes in 30 and 40% methanol, soluble invertase was completely inactivated, while CWI changed its activity within the experimental error. Therefore, soluble invertase and CWI have not shown any substantial difference, but CWI showed better thermal stability and stability in some of the typical protein-denaturing agents.