Comparison of some properties of free and immobilized alpha-amylase by Aspergillus sclerotiorum in calcium alginate gel beads

Some properties of immobilized alpha-amylase by Aspergillus sclerotiorum within calcium alginate gel beads were investigated and compared with soluble enzyme. Optimum pH and temperature were found to be 5.0 and 40 degrees C, respectively, for both soluble and immobilized enzymes. The immobilized enzyme had a better Km value, but kcat/Km values were the same for both enzymes. Entrapment within calcium alginate gel beads improved, remarkably, the thermal and storage stability of alpha-amylase. The half life values of immobilized enzyme and soluble enzyme at 60 degrees C were 164.2, and 26.2 min, respectively. The midpoint of thermal inactivation (Tm) shifted from 56 degrees C (for soluble enzyme) to 65.4 degrees C for immobilized enzyme. The percentages of soluble starch hydrolysis for soluble and immobilized alpha-amylase were determined to be 97.5 and 92.2% for 60 min, respectively.     

Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent.

A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).     

Immobilization of adenosine deaminase onto agarose and casein

In the present study adenosine deaminase (ADA) was immobilized onto two different polymeric materials, agarose and casein. The factors affecting the amount of enzyme attachment onto the polymeric supports such as incubation time were investigated. The maximum amount of enzyme immobilized onto different polymeric supports occurred at incubation pH value 7.5 and ADA concentration 42 units/g and the incubation time needed for the maximum amount of enzyme attachment to the polymeric supports was found to be 8 h. Some phsicochemical properties of the free and immobilized ADA such as operational stability, optimum temperature and thermal stability, pH optimum and stability, storage stability, and the effect of gamma-radiation were studied. The operational stability of the free and immobilized enzyme showed that the enzyme immobilized by a cross-linking technique using gultaric dialdehyde showed poor durability and the relative activity decreased sharply due to the leakage after repeated washing, while the enzymes immobilized by covalent bonds to the carriers showed a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4-6 months, showed that the free enzyme lost its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzyme to a dose of gamma radiation of 0.5-10 Mrad showed complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to gamma radiation up to a dose of 5 Mrad.     

Immobilized Human Plasmins: Preparation and Enzymatic Properties

Plasminogen was immobilized on agarose using either a commercially available substituted gel, or the cyanogen bromide (CNBr) activation procedure as a means of coupling the protein to agarose. Coupling the zymogen to the gel followed by its activation with urokinase yielded an immobilized plasmin. The immobilized enzymes have esterase, amidase and protease activity towards lysine and arginine esters, lysine anilide and casein, respectively. They activate plasminogen by a linear non-autocatalytic process. Both enzyme preparations are stable for extended periods of time in the absence of any stabilizing agents, and are not denatured by high salt concentrations or detergents.     

alpha-Amylase immobilized on bulk acoustic-wave sensor by UV-curing coating

A new method for immobilization of alpha-amylase by UV-curing coating is proposed in this paper. The immobilization procedure of UV-curing coating on piezoelectric quartz crystal is simple and convenient, and causes less loss of enzymatic activity. The activity of the immobilized alpha-amylase is monitored by a technique based on bulk acoustic-wave (BAW) sensor. The frequency shift of BAW sensor can reflect the degree of hydrolysis of starch by the immobilized alpha-amylase. It is appropriate for the immobilized alpha-amylase to hydrolyze the soluble starch under pH 7.0 condition, which is similar to that of the free alpha-amylase. Kinetic parameters (the Michaelis constant, K(m), and the maximum initial rate V(max)) of the enzymatic hydrolysis of starch by the immobilized alpha-amylase are estimated by using a linear method of Lineweaver-Burk plot. K(m)=12.7mgml(-1) and V(max)=15.9Hzmin(-1). And the experimental results show that the immobilized alpha-amylase entrapped by the UV-curing coating retains adequate enzymatic activity and can be reused more than 50 times under certain experimental conditions.     

Immobilization of enzymes based on hydrophobic interaction. I. Preparation and properties of a β-amylase adsorbate

Sweet potato β-amylase (α-1,4 glucan maltohydrolase, EC 3.2.1.2) was immobilized through adsorption onto an agarose gel to which nonpolar side chains had been introduced via ether bridges. The adsorbent showed evidence of saturation at an enzyme content of 35 mg per milliliter of packed gel. The adsorption was rapid and yielded a product whose operational stability depended on the initial content of β-amylase. Activity leakage was low. The relative activity of immobilized enzyme was inversely related to the amount of enzyme adsorbed to a given gel volume, having a maximal value of around 50% at low enzyme contents.     

Stabilization of alanine aminotransferase by consecutive modification and immobilization

Summary A new combination of methodologies for enzyme stabilization has been carried out. Dimethylsuberimidate-modified alanine aminotransferase was covalently immobilized on a preactivated agarose gel. The resulting derivative showed greater residual activity than the immobilized-only counterpart, maintaining the same amount of immobilized enzyme and its stability was greater than the native, modified and immobilized enzymes in several conditions.     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

Purification and covalent coupling of calf brain prolidase

We have investigated methods of stabilizing prolidase by chemical modification and covalent coupling to various supports, for use in protein hydrolysis and possible use in enzyme replacement therapy. Purified acetone powder of calf brain prolidase was further purified by gel filtration on Sephadex G-200 and chromatography on DEAE-Sephadex A25. Polyacrylamide gel electrophoresis showed that the number of bands was reduced from 11 to 2. Since yields were low, the purified (NH₄)₂SO₄ fraction was used in all experiments. Thiolation of the enzyme reduced the amount of protein coupled to AH-or CH-Sepharose 4B. Activities were highest when the protein was linked through its carboxyl groups. The coupled enzyme showed much greater thermal stability than its free counterpart. Of the bound preparations, the thiolated was less stable than the untreated. Untreated and thiolated enzymes bound to either matrix showed higher activity at low pH and less at high pH than the free material. Thiolation shifted the pH maximum from 6.8 to 7.5. The free thiolated enzyme and that bound to activated SH-Sepharose 4B showed greater thermal stability and a broader pH range of optimal activity than the bound untreated enzyme. These results show that prolidase can be immobilized by coupling to an insoluble matrix through various types of covalent bonds with retention of activity and increased stability.     

Digital microfluidic hydrogel microreactors for proteomics

Proteolytic digestion is an essential step in proteomic sample processing. While this step has traditionally been implemented in homogeneous (solution) format, there is a growing trend to use heterogeneous systems in which the enzyme is immobilized on hydrogels or other solid supports. Here, we introduce the use of immobilized enzymes in hydrogels for proteomic sample processing in digital microfluidic (DMF) systems. In this technique, preformed cylindrical agarose discs bearing immobilized trypsin or pepsin were integrated into DMF devices. A fluorogenic assay was used to optimize the covalent modification procedure for enzymatic digestion efficiency, with maximum efficiency observed at 31 μg trypsin in 2-mm diameter agarose gel discs. Gel discs prepared in this manner were used in an integrated method in which proteomic samples were sequentially reduced, alkylated, and digested, with all sample and reagent handling controlled by DMF droplet operation. Mass spectrometry analysis of the products revealed that digestion using the trypsin gel discs resulted in higher sequence coverage in model analytes relative to conventional homogenous processing. Proof-of-principle was demonstrated for a parallel digestion system in which a single sample was simultaneously digested on multiple gel discs bearing different enzymes. We propose that these methods represent a useful new tool for the growing trend toward miniaturization and automation in proteomic sample processing.     

Synthesis and characterization of chitosan-homocysteine thiolactone as a mucoadhesive polymer

Two mucoadhesive thiolated polymers were synthesized by the covalent attachment of homocysteine thiolactone (HT) to chitosan and N,N,N-trimethyl-chitosan (TM-chitosan) at various chitosan:HT ratios. The amount of thiol and disulphide groups immobilized on the chitosan influenced the polymer's mucoadhesion positively and negatively, respectively, with the optimal chitosan:HT (w/w) ratio being found to be 1:0.1. The interaction between mucin and chitosan and its three derivatives was highest for the thiolated chitosan derivatives but was pH dependent. HT-chitosan and TM-HT-chitosan, with the thiol groups of 64.15 and 32.48 μmol/g, respectively, displayed a 3.67- and 6.33-fold stronger mucoadhesive property compared to that of the unmodified chitosan at pH 1.2, but these differences were only ∼1.7-fold at pH 6.4. The swelling properties of TM-HT-chitosan and HT-chitosan were higher than that of chitosan and TM-chitosan, attaining a swelling ratio of up to 240% and 140%, respectively, at pH 1.2 within 2 h.     

Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization

The immobilization of a glutamate dehydrogenase from Thermus thermophilus (GDH) on glyoxyl agarose beads at pH 7 has permitted to perform the immobilization, purification and stabilization of this interesting enzyme. It was cloned in Escherichia coli and a first thermal shock of the crude preparation destroyed most mesophilic multimeric proteins. Glyoxyl agarose can only immobilize enzymes via a multipoint and simultaneous attachment. Therefore, only proteins having several terminal amino groups in a position that permits their interaction with a flat surface can be immobilized. GDH became rapidly immobilized at pH 7 and its multimeric structure became stabilized as evidenced by SDS-PAGE. This derivative was stable at acidic pH value while the non-stabilized enzyme was very unstable under these conditions due to subunit dissociation. After immobilization, a further incubation at pH 10 improved enzyme stability under any inactivating conditions by increasing the enzyme–support bonds. In fact, GDH immobilized at pH 7 and incubated at pH 10 preserved more activity than GDH directly immobilized at pH 10 (50% versus 15% after 24 h of incubation) and was also more stable (1.5- to 3-fold, depending on the conditions).This method could be extended to any other multimeric enzyme expressed in mesophilic hosts.     

Superporous agarose monoliths as mini-reactors in flow injection systems

A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15×5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 m in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 Å) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterised with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 ml min^–1). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular -galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against -galactosidase).     

Enzyme immobilization on tritylagarose

A method is described for the immobilization on tritylated agarose or Sepharose columns of a wide spectrum of enzymes, including types useful in contemporary biochemistry/molecular biology, many of which have never before been reported as immobilized. The method involves the formation of noncovalent hydrophobic bonds between the enzymes and trityl groups which are attached to the agarose by means of ether bonds. The immobilization of calf intestinal and E. coli alkaline phosphatases to tritylagarose is reported in detail. Their binding strength, binding capacity, and long-term stability (greater than six months) are described as a function of the salt concentration, pH, buffer type, and degree of agarose substitution. Homologies are noted between tritylagarose-bound and membrane-bound phosphatases. This method compares favorably with other methods, covalent or otherwise, reported to date, in terms of the enzyme immobilization yield (ca. 100%), the mildness of conditions, resulting, in most cases, in the retention of a high degree of activity, the ease and speed of the manipulations, and the long-term stability of the immobilized enzyme. Further, it is noted that highly tritylated and crosslinked Sephadex G10 selectively and mildly removes detergents from enzyme solutions.     

Stabilization of a highly active but unstable alcohol dehydrogenase from yeast using immobilization and post-immobilization techniques

The alcohol dehydrogenase (ADH) from Baker's yeast is very active but extremely unstable under several different conditions. Mild immobilization methods such as one-point attachment to agarose activated with cyanogen bromide groups or ionic adsorption to agarose activated with charged groups allow high activity recoveries (80–100%) but do not promote protein stabilization. In contrast, immobilization methods that force the enzyme to be covalently attached at multiple points on the support fully inactivate the enzyme. Herein, we propose an interesting solution to address the dichotomy between activity and stability. We have developed a protocol in which the enzyme is immobilized on agarose activated with glyoxyl groups in the presence of acetyl cysteine, which results in the recovery of 25% of the enzyme activity but increases the thermal stability of the soluble enzyme 50-fold. However, this immobilization technique does not stabilize the enzyme quaternary structure. Hence, a post-immobilization technique using functionalized polymers has been used to cross-link all enzyme subunits. In this method, polycationic polymers (polyethylenimine) cross-link the quaternary structure with a negligible effect on catalytic activity, which results in a derivative that is 5-fold more stable than non-cross-linked derivatives under very dilute and acidic conditions that highly favor subunit dissociation. Therefore, the stability was increased 500-fold for this optimal derivative compared to diluted soluble enzyme, although the relative expressed activity was low (25%). However, the low expressed activity may be overcome by designing immobilized biocatalysts with high volumetric activities.     

Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.     

Detection of beta-1,6-glucanase isozymes from Trichoderma strains in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectrofocusing gels.

The filamentous fungus Trichoderma produces, under specific growth conditions, several extracellular fungal cell wall degrading enzymes, amongst them beta-1,6-glucanases. These enzymes seem to play an important role in the antagonistic action of Trichoderma against a wide range of fungal plant pathogens. In this report we describe two different methods for the specific detection of the activity of beta-1,6-glucanase isozymes in gels. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, beta-1,6-glucanase activity can be assayed in the gel by renaturation of the enzyme, incubation with an overlay agarose gel containing solubilized pustulan (a commercially available beta-1,6-glucan), followed by the staining of the agarose gel with Congo Red. In native isoelectrofocusing gels, as little as 1 mU can be detected after incubation with solubilized pustulan followed by a detection reaction of the released reducing sugars with 2,3,5-triphenyltetrazolium chloride. The latter technique has been successfully applied to the screening of beta-1,6-glucanase isozymes from different Trichoderma strains under different growth conditions.     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

Preparation of immobilized alpha-amylase covalently attached to granular polyacrylonitrile

In this report, alpha-Amylase originating from Bacillus subtilis (liquefying type) was immobilized on partially imidoesterized polyacrylonitrile (PAN) by covalent bonding. For the preparation of immobilized alpha-amylase, which has a high activity and high stability to repeated use, the optimum conditions for the preparation reaction were investigated. The optimum conditions for the preparation reaction were quantified on the basis of the enzymatic activity, the preservation of the activity during repeated use in batch process and the protein content on the support. Further-more, enzymatic properties of immobilized alpha-amylase prepared at optimum conditions were compared with the native enzyme. The optimum temperature and reaction time for the imidoes-terification reaction were 30 degrees c and 6 h, respectively, whereas those of the amidinatin reaction were 30-40 degrees C and more than 3 h, respectively; the optimum pH range was 9-10. Immobilized alpha-amylase prepared at the optimum conditions was very stable against the repeated use and had more than 90% of relative to activity of the first use after the tenth procedure. The initial reaction rate of immobilized alpha-amylase was lower than native alpha-amylase, but same amount of reducing sugars were produced after the reaction passed for more than 90 min. The immobilized alpha-amylase was less stabel at the high temperature and the more basic media. However, after long incubation time, immobilized alpha-amylase was more stable than the native enzyme in exposure to heat and a storng base.     

Preparation and characterization of an enzymatically active immobilized derivative of myosin.

Purified skeletal muscle myosin (EC 3.6.1.3) has been covalently bound to Sepharose 4B by the cyanogen bromide procedure. The resulting complex, Sepharose-Myosin, possesses adenosine triphosphatase activity and is relatively stable for long periods of time. Under optimal binding conditions, approximately 33% of the specific ATPase activity of the bound myosin is retained. Polyacrylamide gel electrophoresis of polypeptides released from denatured Sepharose-Myosin indicates that 85% of the myosin is attached to the agarose beads through the heavy chains and the remainder through the light chains, in agreement with predictions of binding and release based upon either the lysine contents or molecular weights of themyosin subunits. The adenosine triphosphatase of the immobilized myosin has been investigated under conditions of varying pH, ionic strength, and cation concentration. The ATPase profiles of immobilized myosin are quite similar to those for free myosin, however subtle differences are found. The Sepharose-Myosin ATPase is not as sensitive as myosin to alterations in salt concentration and the apparent KM is approximately two-fold higher than that of myosin. These differences are probably due to chemical modification in the region of the attachment site(s) to the agarose beads and hydration and diffusion limitations imposed by the polymeric agarose matrix.