Immobilization and stabilization of cephalosporin C acylase on aminated support by crosslinking with glutaraldehyde and further modifying with aminated macromolecules

In this work, cephalosporin C acylase (CA), a heterodimeric enzyme of industrial potential in direct hydrolysis of cephalosporin C (CPC) to 7‐aminocephalosporanic acid (7‐ACA), was covalently immobilized on the aminated support LX1000‐HA (HA) with two different protocols. The stability of CA adsorbed onto the HA support followed by crosslinking with glutaraldehyde (HA–CA–glut) was better than that of the CA covalently immobilized on the glutaraldehyde preactivated HA support (HA–glut–CA). The thermostabilization factors (compared with the free enzyme) of these two immobilized enzymes were 11.2‐fold and 2.2‐fold, respectively. In order to improve the stability of HA–CA–glut, a novel strategy based on postimmobilization modifying with aminated molecules was developed to take advantage of the glutaraldehyde moieties left on the enzyme and support. The macromolecules, such as polyethyleneimine (PEI) and chitosan, had larger effects than small molecules on the thermal stability of the immobilized enzyme perhaps due to crosslinking of the enzymes and support with each other. The quaternary structure of the CA could be much stabilized by this novel approach including physical adsorption on aminated support, glutaraldehyde treatment, and macromolecule modification. The HA–CA–glut–PEI20000 (the HA–CA–glut postmodified with PEI M_w = 20,000) had a thermostabilization factor of 20‐fold, and its substrate affinity (K_m = 14.3 mM) was better than that of HA–CA–glut (K_m = 33.4 mM). The half‐life of the immobilized enzymes HA–CA–glut–PEI20000 under the CPC‐catalyzing conditions could reach 28 cycles, a higher value than that of HA–CA–glut (21 cycles). © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:387–395, 2015     

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (～99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent K_m value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.     

Preparation of a very stable immobilized biocatalyst of glucose oxidase from Aspergillus niger

Glucose oxidase (GOX) has been immobilized on different activated supports, including glyoxyl agarose, epoxy sepabeads and glutaraldehyde-activated supports. Immobilization onto supports pre-activated with glutaraldehyde rendered the most thermo-stable preparation of GOX. Therefore, as the glutaraldehyde chemistry gave a high stabilization of the enzyme, we proposed another technique for improving the multipoint attachment through glutaraldehyde: the enzyme was ionically adsorbed on cationic supports with primary amino groups and then the immobilized preparation was treated with a glutaraldehyde solution. The decrease on enzyme activity was <20%. Following this methodology, we achieved the highest stability of all the immobilization systems analyzed, showing a half-life 100 times higher than the soluble enzyme. Moreover, this derivative showed a higher stability in the presence of organic solvents (for instance methanol) or hydrogen epoxide than the ionically adsorbed enzyme or the soluble one. Therefore, the adsorption of GOX on aminated cationic support and subsequent treatment with glutaraldehyde was presented as a very successful methodology for achieving a very stable biocatalyst.     

Epoxy-amino groups: a new tool for improved immobilization of proteins by the epoxy method

The properties of a new commercially available amino-epoxy support (amino-epoxy-Sepabeads) for immobilizing enzymes have been compared to those of conventional epoxy supports. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. Thus, this support has a great anionic exchanger power and a high number of epoxy groups. We have found a number of advantages to this new heterofunctional support. Immobilization proceeds at low ionic strength using amino epoxy Sepabeads while requiring high ionic strength using conventional monofunctional epoxy supports. Immobilization is much more rapid using amino-epoxy supports than employing conventional epoxy supports. The possibility of achieving immobilized preparations in which the enzyme orientation may be different to that obtained using the traditional hydrophobic supports (with likely effects in terms of activity or stability). Stability of the immobilized enzyme has been found to be much higher using the new support than in preparations using the conventional ones in many cases. Here we show some examples of these advantages using different enzymes (beta-galactosidases, lipase, glutaryl acylase, invertase, and glucoamylase).     

Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment

Sepabeads-EP (a new epoxy support) has been utilized to immobilize-stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 micromol/mL). These features should permit a very intense enzyme-support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three-step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds-fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 degrees C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).     

Immobilization of glycoenzymes through carbohydrate side chains.

Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24, 20, 30, and 15 mol/mol of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an N-hydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 37–50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.     

Invertase covalent grafting onto corn stover

The covalent coupling of an invertase from baker's yeast onto an agricultural by-product, corn grits, has been developed. The optimal conditions for each step of the chemical modification of the support have been determined: oxidation with sodium metaperiodate, amination with ethylenediamine, reduction with sodium cyanoborohydride, and activation with glutaraldehyde. Activities up to 7.2 x 10(4) mumol reducing sugars produced/min g support could thus be achieved. Invertase coupling onto corn grits yields a derivative with a 25 times higher activity than when coupling this enzyme onto porous silica. The operational stability of invertase immobilized onto corn stover was found to be very high, with a half-life of up to 365 days at 40 degrees C when using a 2M sucrose solution as substrate. This immobilization method could be easily scaled up to the preparation of 10 kg of invertase derivative.     

Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii

The ability to convert d-galactose into d-tagatose was compared among a number of bacterial l-arabinose isomerases (araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis–Menten constants of the enzyme determined with l-arabinose, d-galactose and d-fucose also indicated that this enzyme is an unusual, versatile l-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of d-tagatose at 65 °C. Starting from a 30% solution of d-galactose, the yield of d-tagatose was 42% and no sugars other than d-tagatose and d-galactose were detected. Direct conversion of lactose to d-tagatose in a single reactor was demonstrated using a thermostable -galactosidase together with the thermostable l-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.     

Immobilization and stabilization of α-galactosidase on Sepabeads EC-EA and EC-HA

α-Galactosidase from tomato has been immobilized on Sepabead EC-EA and Sepabead EC-HA, which were activated with ethylendiamino and hexamethylenediamino groups, respectively. Two strategy was used for the covalent immobilization of α-galactosidase on the aminated Sepabeads: covalent immobilization of enzyme on glutaraldehyde activated support and cross-linking of the adsorbed enzymes on to the support with glutaraldehyde. By using these two methods, all the immobilized enzymes retained very high activity and the stability of the enzyme was also improved. The obtained results showed that, the most stable immobilized α-galactosidase was obtained with the second strategy. The immobilized enzymes were characterized with respect to free counterpart. Some parameters effecting to the enzyme activity and stability were also analyzed. The optimum temperature and pH were found as 60 °C and pH 5.5 for all immobilized enzymes, respectively. All the immobilized α-galactosidases were more thermostable than the free enzyme at 50 °C. The stabilities of the Sepabead EC-EA and EC-HA adsorbed enzymes treated with glutaraldehyde compared to the stability of the free enzyme were a factor of 6 for Sepabead EC-EA and 5.3 for Sepabead EC-HA. Both the free and immobilized enzymes were very stable between pH 3.0 and 6.0 and more than 85% of the initial activities were recovered. Under the identical storage conditions the free enzyme lost its initial activity more quickly than the immobilized enzymes at the same period of time. The immobilized α-galactosidase seems to fulfill the requirements for different industrial applications.     

Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48

The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.     

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β‐galactosidase

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead‐Glu) or carboxyl groups through acid solution (Immobead‐Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β‐galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead‐Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10–500 mg g^?1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g^?1 support. Gal immobilized on Immobead‐Glu and Immobead‐Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half‐lifes than the soluble enzyme, where the half‐lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:934–943, 2018     

Immobilization of endo-polygalacturonase from Aspergillus niger on various types of macromolecular supports

Endo-polygalacturonase (endo-PG) was immobilized on a wide range of natural and synthetic macromolecular supports and their modified derivatives representing many chemical classes, including esters, amides, phenols, alkyl- and arylamines, and carboxyl derivatives. The immobilization entailed methods of adsorption alone as well as covalent bond formation using glutaraldehyde or carbodiimide or via the diazo-coupling reaction. The most promising system proved to be immobilization on trimalehylchitosan (TMC) via adsorption followed by treatment with glutaraldehyde (GA). The binding capacity of the support is on the order of 13,000 IU/g, half of which is active. Various properties of immobilized endo-PG were evaluated. The optimum pH of the enzyme shifted to the alkaline side. The relative catalytic activity was considerably high even at room temperature and remained so above 70 degrees C. The thermal stability at pH 3-4 was notably improved by immobilization, the half-time doubling. Finally, the apparent K(m) was greater for immobilized endo-PG than for native enzyme, while the V(max) was smaller for the immobilized enzyme.     

Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports

The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.     

An approach for the stable immobilization of proteins

An approach is presented for the stable covalent immobilization of proteins with a high retention of biological activity. First, chemical modification studies were used to establish enzyme structural and functional properties relevant to the covalent immobilization of an enzyme to agarose based supports. Heparinase was used as a model enzyme in this set of studies. Amine modifications result in 75-100% activity loss, but the effect is moderated by a reduction in the degree of derivatization. N-hydroxysuccinimide, 1,1,1-trifluoroethanesulfonic acid, and epoxide activated agarose were utilized to determine the effect of amine reactive supports on immobilized enzyme activity retention. Cysteine modifications resulted in 25-50% loss in activity, but free cysteines were inaccessible to either immobilized bromoacetyl or p-chloromercuribenzoyl groups. Amine reactive coupling chemistries were therefore utilized for the covalent immobilization of heparinase. Second, to ensure maximal stability of the immobile protein-support linkage, the identification and subsequent elimination of the principal sources of protein detachment were systematically investigated. By using high-performance liquid chromatography (HPLC), electrophoresis, and radiolabeling techniques, the relative contributions of four potential detachment mechanisms-support degradation, proteolytic degradation, desorption of noncovalently bound protein, and bond solvolysis-were quantified. The mechanisms of lysozyme, bovine serum albumin, and heparinase leakage from N-hydroxysuccinimide or 1,1,1-trifluoroethanesulfonic acid activated agarose were elucidated. By use of stringent postimmobilization support wash procedures, noncovalently bound protein loss. An effective postimmobilization washing procedure is presented for the removal of adsorbed protein and the complete elimination of immobilized protein loss.     

Covalent Immobilization of Alcohol Dehydrogenase (ADH2) from Haloferax volcanii: How to Maximize Activity and Optimize Performance of Halophilic Enzymes

Alcohol dehydrogenase from halophilic archaeon Haloferax volcanii (HvADH2) was successfully covalently immobilized on metal-derivatized epoxy Sepabeads. The immobilization conditions were optimized by investigating several parameters that affect the halophilic enzyme–support interaction. The highest immobilization efficiency (100 %) and retention activity (60 %) were achieved after 48 h of incubation of the enzyme with Ni-epoxy Sepabeads support in 100 mM Tris–HCl buffer, pH 8, containing 3 M KCl at 5 °C. No significant stabilization was observed after blocking the unreacted epoxy groups with commonly used hydrophilic agents. A significant increase in the stability of the immobilized enzyme was achieved by blocking the unreacted epoxy groups with ethylamine. The immobilization process increased the enzyme stability, thermal activity, and organic solvents tolerance when compared to its soluble counterpart, indicating that the immobilization enhances the structural and conformational stability. One step purification–immobilization of this enzyme has been carried out on metal chelate-epoxy Sepabeads, as an efficient method to obtain immobilized biocatalyst directly from bacterial extracts.     

Tagatose: properties, applications, and biotechnological processes

d-Tagatose has attracted a great deal of attention in recent years due to its health benefits and similar properties to sucrose. d-Tagatose can be used as a low-calorie sweetener, as an intermediate for synthesis of other optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulation. Biotransformation of d-tagatose has been produced using several biocatalyst sources. Among the biocatalysts, l-arabinose isomerase has been mostly applied for d-tagatose production because of the industrial feasibility for the use of d-galactose as a substrate. In this article, the characterization of many l-arabinose isomerases and their d-tagatose production is compared. Protein engineering and immobilization of the enzyme for increasing the conversion rate of d-galactose to d-tagatose are also reviewed.     

Immobilization of enzymes on porous silica supports

Four silica supports differing in pore dimensions were activated by treatment with SiCl₄ and then with ethylenediamine to obtain alkylamine groups on the silica surface. Three enzymes, peroxidase from cabbage, glucoamylase from Aspergillus niger C and urease from soybean were immobilized on these supports using glutaraldehyde as coupling agent. It was found that the protein content, the retained enzymatic activity and the storage stability of the silica supported enzymes were considerably affected by support pore size and enzyme molecular weight, the factors which are supposed to alter protein distribution inside the support pores. The highest activity was found for peroxidase and glucoamylase attached to the silica with the widest pores, but their loss in activity during storage was considerable. The urease retained less activity after immobilization, but its storage stability was excellent.     

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.     

Characterization of rifamycin oxidase immobilized on nylon fibers

Summary Rifamycin oxidase, an enzyme used in the biotransformation of rifamycin B to S was immobilized on nylon fibers using glutaraldehyde as the cross linking agent. An activity of 18 U/g of nylon fiber with a binding efficiency of 37% was achieved. The immobilized enzyme showed an operational stability of 7 days and was also protected against thermal inactivation. It exhibited a K_m(app.) of 2.0mM.     

Immobilization of penicillin G acylase on aminoalkylated polyacrylic supports

A procedure is described for the immobilization of penicillin G acylase (PA) on Amberlite XAD7 modified by transamidation with 1,2-ethylenediamine and activated with glutaraldehyde. Reduction with sodium borohydride of the Schiff's bases formed between the amino groups of the protein and glutaraldehyde results in a dramatic improvement of the operational stability of the immobilized enzyme without affecting the catalytic activity. The enzyme kept in presence of the substrate, penicillin G, displays an increased stability with respect to that stored in pure phosphate buffer solution. The inactivation kinetics of the immobilized preparations of PA, determined in a continuous fixed bed reactor, as well as a discontinuous batch reactor, are reported.