Modulation of phosphofructokinase behavior by chemical modifications during the immobilization process

Phosphofructokinase was immobilized within a protein membrane or on soluble protein polymers using glutaraldehyde as cross-linking reagent. The native enzyme was also modified chemically, using the cross-linking reagent alone. A comparative kinetic investigation of these preparations was carried out. The catalytic activity of the chemically modified enzyme and its affinity towards fructose 6-phosphate decreased significantly; the modified enzyme lost its cooperative properties and the allosteric regulation by AMP was affected. When the chemical treatment was performed in the presence of effectors (AMP or ATP) the allosteric transition induced by AMP was restored, suggesting that the cross-linking reagent modified the AMP regulatory sites, albeit no higher-substrate-affinity enzyme conformation was frozen. Molecular data showed that glutaraldehyde produced intramolecular then intermolecular bonds as its concentration increased. When the enzyme was immobilized into protein membranes or on soluble polymers, the enzyme behavior was quite similar: decrease of affinity towards fructose 6-phosphate but no changes in cooperative properties and modifications of allosteric transition induced by AMP. When AMP was present during the immobilisation process, the enzyme immobilized in this way was no longer sensitive to effectors, either AMP or ATP. It showed Michaelian behavior and higher substrate affinity quite similar to that of the native enzyme. The data suggested that a higher-substrate-affinity enzymatic form was most probably stabilized by immobilization.     

Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization

Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.     

Use of hexadecyl Fractosil as a hydrophobic carrier for adsorptive immobilization of proteins

Fractosil, a porous form of silica, has been used for the preparation of a hydrophobically derivatized carrier for protein immobilization. Interaction of a number of arbitrarily chosen proteins with hexadecyl-substituted Fractosil has been investigated. Binding of proteins was found to take place with retention of their native properties. Glutamate dehydrogenase, used as a model allosteric protein, was found to retain its catalytic and allosteric properties upon binding to the adsorbent in the form of suspension or column. Positive cooperative interactions for binding of bovine serum albumin and glutamate dehydrogenase to the matrix were observed. These findings are discussed in terms of hydrophobic interactions occurring between various residues of the protein molecules and the hydrophobic ligands in addition to those interactions which may occur with the unsubstituted gel. Results presented on immobilized glutamate dehydrogenase, trypsin, alpha-chymotrypsin, alpha-amylase, and amyloglucosidase clearly indicate possible potential of the support for continuous catalytic transformations.     

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.     

A smart bioconjugate of chymotrypsin

alpha-Chymotrypsin was immobilized on Eudragit S-100 via covalent coupling with 93% retention of proteolytic activity. The conjugate behaved as a smart biocatalyst and functioned as a pH-dependent reversibly soluble-insoluble biocatalyst. The pH optimum of chymotrypsin broadened on immobilization, and the immobilized preparation showed better stability at and above pH 6.5 as compared to the free enzyme. The immobilized enzyme showed a slight shift in the temperature optimum and enhanced thermal stability retaining 70% of its original activity after 1 h of exposure to 40 degrees C as compared to the 25% residual activity for the free enzyme under identical conditions. K(m) and V(max) values did not change on immobilization. Also, the immobilized preparation was quite stable to reuse, it retained almost 85% of its original activity even after a fifth precipitation cycle. UV spectroscopy and circular dichroism were used to probe structural changes in the enzyme upon immobilization.     

Sucrose hydrolysis by invertase immobilized on functionalized porous silicon

A successful recipe for the production of immobilized invertase/porous silicon layer with appropriate catalytic behavior for the sucrose hydrolysis reaction is presented. The procedure is based on support surface chemical oxidation, silanization, activation with glutaraldehyde and finally covalent bonding of the free enzyme to the functionalized surface. The catalytic behavior of the composite layer as a function of pH, temperature, and the current density applied in the porous silicon (PS) preparation is investigated. Interestingly, V_max undergoes a substantial increase (ca. 30%) upon immobilization. The value of K_m increases by a factor of 1.53 upon immobilization. The initial activity is still preserved up to 28 days while the free enzyme undergoes a 26% loss of activity after the same period. Based on the outcomes of this study, we believe that tailored PS layers may be used for the development of new bioreactors in which the active enzyme is immobilized on the internal walls and is not lost during the process.     

Polyaniline as a support for urease immobilization

Polyaniline synthesized by chemical oxidative polymerization was used as an immobilization support for jack bean urease. Such immobilized enzyme has a good catalytic activity, storage stability, and reusability. Properties of free and immobilized urease were compared. Blends of polystyrene, cellulose acetate and poly(methyl methacrylate) with polyaniline were used for urease immobilization as well.     

Immobilization of Aspergillus oryzae tannase and properties of the immobilized enzyme

Tannase enzyme from Aspergillus oryzae was immobilized on various carriers by different methods. The immobilized enzyme on chitosan with a bifunctional agent (glutaraldehyde) had the highest activity. The catalytic properties and stability of the immobilized tannase were compared with the corresponding free enzyme. The bound enzyme retained 20·3% of the original specific activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme. The optimum temperature of the reaction was determined to be 40 °C for the free enzyme and 55 °C for the immobilized form. The stability at low pH, as well as thermal stability, were significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilized enzyme retained about 85% of the initial catalytic activity, even after being used 17 times.     

Immobilized phospholipase A2 from cobra venom. Prevention of substrate interfacial and activator effects

The activation of cobra venom phospholipase A2 by activators (containing phosphorylcholine moieties) appears to depend upon the aggregation state of the enzyme, and the presence of a lipid-water interface. The characteristics of this activation were studied by comparing the behavior of the enzyme immobilized on an agarose gel to that of the soluble enzyme. The immobilized enzyme displays only a few per cent of the soluble enzyme activity toward micellar dipalmitoyl-phosphatidylcholine (PC). However, the relative loss of activity is much less with micellar dipalmitoylphosphatidylethanolamine or soluble diheptanoyl-PC. The affinity for Ca2+ is increased about 10-fold by immobilization while the apparent pKa of the enzyme is decreased by 0.5-0.8 pH units. Activation energies are similar for the two enzyme forms and are independent of the physical state of the substrate used. Catalytic constants of the enzyme toward monomeric PC are not changed by immobilization. Yet, activators of the soluble enzyme have negligible effect on the immobilized enzyme, either in the presence or absence of an interface. Monomeric activators promote the binding of the soluble enzyme to the immobilized form. Apparently, immobilization mainly produces monomerically constrained enzyme which cannot be activated under any condition, whereas normally, activators in the presence of lipid-water interfaces induce the formation of enzyme dimers or possibly higher order aggregates.     

Polymer materials for enzyme immobilization and their application in bioreactors

Enzymatic catalysis has been pursued extensively in a wide range of important chemical processes for their unparalleled selectivity and mild reaction conditions. However, enzymes are usually costly and easy to inactivate in their free forms. Immobilization is the key to optimizing the in-service performance of an enzyme in industrial processes, particularly in the field of non-aqueous phase catalysis. Since the immobilization process for enzymes will inevitably result in some loss of activity, improving the activity retention of the immobilized enzyme is critical. To some extent, the performance of an immobilized enzyme is mainly governed by the supports used for immobilization, thus it is important to fully understand the properties of supporting materials and immobilization processes. In recent years, there has been growing concern in using polymeric materials as supports for their good mechanical and easily adjustable properties. Furthermore, a great many work has been done in order to improve the activity retention and stabilities of immobilized enzymes. Some introduce a spacer arm onto the support surface to improve the enzyme mobility. The support surface is also modified towards biocompatibility to reduce non-biospecific interactions between the enzyme and support. Besides, natural materials can be used directly as supporting materials owning to their inert and biocompatible properties. This review is focused on recent advances in using polymeric materials as hosts for lipase immobilization by two different methods, surface attachment and encapsulation. Polymeric materials of different forms, such as particles, membranes and nanofibers, are discussed in detail. The prospective applications of immobilized enzymes, especially the enzyme-immobilized membrane bioreactors (EMBR) are also discussed.     

Protected immobilization of Taq DNA polymerase by active site masking on self-assembled monolayers of ω-functionalized thiols

A new efficient immobilization method that enables oriented immobilization of biologically active proteins was developed based on concepts of active site masking and kinetic control. Taq DNA polymerase was immobilized covalently on mixed self-assembled monolayers (SAMs) of ω-carboxylated thiol and ω-hydroxylated thiol through amide bonds between the protein and the carboxyl group in SAMs. Activity of the immobilized enzyme as large as 70% of solution-phase enzyme was achieved by masking the active site of the Taq DNA polymerase prior to the immobilization. In addition, the number of immobilization bonds was controlled by optimizing the carboxyl group concentration in the mixed monolayer. The maximum activity of immobilized Taq DNA polymerase was achieved at 5% of 12-mercaptododecanoic acid. The activity observed with protected immobilized enzyme was approximately 20 times higher than that observed with randomly immobilized enzyme. The maximum activity was acquired at a 1:1 DNA/enzyme masking ratio, immobilization pH 8.3, and within 10 min of reaction time. This concept of the active site masking and kinetic control of the number of covalent bonds between proteins and the surface can be generally applicable to a broad range of proteins to be immobilized on the solid surface with higher activity.     

Electrospun polyvinyl alcohol/bovine serum albumin biocomposite membranes for horseradish peroxidase immobilization

Electrospinning, a simple and versatile method to fabricate nanofibrous supports, has attracted attention in the field of enzyme immobilization. Biocomposite nanofibers were fabricated from mixed PVA/BSA solution and the effects of glutaraldehyde treatment, initial BSA concentration and PVA concentration on protein loading were investigated. Glutaraldehyde cross-linking significantly decreased protein release from nanofibers and BSA loading reached as high as 27.3% (w/w). In comparison with the HRP immobilized into the nascent nanofibrous membrane, a significant increase was observed in the activity retention of the enzyme immobilized into the PVA/BSA biocomposite nanofibers. The immobilized HRP was able to tolerate much higher concentrations of hydrogen peroxide than the free enzyme and thus the immobilized enzyme did not demonstrate substrate inhibition. The immobilized HRP retained ⿼50% of the free enzyme activity at 6.4 mM hydrogen peroxide and no significant variation was observed in the KM value of the enzyme for hydrogen peroxide after immobilization. In addition, reusability tests showed that the residual activity of the immobilized HRP were 73% after 11 reuse cycles. Together, these results demonstrate efficient immobilization of HRP into electrospun PVA/BSA biocomposite nanofibers and provide a promising immobilization strategy for biotechnological applications.     

Optimization of invertase immobilization by adsorption in ionic exchange resin for sucrose hydrolysis

This work presents as a main objective to study the immobilization process of yeast invertase by adsorption in the ion exchanging resin Duolite A-568 for invert sugar production. Initially, a kinetic study of the soluble form of the enzyme was carried out. At the sequence was studied the immobilization process of yeast invertase in the weakly exchanging anionic resin Duolite A-568. The influences of the pH, enzyme concentration and temperature in the enzyme immobilization were analyzed through a central composite design (CCD). The results indicated that the retention of the catalytic activity in immobilization was strongly dependent of these variables, being maximum in a pH value of 5.0, with an enzyme concentration of 12.5 g/L (1.875 g of protein per liter) and temperature of 30 °C. The simultaneous influence of pH and temperature on the free and immobilized invertase activity was also studied through a CCD.     

Immobilized diol dehydrase and its use in studies of cobalamin binding and subunit interaction.

Coenzyme B12 dependent diol dehydrase from Aerobacter aerogenes was immobilized by covalent binding to CNBr-activated Sepharose 4B. The Sepharose-bound enzyme exhibited a markedly high catalytic activity, viz., 75-95% of the specific activity of the original free enzyme. The apoenzyme acquired much greater stability to heat by immobilization. No significant difference between the immobilized and free enzymes was observed in the following properties: the affinity for coenzyme B12; the sensitivity to a sulfhydryl-modifying agent; the absolute requirement for a certain monovalent cation, such as K+, for catalysis; the susceptibility toward oxygen upon incubation with coenzyme B12 in the absence of substrate. These results suggest that the structure and function of the enzyme are not significantly influenced by immobilization on Sepharose. The immobilized enzyme was found to provide a convenient method for a study of ligand interaction with the enzyme. The subunit interaction between two dissimilar subunits, components F and S, was investigated using the component S immobilized on CNBr-activited Sepharose and free component F, and it was demonstrated that the substrate (1,2-propanedoil) promotes the hybrid formation between component F and component S, but K+ alone rather retarded the subunit association to some extent. Na+ markedly weakens the forces which bind the subunits together. The relationship between cobalamin binding and subunit structure is also discussed.     

γ-Fe_2O_3-凹土超顺磁性纳米复合材料固载猪胰脂肪酶

以凹凸棒石黏土为原料,制备γ-Fe2O3-凹土超顺磁性纳米复合材料(γ-Fe2O3-ATP)作为猪胰脂肪酶(PPL)固定化的载体,利用透射电子显微镜(TEM)、N2吸附脱附等温图(BET)、振动试样磁强计(VSM)等对材料进行表征,同时对固定化条件和固定化酶的相关性质进行了研究。结果表明:制备的γ-Fe2O3-ATP是介孔材料,比表面积为102.63 m2/g,平均孔径为10.862 nm,饱和磁化强度为8.915 emu/g,其作为载体能实现固定化酶与反应介质简单、快速分离回收和重复利用。在固定化时间为4 h及pH 6.0时制备的固定化酶效果最佳;经过6 h高温保存后固定化酶可保留初始酶活的52%,而游离酶仅保留初始酶活的19%,同时固定化酶在重复使用5次后酶活仍保留初始酶活的60%。     

pheA (Rv3838c) of Mycobacterium tuberculosis encodes an allosterically regulated monofunctional prephenate dehydratase that requires both catalytic and regulatory domains for optimum activity

Prephenate dehydratase (PDT) is a key regulatory enzyme in l-phenylalanine biosynthesis. In Mycobacterium tuberculosis, expression of pheA, the gene encoding PDT, has been earlier reported to be iron-dependent (1, 2). We report that M. tuberculosis pheA is also regulated at the protein level by aromatic amino acids. All of the three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) are potent allosteric activators of M. tuberculosis PDT. We also provide in vitro evidence that M. tuberculosis PDT does not possess any chorismate mutase activity, which suggests that, unlike many other enteric bacteria (where PDT exists as a fusion protein with chorismate mutase), M. tuberculosis PDT is a monofunctional and a non-fusion protein. Finally, the biochemical and biophysical properties of the catalytic and regulatory domains (ACT domain) of M. tuberculosis PDT were studied to observe that, in the absence of the ACT domain, the enzyme not only loses its regulatory activity but also its catalytic activity. These novel results provide evidence for a monofunctional prephenate dehydratase enzyme from a pathogenic bacterium that exhibits extensive allosteric activation by aromatic amino acids and is absolutely dependent upon the presence of catalytic as well as the regulatory domains for optimum enzyme activity.     

Conformational transitions and redox potential shifts of cytochrome P450 induced by immobilization

Cytochrome P450 (P450) from Pseudomonas putida was immobilized on Ag electrodes coated with self-assembled monolayers (SAMs) via electrostatic and hydrophobic interactions as well as by covalent cross-linking. The redox and conformational equilibria of the immobilized protein were studied by potential-dependent surface-enhanced resonance Raman spectroscopy. All immobilization conditions lead to the formation of the cytochrome P420 (P420) form of the enzyme. The redox potential of the electrostatically adsorbed P420 is significantly more positive than in solution and shows a steady downshift upon shortening of the length of the carboxyl-terminated SAMs, i.e., upon increasing the strength of the local electric field. Thus, two opposing effects modulate the redox potential of the adsorbed enzyme. First, the increased hydrophobicity of the heme environment brought about by immobilization on the SAM tends to upshift the redox potential by stabilizing the formally neutral ferrous form. Second, increasing electric fields tend to stabilize the positively charged ferric form, producing the opposite effect. The results provide insight into the parameters that control the structure and redox properties of heme proteins and contribute to the understanding of the apparently anomalous behavior of P450 enzymes in bioelectronic devices.     

Affinity and stability modifications of immobilized alcohol dehydrogenase through multipoint copolymerization

Yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), a potentially useful enzyme for cofactor regeneration processes, was covalently immobilized in a multipoint fashion by activation with acryloyl chloride and subsequent copolymerization in a polyacrylamide gel. Several properties such as the activity and stability were systematically studied for the free enzyme, the acryloate-enzyme and the immobilized enzyme. The activation energy was significantly lowered upon immobilization. The thermal stability of the immobilized enzyme was, however, greatly increased. But its maximum activity was observed at a lower temperature. These results suggest an important effect of the diffusional restrictions and of the mode of activation and immobilization on the activity and the stability of the enzyme.     

Multi-point enzyme immobilization,surface chemistry,and novel platforms: a paradigm shift in biocatalyst design

Engineering enzymes with improved catalytic properties in non-natural environments have been concerned with their diverse industrial and biotechnological applications. Immobilization represents a promising but straightforward route, and immobilized biocatalysts often display higher activities and stabilities compared to free enzymes. Owing to their unique physicochemical characteristics, including the high-specific surface area, exceptional chemical, electrical, and mechanical properties, efficient enzyme loading, and multivalent functionalization, nano-based materials are postulated as suitable carriers for biomolecules or enzyme immobilization. Enzymes immobilized on nanomaterial-based supports are more robust, stable, and recoverable than their pristine counterparts, and are even used for continuous catalytic processes. Furthermore, the unique intrinsic properties of nanomaterials, particularly nanoparticles, also confer the immobilized enzymes to be used for their broader applications. Herein, an effort has been made to present novel potentialities of multi-point enzyme immobilization in the current biotechnological sector. Various nano-based platforms for enzyme/biomolecule immobilization are discussed in the second part of the review. In summary, recent developments in the use of nanomaterials as new carriers to construct robust nano-biocatalytic systems are reviewed, and future trends are pointed out in this article.     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.