Evaluation of man-tailored cellulose-based carriers in glucoamylase immobilization

Covalent immobilization of glucoamylase on the cellulose-based carrier Granocel was optimized by changing the anchor groups and the methods of activation/immobilization. Binding of the enzyme was via its primary amino groups. It was shown that using carbodiimide and divinyl sulfone for the activation of -COOH and -OH groups on the carrier resulted in the preparations with very low activity. A third method, using pentaethylenehexamine with glutaraldehyde, led to the attachment through a long spacer arm and to the preparations with the highest activity. Further optimization of the carrier's structure consisted of changing pore diameters and amount of functional groups on the carrier surface. The highest activity of bound glucoamylase was obtained by linking the protein via glutaraldehyde on NH(2)-Granocel having high pore size and high number of functional groups. The immobilized enzyme was stable throughout extended storage and possessed higher thermal stability.     

Comparative studies on immobilized laccase behaviour in packed-bed and batch reactors

Extracellular laccase produced by the wood-rotting fungus Cerrena unicolor was immobilized covalently via glutaraldehyde to cellulose-based carrier Granocel. Laccase was partially purified by membrane concentration and diafiltration followed by precipitation with acetone. Five-fold increase in the measured activity of immobilized enzyme was obtained when six times purer laccase was used for immobilization. For the best preparation, with very high activity of 2053 U per 1 mL of the carrier, thermal- and pH-stability, and activity profiles were determined. Experiments carried out in a batch reactor showed that k_cat/K_m for immobilized enzyme (0.65) is three times lower than the value obtained for the native laccase (2.19) whereas k_cat/K_m estimated from continuous reactor (1.50) is notably closer to that for the native enzyme. Continuous process probably reflects more precisely kinetics of the reaction accompanied by simultaneous product precipitation on the carrier’s surface. Operational stability of immobilized laccase was tested in continuous mode operation with ABTS, guaiacol and trichlorophenol as substrates and showed that packed-bed reactor is unprofitable system for laccase immobilized on Granocel carrier due to the high bed compaction. However, excellent stability of the preparation was noted under 20 successive runs in the well mixed tank reactor and better ability towards trichlorophenol biotransformation was observed in the case of immobilized laccase.     

Immobilized lipase on porous silica particles: Preparation and application for biodegradable polymer syntheses in ionic liquid at higher temperature

Porous silica particles (PSP) modified with different surface active groups were prepared for covalent immobilization of porcine pancreas lipase (PPL). Organosilanes combined with reactive end amino-group or epoxy-group were employed for the modification through silanization process. Polyethylenimine and long chain alkyl silane coupling agent were also used in the modification process. Several modification-immobilization strategies were performed, while good coupling yield could be achieved within the range of 86.2–158.2 mg of native PPL per gram of the carrier. Furthermore, at higher temperature, the resulting immobilized PPL (IPPL) could successfully perform the syntheses of polycaprolactone (PCL) and poly(5,5-dimethyl-1,3-dioxan-2-one) (PDTC) in ionic liquid medium. No polymers could be obtained catalyzed by native PPL, suggesting that IPPL showed much higher catalytic activity than native PPL. Effect of different treatments on the activity of IPPL also showed the long time high temperature stability in ionic liquid medium, contributing to a good combination of immobilization and ionic liquids effect. The catalytic activity of IPPL for polymerization was closely related to both the properties of immobilized enzyme and cyclic monomer. This work would be expected to highlight further careful design of immobilized enzyme for a wide range of application, especially in biodegradable polymers syntheses.     

双醛淀粉柔性固定木瓜蛋白酶研究

提出“柔性固定化酶”的模型,即：用一亲水、柔性高分子链接枝于载体表面制得柔性固定化载体,再用其以共价键合的方式进行酶的柔性固定化。其特点是：柔性固定可改善因直接固定化及手臂固定化使酶失活的缺陷,并提高固定化酶的自由度;如选用粒径单分散微球可改善固定化反应及固定化酶催化反应的均一性。以双醛淀粉(DAS)为柔性链对羧基化聚苯乙烯载体进行柔性化修饰后,固定木瓜蛋白酶,其活力回收率可达50％．相当于用戊二醛进行手臂固定化的活力回收率的2倍。     

Covalent enzyme immobilization onto glassy carbon matrix-implications in biosensor design

The aim of the present work is to design an electrode for biosensors by covalent immobilization of the redox enzyme. In the covalently modified electrode, the biocatalyst is located close to the electrode surface and this is expected to enhance the electron transfer rate from the enzyme to the electrode. Several methods of covalent immobilization of enzymes onto a glassy carbon surface are described. We have chosen horse radish peroxidase enzyme in our study but any other suitable enzyme can be immobilized depending on the intended use. A three step procedure that includes (i) heat treatment of matrix at l00-l10°C to remove volatiles and absorbates, (ii) chemjcal pretreatment to introduce functional groups like -OH, -NO₂, -Br etc. followed by (iii) glutaraldehyde coupling of the enzyme (for the nitrated matix after subsequent reduction) or modification of the matrix by carboxymethylation and enzyme coupling using carbodiimide (for hydroxylated matrix) was followed. The amount of enzyme immobilized onto the carbon surface was estimated by spectrophotometric enzymatic activity assay, commonly used for the soluble enzyme. We found that simple nitration did not introduce any significant amount of functional groups and the matrix with hydrogen peroxide pretreatment showed the highest enzyme loading of 0.05 U/mg of carbon matrix. The HRP enzyme electrode was tested in a rotating disk experiment for its response with the substrate.     

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.     

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.     

Enzyme immobilization techniques on poly(glycidyl methacrylate-co-ethylene dimethacrylate) carrier with penicillin amidase as model.

Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.     

Development of Silica Gel-Supported Modified Macroporous Chitosan Membranes for Enzyme Immobilization and Their Characterization Analyses

The present work was aimed at developing stability enhanced silica gel-supported macroporous chitosan membrane for immobilization of enzymes. The membrane was surface modified using various cross-linking agents for covalent immobilization of enzyme Bovine serum albumin. The results of FT-IR, UV–vis, and SEM analyses revealed the effect of cross-linking agents and confirmed the formation of modified membranes. The presence of silica gel as a support could provide a large surface area, and therefore, the enzyme could be immobilized only on the surface, and thus minimized the diffusion limitation problem. The resultant enzyme immobilized membranes were also characterized based on their activity retention, immobilization efficiency, and stability aspects. The immobilization process increased the activity of immobilized enzyme even higher than that of total (actual) activity of native enzyme. Thus, the obtained macroporous chitosan membranes in this study could act as a versatile host for various guest molecules.     

Improved stabilization of chemically aminated enzymes via multipoint covalent attachment on glyoxyl supports

The surface carboxylic groups of penicillin G acylase and glutaryl acylase were chemically aminated in a controlled way by reaction with ethylenediamine via the 1-ethyl-3-(dimethylamino-propyl) carbodiimide coupling method. Then, both proteins were immobilized on glyoxyl agarose. In both cases, the immobilization of the chemically modified enzymes improved the enzyme stability compared to the stability of the immobilized but non-modified enzyme (by a four-fold factor in the case of PGA and a 20-fold factor in the case of GA). The chemical modification presented a deleterious effect on soluble enzyme stability. Therefore, the improved stability should be related to a higher multipoint covalent attachment, involving both the lysine amino groups and also the new amino groups chemically introduced on the enzyme. Moreover, the lower pK(a) of the new amino groups permitted to immobilize the enzyme under milder conditions. In fact, the aminated proteins could be immobilized even at pH 9, while the non-modified enzymes could only be immobilized at pH over 10.     

d-Galacturonandigalacturonohydrolase covalently bound on to a polyacrylamide-type support

d -Galacturonandigalacturonohydrolase was immobilized by covalent coupling on to a polyacrylamide-type carrier BIO Gel CM100, activated by water-soluble carbodiimide. Catalytic properties, stability and action pattern of the immobilized enzyme are reported.     

Immobilization of the α-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance

The α-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K _m increased, while V _max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized α-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized α-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 α-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.     

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.     

Stabilization of alpha-chymotrypsin by covalent immobilization on amine-functionalized superparamagnetic nanogel

Stabilization of alpha-chymotrypsin (CT) by covalent immobilization on the amine-functionalized magnetic nanogel was studied. The amino groups containing superparamagnetic nanogel was obtained by Hoffman degradation of the polyacrylamide (PAM)-coated Fe(3)O(4) nanoparticles prepared by facile photochemical in situ polymerization. CT was then covalently bound to the magnetic nanogel with reactive amino groups by using 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide as coupling reagent. The binding capacity was determined to be 61mg enzyme/g nanogel by BCA protein assay. Specific activity of the immobilized CT was measured to be 0.93U/(mgmin), 59.3% as that of free CT. The obtained immobilized enzyme had better resistance to temperature and pH inactivation in comparison to free enzyme and thus widened the ranges of reaction pH and temperature. The immobilized enzyme exhibited good thermostability, storage stability and reusability. Kinetic parameters were determined for both the immobilized and free enzyme. The value of K(m) of the immobilized enzyme was larger than did the free form, whereas the V(max) was smaller for the immobilized enzyme.     

Specific immobilization of laccase onp-benzoquinone-activated supports

Summary A specific immobilization of laccase (EC 1.10.3.2) onto a ready-to-usep-benzoquinone-activated agarose support is described. The single-step procedure leads to a laccase protein coupling of I8% and an enzyme activity immobilization yield of 27%, while the retained specific activity of the immobilized enzyme was 150% of the specific activity of the free laccase. This peculiar result is thought to be related to the fact that during the process of support activation byp-benzoquinone, a significant amount of the hydroquinone by-product of the activation process is coupled to the support. These coupled derivatives constitute substrate (hydroquinone) analogues for which laccase exhibits a high affinity. Therefore, simultaneous affinity retention on the hydroquinone groups and covalent coupling on the p-benzoquinone groups allow the binding of the enzyme in an advantageous conformation which can generate this increase specific activity by immobilization. The entire process can be considered as an affinity immobilization. The immobilized enzyme is much more stable to the inhibitory action of chloride and azide ions, with a recovery of 100% of the activity, than the free laccase, with a recovery of 67% and 32%, respectively, after removal of the inhibitors by dialysis. The stability was 95% after storage for 14 months at 4° C.Abbreviations HQ hydroquinone - p-BQ p-benzoquinone - U enzyme units Part of the work was presented at the Satellite FEBS 1989 Symposium onBiochemical and biophysical approaches to the study of copper proteins, Camerino, Italy.     

固定化酶的空间取向控制策略

阐述了固定化酶的空间取向控制的方法和应用研究。     

One-step purification,covalent immobilization,and additional stabilization of a thermophilic poly-His-tagged beta-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate-epoxy Sepabeads

Using the poly-His-tagged-beta-galactosidase from Thermus sp. strain T2 overexpressed in Escherichia coli (MC1116) as a model enzyme, we have developed a strategy to purify and immobilize proteins in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification. The aforementioned enzyme could not be immobilized onto standard epoxy supports with good yields, and after purification and storage, it exhibited a strong trend to yield very large aggregates as shown by ultracentrifugation experiments. That preparation could not be immobilized in any support, very likely because the pores of the solid became clogged by the large aggregates. These novel epoxy-metal chelate heterofunctional supports contain a low concentration of Co(2+) chelated in IDA groups and a high density of epoxy groups. This enabled the selective adsorption of poly-His-tagged enzymes, and as this adsorption step is necessary for the covalent immobilization procedure, the selective covalent immobilization of the target enzyme could take place. This strategy allowed similar maximum loadings of the target enzyme using either pure or crude preparations of the enzyme. The enzyme derivative presented a very high activity at 70 degrees C (over 1000 IU in the hydrolysis of lactose) and very high stability and stabilization when compared to its soluble counterpart (activity remained unaltered after several days of incubation at 50 degrees C). In fact, this preparation was much more stable than when the same enzyme was immobilized onto standard epoxy Sepabeads.     

Bioactive polymers. 56: urease immobilization on carboxymethylcellulose

This article reports on the reaction of urease immobilization through its covalent bonding on carboxymethylcellulose. The reaction is activated by dicyclohexylcarbodiimide. The coupling reaction is influenced by the enzyme-support and activator-support ratios, as well as by duration. Starting from a rotating, composed experimental program of the second order, the function correlating the activity of the immobilized enzyme with the reaction parameters is established. Immobilized urease exhibits thermal stability higher than that of free enzyme, regarding both pH and the inhibiting action of some metal ions or organic substances. The stability over time of the immobilized urease is high, its enzymatic activity being maintained at over 85% of the initial value three months after synthesis.     

Immobilized lipase on macroporous polystyrene modified by PAMAM-dendrimer and their enzymatic hydrolysis

The novel enzyme carrier, polyamidoamine (PAMAM) dendrimers modified macroporous polystyrene, has been synthesized by Michael addition and firstly used in the immobilization of porcine pancreas lipase (PPL) effectively by covalent attachment. The resulting carrier was characterized with the Fourier transform infrared spectra (FT-IR), scanning electron microscopy (SEM), elemental analysis and thermogravimetric (TG) analysis. Meanwhile, the amount of immobilized lipase was up to 100 mg g⁻¹ support and the factors related with the enzyme activity were investigated. The immobilization of the PPL improved their performance in wider ranges of pH and temperature. Thermal stability of the immobilized lipase also increased dramatically in comparison with the free ones and the immobilized lipase exhibited a favorable denaturant tolerance. As a biocatalyst, the immobilized lipase for batch hydrolysis of olive oil emulsion retained 85% activity after 10 times of recycling. This well-reusability of immobilized lipase was very valuable and meaningful in enzyme technology.     

Surface functionalized electrospun biodegradable nanofibers for immobilization of bioactive molecules

A blend mixture of biodegradable poly(epsilon-caprolactone) (PCL) and poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol)-NH(2) (PLGA-b-PEG-NH(2)) block copolymer was electrospun to produce surface functionalized nanofibers. The resulting nanofibrous mesh with primary amine groups on the surface was applied for immobilization of biologically active molecules using lysozyme as a model enzyme. Lysozyme was immobilized via covalent conjugation by using a homobifunctional coupling agent. The nanofibrous mesh could immobilize a far greater amount of lysozyme on the surface with concomitantly increased activity, primarily due to its larger surface area, compared to that of the solvent casting film. It was also found that the enzyme immobilization process slightly altered thermal and pH-dependent catalytic activity profiles compared to those of native lysozyme. The results demonstrated the surface functionalized electrospun nanofibrous mesh could be used as a promising material for immobilizing a wide range of bioactive molecules.