Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP?g^?1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^?1 µg^?1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Immobilization of lipase from Candida rugosa on Eupergit C supports by covalent attachment

The present study compares the results of three different covalent immobilization methods employed for immobilization of lipase from Candida rugosa on Eupergit^® C supports with respect to enzyme loadings, activities and coupling yields. It seems that method yielding the highest activity retention of 43.3% is based on coupling lipase via its carbohydrate moiety previously modified by periodate oxidation. Study of thermal deactivation kinetics at three temperatures (37, 50 and 75 °C) revealed that the immobilization method also produces an appreciable stabilization of the biocatalyst, changing its thermal deactivation profile. By comparison of the t_1/2 values obtained at 75 °C, it can be concluded that the lipase immobilized via carbohydrate moiety was almost 2-fold more stable than conventionally immobilized one and 18-fold than free lipase. The immobilization procedure developed is quite simple, and easily reproduced, and provides a promising solution for application of lipase in aqueous and microaqueous reaction system.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

Entrapment of lipase into K-carrageenan beads and its use in hydrolysis of olive oil in biphasic system

The porcine pancrease lipase was immobilized by entrapment in the beads of K-carrageenan and cured by treatment with polyethyleneimine (PEI) in the phosphate buffer. The retention of hydrolytic activity of lipase and compressive strength of the beads were examined. The activity of free and immobilized lipase was assessed by using olive oil as the substrate. The immobilized enzyme exhibited a little shift towards acidic pH for its optimal activity and retained 50% of its activity after 5 cycles. When the enzyme concentration was kept constant and substrate concentration was varied the K_m and V_max were observed to be 0.18 × 10⁻² and 0.10, and 0.10 × 10⁻² and 0.09 respectively, for free and for entrapped enzymes. When the substrate concentration was kept constant and enzyme concentration was varied, the values of K_m and V_max were observed to be 0.19 × 10⁻⁷ and 0.41, and 0.18 × 10⁻⁷ and 0.41 for free and entrapped enzymes. Though this indicates that there is no conformational change during immobilization, it also shows that the reaction velocity depends on the concentration. Immobilized enzyme showed improved thermal and storage stability. Hydrolysis of olive oil in organic–aqueous two-phase system using fixed bed reactor was carried out and conditions were optimized. The enzyme in reactor retained 30% of its initial activity after 480 min (12 cycles).     

Coenzyme production using immobilized enzymes. I. Preparation, characterization, and laboratory-scale application of an immobilized NAD kinase

NAD⁺ kinase (ATP: NAD⁺ 2-phosphotransferase, EC2.7.1.23) isolated from chicken liver was immobilized on a silica-based support possessing aldehyde functional groups. The highest catalytic activity achieved was 16 U g⁻¹ solid. The optimal pH for the catalytic activity of the immobilized NAD⁺ kinase was pH 7.1–7.3. The apparent optimum temperature for the immobilized enzyme was about 5°C higher than that of the soluble enzyme. There were no significant differences in the K_{m app} values. The immobilization improved the conformational stability of the enzyme. In preliminary experiments, a 95% conversion of NAD⁺ to NADP⁺ was achieved with use of the immobilized NAD⁺ kinase, which preserved its starting activity practically unchanged up to 36 days.     

Stability of free and immobilised peroxidase in aqueous–organic solvents mixtures

The activity and stability of horseradish (Amoracia rusticana) peroxidase (HRP) free in solution and immobilised onto silica microparticles was studied in the presence of organic co-solvents.

The effect of several hydrophilic organic solvents, namely dimethyl sulfoxide, dimethylformamide, dioxan, acetonitrile and tetrahydrofuran, in the activity and stability of free HRP was studied. From the solvents tested, DMSO led to the highest activities and stabilities. After 2 h of incubation at 35°C, the remaining activity of the enzyme in the presence of 30% of each solvent was less than 30%, with exception of DMSO for which the enzyme remained fully active.

In order to increase stability, HRP was covalently immobilised onto silica microparticles. The half-life of the enzyme in buffer at 50°C increased from 2 to 52 h when the enzyme was immobilised. The stability of both free and immobilised HRP was also studied at 50°C in aqueous mixtures of 3.5, 20, 35 and 50% (v/v) DMSO. Free HRP stability was not affected by the presence of 3.5 and 20% DMSO, but higher contents lead to a more pronounced deactivation. Immobilised HRP stability increased with DMSO content up to 20%, decreasing for higher contents. The enzyme half-life increased more than 300% when changing from buffer to 20% DMSO.

The deactivation of free HRP was modelled using the simple exponential decay, and the deactivation of immobilised HRP was described by a two-step inactivation model.     

A comparative study of free and immobilized soybean and horseradish peroxidases for 4-chlorophenol removal: protective effects of immobilization

Horseradish peroxidase (HRP) and soybean peroxidase (SBP) were covalently immobilized onto aldehyde glass through their amine groups. The activity yield and the protein content for the immobilized SBP were higher than for the immobilized HRP. When free and immobilized peroxidases were tested for their ability to remove 4-chlorophenol from aqueous solutions, the removal percentages were higher with immobilized HRP than with free HRP, whereas immobilized SBP needs more enzyme to reach the same conversion than free enzyme. In the present paper the two immobilized derivatives are compared. It was found that at an immobilized enzyme concentration in the reactor of 15 mg l(-1), SBP removed 5% more of 4-chlorophenol than HRP, and that a shorter treatment was necessary. Since immobilized SBP was less susceptible to inactivation than HRP and provided higher 4-chlorophenol elimination, this derivative was chosen for further inactivation studies. The protective effect of the immobilization against the enzyme inactivation by hydrogen peroxide was demonstrated.     

Immobilization of the Solanum tuberosum epoxide hydrolase and its application in an enantioconvergent process

Five supports have been evaluated for the immobilization of the epoxide hydrolase from Solanum tuberosum (StEH) by adsorption. The highest immobilization yield (90-99%) and the maximum EH (epoxide hydrolase) activity (0.6 U g^-1 wet support) were obtained by ionic adsorption onto DEAE-cellulose. Although the activity recovered upon immobilization of StEH onto DEAE-cellulose was low, a notable stabilization factor of 6.9 at 65°C was obtained. In addition, the immobilized StEH showed a higher temperature for maximal activity (57°C) and the optimal pH (5.0) was shifted one unit towards the acidic region as compared to the free enzyme. Immobilized StEH was successfully reused in six consecutive hydrolytic kinetic resolutions of rac-pCSO without noticeable loss in activity. Finally, the sequential use of immobilized StEH with the immobilized EH from Aspergillus niger (AnEH) in a repeated batch reactor, operated for five cycles, enabled the enantioconvergent preparation of the corresponding (R)-diol, which was thus obtained with an ee of 89% and an overall yield of 100%.     

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.     

Immobilization of catalase into chemically crosslinked chitosan beads

Bovine liver catalase was immobilized into chitosan beads prepared in crosslinking solution. Various characteristics of immobilized catalase such as the pH–activity curve, the temperature–activity curve, thermal stability, operational stability, and storage stability were evaluated. Among them the pH optimum and temperature optimum of free and immobilized catalase were found to be pH 7.0 and 35 °C. The K_m value of immobilized catalase (77.5 mM) was higher than that of free enzyme (35 mM). Immobilization decreased in V_max value from 32,000 to 122 μmol (min mg protein)⁻¹. It was observed that operational, thermal and storage stabilities of the enzyme were increased with immobilization.     

Production of cyclodextrin by poly-lysine fused Bacillus macerans cyclodextrin glycosyltransferase immobilized on cation exchanger

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the V_m of the immobilized CGTase by 40% without a change in K_m. The activation energies of thermal deactivation (E_a) were 41.4, 28.1, and 25.9 kcal mol⁻¹, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l⁻¹ h⁻¹ was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min⁻¹ (D = 240 h⁻¹) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0.     

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g^-1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8-9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a K_m of 6.6 mM for H₂O₂ and V_max of 707 mM H₂O₂ min^-1 g^-1 wet wt. cells, and showed saturation kinetics at 50 mM H₂O₂. The cells were entrapped in calcium alginate and used for H₂O₂ degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.     

Preparation and characterization of immobilized phospholipase A2 on chitosan beads for lowering serum cholesterol concentration

Phospholipase A₂ (PLA₂) from cobra venom, which can hydrolyze the S_N2 ester bond of 1,2-diacylphosphatides, was immobilized by covalent binding to porous chitosan beads. Immobilization has to be carried out by using the carboxylic groups instead of the amine groups of the enzyme to get reasonable activity retention (higher than 50%). The effects of amount of activating reagent EDC and enzyme loading during the immobilization step were investigated. Since EDC could modify important Asp groups in the enzyme, the EDC/enzyme weight ratio should be less than 10. Although the activity retention of immobilized enzyme increased with enzyme/bead weight ratio, this ratio should be kept to a minimum at 1×10⁻³ to optimize coupling yield of enzyme activity and reduce internal diffusion resistance. The kinetic properties and stability of the immobilized enzyme were determined. The immobilized PLA₂ was packed into a column to hydrolyze phospholipid in a circulating packed-bed reactor. The flow rate of the substrate solution should be set at 37.5 cm/min (superficial velocity) to eliminate external diffusion resistance, under which condition the column reactor could be reused up to 10 times with less than 20% loss of activity. Since enzymatic hydrolysis of phospholipid on low density lipoprotein (LDL) particle surface with PLA₂ could result in faster plasma clearance of the modified LDL particles, an in vitro bioreactor containing immobilized PLA₂ should be able to lower serum cholesterol concentration. A significant decrease in total serum cholesterol concentration in hypercholesterolemic rabbits was observed after 90-min treatment.     

Immobilization of β-galactosidase from Kluyveromyces lactis on silica and agarose: comparison of different methods

The covalent immobilization of β-galactosidase from Kluyveromyces lactis (β-gal) on to two different porous carriers, CPC-silica and agarose, is reported. CPC-silica was silanizated and activated with glutaraldehyde. The activation of agarose via a cyanylating agent (CDAP) was optimized. Gel-bound protein and gel-bound activity were both measured directly, allowing the determination of apparent specific activities (S.A.). Higher amounts of β-gal were immobilized on the activated CPC-silica (maximum capacity, 23 mg ml⁻¹ of packed support) than on the CDAP-activated agarose. For the lower enzyme loading assayed (12.6 mg ml⁻¹ packed support), 100% of the enzyme was immobilized but only 34% of its activity was expressed. This inactivation during immobilization was confirmed by the S.A. values (22–29 EU mg⁻¹ for the CPC-derivatives and 80 EU mg⁻¹ for soluble β-gal). The K_app (3.4 mM) for the CDAP-derivative with ONPG as substrate was higher than the K_M value for soluble β-gal (2 mM). When the enzyme loading was increased five-fold, the K_app increased four-fold, to 13 mM. The V_app values for the CPC-derivatives were remarkably lower than the V_max for soluble β-galactosidase. CDAP-derivatives showed better thermal stabilities than CPC-derivatives but neither of them enhanced the stability of the soluble enzyme. When stored at 4°C, the activity of both derivatives remained stable for at least 2 months. Both derivatives displayed high percentages of lactose conversion (90%) in packed bed mini-reactors. Glucose production was 3.3-fold higher for the CPC-derivative than for the CDAP-derivative, as a consequence of the higher flow rates achieved.     

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.     

Bioconversion of phospholipids by immobilized phospholipase A2

Phospholipase A₂ selectively hydrolyses the ester linkage at the sn-2 position of phospholipids forming lysocompounds. This bioconversion has importance in biotechnology since lysophospholipids are strong bioemulsifiers. The aim of the present work was to study the kinetic behaviour and properties of immobilized phospholipase A₂ from bee venom adsorbed into an ion exchange support. The enzyme had high affinity for CM-Sephadex^® support and the non-covalent interaction was optimum at pH 8. The activity of immobilized phospholipase A₂ was comparatively evaluated with the soluble enzyme using a phospholipid/Triton X-100 mixed micelle as assay system. The immobilized enzyme showed high retention activity and excellent stability under storage. The activity of the immobilized system remained almost constant after several cycles of hydrolysis. Immobilized phospholipase A₂ was less sensitive to pH changes compared to soluble form. The kinetic parameters obtained (V_max 883.4 μmol mg⁻¹ min⁻¹ and a K_m 12.9 mM for soluble form and V_max = 306 μmol mg⁻¹ min⁻¹ and a K_m = 3.9 for immobilized phospholipase A₂) were in agreement with the immobilization effect. The results obtained with CM-Sephadex^®-phospholipase A₂ system give a good framework for the development of a continuous phospholipid bioconversion process.     

Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber

Having been activated with glutaraldehyde, modified poly(ethylene terephthalate) grafted acrylamide fiber was used for the immobilization of horseradish peroxidase (HRP). Both the free HRP and the immobilized HRP were characterized by determining the activity profile as a function of pH, temperature, thermal stability, effect of organic solvent and storage stability. The optimum pH values of the enzyme activity were found as 8 and 7 for the free HRP and the immobilized HRP respectively. The temperature profile of the free HRP and the immobilized HRP revealed a similar behaviour, although the immobilized HRP exhibited higher relative activity in the range from 50 to 60 °C. The immobilized HRP showed higher storage stability than the free HRP.     

Effects of calcium on the thermal stability, stability in organic solvents and resistance to hydrogen peroxide of artichoke (Cynara scolymus L.) peroxidase: A potential method of enzyme control

The effects of calcium ions (Ca²⁺) on the stability of artichoke (Cynara scolymus L.) peroxidase (AKPC) have been studied. The thermal stability of AKPC was improved by the addition of Ca²⁺; the melting temperature increased by 20 °C and the deactivation energy by 26 kJ mol⁻¹. AKPC was stable in a selection of organic solvents but was less active with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) than under aqueous conditions. Ca²⁺-free AKPC retained more activity in the presence of organic solvents due to its better maintenance of the rate of compound I formation with hydrogen peroxide (H₂O₂) compared to AKPC-Ca²⁺. AKPC retained at least 75% activity over 24 h in the pH range 3.0–10.5 and about 50% over 1 month at pH 7.0 or 5.5, irrespective of the Ca²⁺ content. AKPC-Ca²⁺ was considerably more resistant to inactivation by H₂O₂ than Ca²⁺-free AKPC suggesting that the presence of Ca²⁺ boosts turnover under oxidizing conditions. AKPC has been applied as an alternative to horseradish peroxidase (HRP) in glucose concentration assays; the presence of Ca²⁺ or of the Ca²⁺ chelating agent ethylenediaminetetraacetic acid made no difference to the final result. The possibility is discussed that addition and removal of a labile Ca²⁺ from AKPC could be used to control enzyme activity both in vivo and in vitro.     

Enhanced activity and stability of immobilized lipases by treatment with polar solvents prior to lyophilization

Lipases from Candida rugosa, Mucor javanicus and Rhizopus oryzae were respectively adsorbed on Amberlite XAD-7 followed by incubation in 2-propanol and then lyophilization. The activities of the immobilized enzymes were 1.6–3.4 times higher than those of the immobilized enzymes without incubation in the organic solvent before lyophilization for esterification of lauric acid (0.1 M) and 1-propanol (0.1 M) in isooctane at 37 °C. The immobilized C. rugosa lipase (Sigma) without the incubation did not show any activity but displayed considerable activity (19.8 μmol h⁻¹ mg⁻¹) after the incubation before lyophilization. Besides 2-propanol, acetone, 1-propanol and ethyl acetate were also found to be good solvents for treating M. javanicus lipase immobilized on Amberlite XAD-7 and acetone was the best among them. When incubated in isooctane at 25 °C for 120 h, the immobilized M. javanicus lipase prepared by incubation in acetone for 1 h before lyophilization retained 70% of its initial activity while the immobilized enzyme without the solvent treatment kept only 50% of its initial activity.     

BSA和PEG影响固定化辣根过氧化物酶HRP在有机相中活力

ＢＳＡ和ＰＥＧ可以有效地提高固定化辣根过氧化物酶（ＨＲＰ）在有机相中的活力。固定化酶活力的提高与试剂加入的顺序有密切的联系;不同载体对酶的影响不同,Ｇｅｌｉｔｅ,ａｌｕｍｉｎａ,ＸＡＤ－７,Ｋｉｓｅｌｇｅｌ和Ｆｌｏｒｉｓｉｌ为载体,分别以吸附法制备固定化酶。实验表明固定化过程中保护剂和酶的加入顺序与国家化酶活力密切相关,而这些载体的固定化效果又以Ｃｅｌｉｔｅ最佳,Ｆｌｏｒｉｓｉｌ最差。Ｆｌｏｒｉｓ