Hybrid Magnetic Cross-Linked Enzyme Aggregates of Phenylalanine Ammonia Lyase from Rhodotorula glutinis

Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in K_m of enzyme in CLEAs and HM-PAL-CLEAs, V_max of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.     

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.     

Cross-linking enzyme aggregates in the macropores of silica gel: A practical and efficient method for enzyme stabilization

Cross-linked enzyme aggregates of papain were prepared in commercial macroporous silica gel (CLEAs-MSG) in order to improve the operability and mechanical stability of CLEAs. CLEAs-MSG was obtained from simple adsorption, precipitation and one-step-cross-linking. CLEAs-MSG was characterized by stable structure that did not leak out enzyme from the macropores because of covalent bonding between CLEAs and MSG. The optimal temperature of papain CLEAs in MSG was 40–90 °C and the optimal pH was 7.0, which were improved compared to free papain and CLEAs. The CLEAs-MSG also enhanced the storage stability and thermal stability. Moreover, the CLEAs-MSG exhibited good reusability due to its suitable size and active properties. By using CLEAs-MSG of papain as biocatalyst, the kinetically controlled z-Ala-Gln synthesis was achieved with the yield of 32.9%, which was almost equal to that by using free papain as biocatalyst.     

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.     

Evaluation of biodiesel production using lipase immobilized on magnetic silica nanocomposite particles of various structures

Nonporous and mesoporous silica-coated magnetite cluster nanocomposites particles were fabricated with various silica structures in order to develop a desired carrier for the lipase immobilization and subsequent biodiesel production. Lipase from Pseudomonas cepacia was covalently bound to the amino-functionalized particles using glutaraldehyde as a coupling agent. The hybrid systems that were obtained exhibited high stability and easy recovery regardless of the silica structure, following the application of an external magnetic field. The immobilized lipases were then used as the recoverable biocatalyst in a transesterification reaction to convert the soybean oil to biodiesel with methanol. Enzyme immobilization led to higher stabilities and conversion values as compared to what was obtained by the free enzyme. Furthermore, the silica structure had a significant effect on stability and catalytic performance of immobilized enzymes. In examining the reusability of the biocatalysts, the immobilized lipases still retained approximately 55% of their initial conversion capability following 5 times of reuse.     

Immobilization of Paenibacillus macerans NRRL B-3186 cyclodextrin glucosyltransferase and properties of the immobilized enzyme

Cyclodextrin glucosyltransferase from Paenibacillus macerans NRRL B-3186 was immobilized on aminated polyvinylchloride (PVC) by covalent binding with a bifunctional agent (glutaraldehyde). The immobilized activity was affected by the length of the hydrocarbon chain attached to the PVC matrix, the amount of the protein loaded on the PVC carrier, and glutaraldehyde concentration. The activity of the immobilized enzyme was 121 units/gram carrier, the specific activity calculated on bound protein basis was 48% of the soluble enzyme. Compared to the free enzyme, the immobilized form exhibited: a higher optimal reaction temperature and energy of activation, a higher K_m (Michaelis constant) and lower V_max (maximal reaction rate), improved thermal stability and resistance to chemical denaturation. The operational stability was evaluated in repeated batch process and the immobilized enzyme retained about 85% of the initial catalytic activity after being used for 14 cycles.     

The potential of immobilized bacterial α-amylase on coconut coir,a smart carrier for biocatalysts

Purified α-amylase from a soil bacterium Bacillus sp. SKB4 was immobilized on coconut coir, an inexpensive cellulosic fiber, with the cross-linking agent glutaraldehyde. The catalytic properties and stability of the immobilized enzyme were compared with those of its soluble form. The enzyme retained 97.2% of its activity and its catalytic properties were not drastically altered after immobilization. The pH optimum and stability of the immobilized enzyme were shifted towards the alkaline range compared to the free enzyme. The optimum temperature for enzymatic activity was 90°C in both forms of the enzyme. The soluble and immobilized enzyme retained 19% and 70% of original activity, respectively, after pre-incubation for 1 h at 90°C. Immobilized amylase was less susceptible to attack by heavy metal ions and showed higher K_m and V_max values than its free form. The bound enzyme showed significant activity and stability after 6 months of storage at 4°C. All of these characteristics make the new carrier system suitable for use in the bioprocess and food industries.     

Improved biochemical characteristics of crosslinked β-glucosidase on nanoporous silica foams

Large mesoporous cellular foam (LMCF) materials were synthesized using the microemulsion templating route. For the enzyme stabilization, β-glucosidase was immobilized onto mesocellular silica foams (MCFs) in a simple and effective way, a process achieved using enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of crosslinked enzyme aggregates (CLEAs) of nanometer scale. The structural and chemical properties of these prepared materials were characterized by TG, CPMAS NMR and nitrogen adsorption measurements. The crosslinked immobilizates retained activity over wider ranges of temperature and pH than those of the free enzyme. Kinetic parameter (K_m) of the immobilized β-glucosidase is lower than that of its free counterpart. The resulting CLEA was proved to be active and recyclable up to 10 cycles without much loss in activity. This demonstrates its prospects for commercial applications. The immobilizate exhibited enhanced storage stability characteristics than the native enzyme. In contrast to adsorbed GL and covalently bound glucosidase, the resulting crosslinked enzyme aggregates (CLEAs) showed an impressive stability with high enzyme loadings.     

Comparative study of thermostabilty and ester synthesis ability of free and immobilized lipases on cross linked silica gel

A novel support has been utilized for immobilization of lipase, which was prepared by amination of silica with ethanolamine followed by cross linking with glutaraldehyde. Lipases from Rhizopus oryzae 3562 and Enterobacter aerogenes were immobilized on activated silica gel, where they retained 60 and 50% of respective original activity. The thermal stability of the immobilized lipases was significantly improved in comparison to the free forms while the pH stability remained unchanged. E. aerogenes and R. oryzae 3562 lipases retained 75 and 97% of respective initial activity on incubation at 90 degrees C, whereas both the free forms became inactive at this temperature. The conversion yield of isoamyl acetate was found to be higher with the immobilized fungal (90 vs. 21%) and bacterial lipases (64 vs. 18%) than the respective free forms. Immobilized R. oryzae 3562 lipases retained 50% activity for isoamyl acetate synthesis up to ten cycles whereas it was eight cycles for E. aerogenes.     

Enzymatic hydrolysate of geniposide directly acts as cross-linking agent for enzyme immobilization

Enzyme immobilization is a routine biotechnology of many industries such as pharmaceutical, chemical and food. Among the different techniques of enzyme immobilization, cross-linking methods are often used. Geniposide is a natural product extracted from gardenia and its hydrolysate genipin is one of green cross-linking agent for enzyme immobilization, but the environmental pollution and cost of the genipin extraction process have become the main obstacle to its wide application. Enzyme β-glucosidase was immobilized on chitosan by self-catalysis and further used to hydrolyze geniposide. The laccase was immobilized on Nano-SiO₂ through the hydrolysate of geniposide directly acts as cross-linking agent. The simplification of the extraction steps overcomes the obstacles to the widespread use of genipin. Compared with the free laccase, the Nano-SiO₂@laccase exhibited better pH stability and thermal stability. The Nano-SiO₂@laccase was used to degrade Bisphenol A (BPA) and the biodegradation efficiency of the Nano-SiO₂@laccase was 84.3 % after 10 cycles of reusing.     

Immobilization of Aspergillus oryzae β-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking

The main objective of the present work is to study the immobilization process of Aspergillus oryzae β-galactosidase using the ionic exchange resin Duolite A568 as carrier. Initially, the immobilization process by ionic binding was studied through a central composite design (CCD), by analyzing the simultaneous influences of the enzyme concentration and pH on the immobilization medium. The results indicate that the retention of enzymatic activity during the immobilization process was strongly dependant of those variables, being maximized at pH 4.5 and enzyme concentration of 16 g/L. The immobilized enzyme obtained under the previous conditions was subjected to a cross-linking process with glutaraldehyde and the conditions that maximized the activity were a glutaraldehyde concentration of 3.83 g/L and cross-linking time of 1.87 h. The residual activity of the immobilized enzyme without glutaraldehyde cross-linking was 51% of the initial activity after 30 uses, while the enzyme with cross-linking immobilization was retained 90% of its initial activity. The simultaneous influence of pH and temperature on the immobilized β-galactosidase activity was also studied through a central composite design (CCD). The results indicate a greater stability on pH variations when using the cross-linking process.     

Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads

The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4 mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1 unit (pH 8.0) and 5 °C higher (45 °C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30 min of each reaction time, indicating stable reusability under aqueous medium.     

Immobilization and biocatalysis of chlorophyllase in selected organic solvent systems

Chlorophyllase extract from Phaeodactylum tricornutum was immobilized by physical adsorption on DEAE-cellulose and silica gel as well as by covalent binding on Eupergit C, Eupergit C250L, Eupergit C/ethylenediamine (EDA) and Eupergit C250L/EDA. Although the highest immobilization yield (83-93%) and efficiency (51-53%) were obtained when chlorophyllase extract was immobilized on DEAE-cellulose and silica gel, there was no improvement in the thermal stability of chlorophyllase as compared to that of the free one. The immobilization of chlorophyllase extract on Eupergit C250L/EDA resulted by a high recovery of enzymatic activity, with an immobilization efficiency of 44%, and promoted a higher stabilization of chlorophyllase (four times) in the aqueous/miscible organic solvent medium. On the other hand, the inhibitory effect of refined bleached deodorized (RBD) canola oil was reduced by immobilization of chlorophyllase extract onto silica gel as compared to those obtained with other enzyme preparations. However, the re-cycled chlorophyllase extract immobilized on Eupergit C250L/EDA retained more than 75% of its initial enzyme activity after 6 cycles, whereas that immobilized on silica gel was completely inactivated. The highest catalytic efficiency, for both free and immobilized chlorophyllase on Eupergit C250L/EDA, was obtained in the ternary micellar system as compared to the aqueous/miscible organic solvent and biphasic media.     

Adsorption of peroxidase on Celite 545 directly from ammonium sulfate fractionated white radish (Raphanus sativus) proteins

This paper demonstrates the direct immobilization of peroxidase from ammonium sulfate fractionated white radish proteins on an inorganic support, Celite 545. The adsorbed peroxidase was crosslinked by using glutaraldehyde. The activity yield for white radish peroxidase was adsorbed on Celite 545 was 70% and this activity was decreased and remained 60% of the initial activity after crosslinking by glutaraldehyde. The pH and temperature-optima for both soluble and immobilized peroxidase was at pH 5.5 and 40°C. Immobilized peroxidase retained higher stability against heat and water-miscible organic solvents. In the presence of 5.0 mM mercuric chloride, immobilized white radish peroxidase retained 41% of its initial activity while the free enzyme lost 93% activity. Soluble enzyme lost 61% of its initial activity while immobilized peroxidase retained 86% of the original activity when exposed to 0.02 mM sodium azide for 1 h. The K_m values were 0.056 and 0.07 mM for free and immobilized enzyme, respectively. Immobilized white radish peroxidase exhibited lower V_max as compared to the soluble enzyme. Immobilized peroxidase preparation showed better storage stability as compared to its soluble counterpart.     

Calcium alginate–starch hybrid support for both surface immobilization and entrapment of bitter gourd (Momordica charantia) peroxidase

Calcium alginate–starch hybrid gel was employed as an enzyme carrier both for surface immobilization and entrapment of bitter gourd peroxidase. Entrapped crosslinked concanavalin A–bitter gourd peroxidase retained 52% of the initial activity while surface immobilized and glutaraldehyde crosslinked enzyme showed 63% activity. A comparative stability of both forms of immobilized bitter gourd peroxidase was investigated against pH, temperature and chaotropic agent; like urea, heavy metals, water-miscible organic solvents, detergent and inhibitors. Entrapped peroxidase was significantly more stable as compared to surface immobilized form of enzyme. The pH and temperature-optima for both immobilized preparations were the same as for soluble bitter gourd peroxidase. Entrapped crosslinked concanavalin A–bitter gourd peroxidase showed 75% of the initial activity while the surface immobilized and crosslinked bitter gourd peroxidase retained 69% of the original activity after its seventh repeated use.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.     

Synthesis of Protein-Inorganic Nanohybrids with Improved Catalytic Properties Using Co<Subscript>3</Subscript>(PO<Subscript>4</Subscript>)<Subscript>2</Subscript>

In the present study, a method for easy and rapid synthesis of lipase nanohybrids was evaluated using cobalt chloride as an encapsulating agent. The synthesized nanohybrids exhibited higher activity (181%) compared to free lipase and improved catalytic properties at higher temperature and in harsh conditions. The nanohybrids retained 84% of their residual activity at 25 °C after 10 days. In addition, these nanohybrids also exhibited high storage stability and reusability. Collectively, the synthesis of carrier-free immobilized biocatalysts was performed rapidly within 24 h at 4 °C. Their high reusability and catalytic activities highlight the broad applicability of this method for catalysis in organic and aqueous media.     

Preparation of nano-enzyme aggregates by crosslinking lipase with sodium tripolyphosphate

Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization of enzyme. The ionic cross-linking agent-sodium tripolyphosphate (TPP) was first used in preparing CLEAs. Aspergillus niger lipase was precipitated with ammonium sulfate and further cross-linked by TPP. The factors including enzyme concentration, pH of cross-linking medium, TPP dosage and cross-linking time were optimized. Maximum recovery activity (99.5 ± 0.634 %) and cross-linking yield (88.4 ± 0.46 %) can be obtained under the optimal process conditions, which can illustrate TPP had little effect on enzyme activity. CLEAs showed improved activity over broad pH and temperature range compared to the free enzyme. The thermal stability was obviously improved compared to free enzyme under the optimal temperature (40℃) and the half-life was 7.5-fold higher than that of free enzyme. Moreover, scanning electron microscopy (SEM) revealed that CLEAs had a cavity with porous structure and the particle size was 249 ± 3.98 nm. X-ray diffraction (XRD) showed the crystallinity of the CLEAs decreased. The changes in secondary structures of CLEAs revealed the increment in conformational rigidity. Such results suggested that the CLEAs has ideal application prospects.     

Efficient immobilization of epoxide hydrolase onto silica gel and use in the enantioselective hydrolysis of racemic para-nitrostyrene oxide

Epoxide hydrolase from Aspergillus niger (E.C. 3.3.2.3) was immobilized by covalent linking to epoxide-activated silica gel under mild conditions. A very easy procedure allowed to prepare an immobilized biocatalyst with more than 90% retention of the initial enzymatic activity. Immobilized and free enzyme showed very similar behaviour with respect to the effect of pH on activity and stability. One benefit of immobilizing epoxide hydrolase from A. niger on silica gel was the enhanced enzyme stability in the presence of 20% DMSO. The kinetic resolution of racemic para-nitrostyrene oxide was investigated by using this new immobilized biocatalyst. The enantioselectivity of the enzyme was not altered by the immobilization reaction: both unreacted epoxide and formed diol were obtained with very high ee (99 and 92%, respectively). In addition, the biocatalyst could be easily separated from the reaction mixture and re-used for over nine cycles without any noticeable loss of enzymatic activity or change in the enantioselectivity extent. The activity of immobilized AnEH was retained for several months.