Immobilization of lipase from Pseudomonas fluorescens on glyoxyl-octyl-agarose beads: Improved stability and reusability

The lipase from Pseudomonas fluorescens (PFL) has been immobilized on glyoxyl-octyl agarose and compared to the enzyme immobilized on octyl-agarose. Thus, PFL was immobilized at pH 7 on glyoxyl-octyl support via lipase interfacial activation and later incubated at pH 10.5 for 20 h before reduction to get some enzyme-support covalent bonds. This permitted for 70% of the enzyme molecules to become covalently attached to the support. This biocatalyst was slightly more stable than the octyl-PFL at pH 5, 7 and 9, or in the presence of some organic solvents (stabilization factor no higher than 2). The presence of phosphate anions produced enzyme destabilization, partially prevented by the immobilization on glyoxyl-octyl (stabilization factor became 4). In contrast, the presence of calcium cations promoted a great PFLstabilization, higher in the case of the glyoxyl-octyl preparation (that remained 100% active when the octyl-PFL preparations had lost 20% of the activity). However, it is in the operational stability where the new biocatalyst showed the advantages: in the hydrolysis of 1 M triacetin in 60% 1.4 dioxane, the octyl biocatalyst released >60% of the enzyme in the first cycle, while the covalently attached enzyme retained its full activity after 5 reaction cycles.     

Improvement of the stability and selectivity of Rhizomucor miehei lipase immobilized on silica nanoparticles: Selective hydrolysis of fish oil using immobilized preparations

We report the effect of random and oriented immobilization of Rhizomucor miehei lipase (RML) on its functional properties. For this purpose, silica nanoparticles (MCM-41 and SBA-15) were prepared, characterized and functionalized by glycidyloxypropyl trimethoxysilane. Direct immobilization of RML on these supports was performed via the variety of amino acid residues on the surface of RML which promotes random immobilization. To perform oriented immobilization, partial modification of epoxy functionalized supports was carried out by introducing iminodiacetic acid groups followed by addition of Cu²⁺. In this way, immobilization is mainly directed via the most accessible histidine group, followed by intramolecular reaction of the other nucleophilic residues of the enzyme and the remaining epoxy groups on the support. The results showed higher thermal stability for immobilized derivatives compared to the soluble enzyme. Co-solvent stability of the derivatives was also studied in presence of six polar organic solvents (DMSO, THF, acetonitrile, 1-propanol, 2-propanol and dioxane). Influence of the immobilization procedure on activity and selectivity of the immobilized preparations was studied in selective hydrolysis of fish oil. All the derivatives discriminate between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. Remarkable improvement in selectivity was obtained using oriented immobilization of RML.     

Enzymatic transesterification in a solvent-free system: synthesis of sn-2 docosahexaenoyl monoacylglycerol

Abstract

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was carried out by using several immobilized lipases in a solvent-free system. This reaction involves the initial formation of sn-2 docosahexaenyl monoacylglycerol. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp. (AQ) and Candida antarctica-fraction B (CALB) were immobilized by interfacial adsorption on a commercial hydrophobic support (a methacrylate resin containing octadecyl groups, Sepabeads C-18) and tested for glycerolysis of DHA ethyl ester. In certain cases (e.g. immobilized CALB), the transesterification reaction continues to the formation of triacylglycerol (80%) by using a very high excess of DHA ethyl ester ((115 mmols versus 1.24 mmols of glycerol and high temperatures (50?°C). However, the same biocatalyst working at lower temperatures, 37?°C, synthetizes a 90% of sn-2 monoacylglycerol even in the presence of that a high excess of DHA ethyl ester. Interestingly, immobilized RML derivative synthesizes a 98% of sn-2 monoacylglyceride (2-MG) in 15?min at 37?°C with a 4% of immobilized biocatalyst. These high activity and regioselectivity under very mild reaction conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid as well as for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate commercial markers, the formation of the 2-MG was confirmed.     

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.     

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

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

Resolution of d,l-menthol by interesterification with triacetin using the free and immobilized lipase of Candida cylindracea

Summary Interesterification in isooctane with triacetin as an acyl donor was found to be a new and effective method of racemic resolution of d,l-menthol, when using the free and immobilized lipase of Candida cylindracea. No water was produced by this highly stereoselective type of reaction in contrast to ester synthesis with acetic acid as an acyl donor. Even with diacetin no possible back reaction occurred and the enzyme was easily separated from the reaction solution as opposed to ester hydrolysis in aqueous systems. Inhibition of interesterification was caused by increasing concentrations of the acyl donor triacetin by more than 10 mmol·l^-1 on the one hand, and especially by diacetin on the other hand. The reaction product menthyl acetate had no influence. By adding water the interesterification activity of the lipase was reduced significantly. An alteration of the acyl donor triacetin to longerchained triglycerides caused changes in higher specific activities but poor enantioselectivities of the products, as in the case of ester synthesis starting from longer-chained organic acids.Dedicated to Prof. Dr. Fritz Wagner on the occasion of his 60th birthday     

Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine

Glucoamylase (GA) from Aspergillus niger was immobilized via ionic adsorption onto DEAE-agarose, Q1A-Sepabeads, and Sepabeads EC-EP3 supports coated with polyethyleneimine (PEI). After optimization of the immobilization conditions (pH, polymer size), it was observed that the adsorption strength was much higher in PEI-Sepabeads than in Q1A-Sepabeads or DEAE-supports, requiring very high ionic strength to remove glucoamylase from the PEI-supports (e.g., 1 M NaCl at pH 5.5). Thermal stability and optimal temperature was marginally improved by this immobilization. Recovered activity depended on the substrate used, maltose or starch, except when very low loading was used. The optimization of the loading allowed the preparation of derivatives with 750 IU/g in the hydrolysis of starch, preserving a high percentage of immobilized activity (around 50%).     

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

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

Optimization of an industrial biocatalyst of glutaryl acylase: stabilization of the enzyme by multipoint covalent attachment onto new amino-epoxy Sepabeads

Glutaryl-7-aminocephalosporanic acid acylase (GA), an industrially relevant enzyme, has been immobilized onto very different supports, including glyoxyl agarose, heterofunctional epoxy Sepabeads, glutaraldehyde and cyanogen bromide (CNBr) activated supports. Immobilization onto amino-epoxy Sepabeads rendered the most thermo stable preparation of GA, with a half-life time eight times higher than the soluble enzyme, keeping 80% of the enzyme activity. Several parameters that affect the enzyme-support interaction (pH and incubation time) were studied. It was found that after immobilization onto amino-epoxy Sepabeads, incubation at alkaline pH and low temperature exerted dramatic stabilizing effects, increasing the half-life time of the derivative 130 times with respect to the soluble enzyme, while keeping unaltered its intrinsic activity. The loading capacity of the amino-epoxy Sepabeads proved to be very good with a maximum load of 62 mg of protein per g of support with 85 IU/g at 25 degrees C and 200 IU/g at 37 degrees C which makes it a biocatalyst of possible industrial application.     

Hydrolysis of fish oil by hyperactivated rhizomucor miehei lipase immobilized by multipoint anion exchange

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Tuning of Lecitase features via solid-phase chemical modification: Effect of the immobilization protocol

Lecitase Ultra (a quimeric fosfolipase commercialized by Novozymes) has been immobilized via two different strategies: mild covalent attachment on cyanogen bromide agarose beads and interfacial activation on octyl-agarose beads. Both immobilized preparations have been submitted to different individual or cascade chemical modifications (amination, glutaraldehyde or 2,4,6-trinitrobenzensulfonic acid (TNBS) modification) in order to check the effect of these modifications on the catalytic features of the immobilized enzymes (including stability and substrate specificity under different conditions). The first point to be remarked is that the immobilization strongly affects the enzyme catalytic features: octyl-Lecitase was more active versus p-nitrophenylbutyrate but less active versus methyl phenylacetate than the covalent preparations. Moreover, the effects of the chemical modifications strongly depend on the immobilization strategy used. For example, using one immobilization protocol a modification improves activity, while for the other immobiled enzyme is even negative. Most of the modifications presented a positive effect on some enzyme properties under certain conditions, although in certain cases that modification presented a negative effect under other conditions. For example, glutaraldehyde modification of immobilized or modified and aminated enzyme permitted to improve enzyme stability of both immobilized enzymes at pH 7 and 9 (around a 10-fold), but only the aminated enzyme improved the enzyme stability at pH 5 by glutaraldehyde treatment. This occurred even though some intermolecular crosslinking could be detected via SDS-PAGE. Amination improved the stability of octyl-Lecitase, while it reduced the stability of the covalent preparation. Modification with TNBS only improved enzyme stability of the covalent preparation at pH 9 (by a 10-fold factor).     

Advantages of the pre-immobilization of enzymes on porous supports for their entrapment in sol-gels

In this work, we have compared the entrapment of free or previously immobilized glucose oxidase using a sol-gel technique. The preimmobilization was carried out on Sepabeads (a porous support) derivatized with glutaraldehyde as the functional group. The prior immobilization of the enzyme permitted to maintain the enzyme activity intact after the formation of the sol-gel. In fact, only 10% of the enzyme activity was lost whereas the soluble enzyme lost 60% of its initial activity. Additionally, enzyme leakage from the sol-gel matrix was avoided, which was relatively high when entrapping the soluble enzyme (39% of the enzyme activity was released after 16 h of incubation in a buffered solution). Moreover, the immobilized enzyme, inside the porous support, cannot be in contact with the sol-gel, and, therefore, it maintained the stability achieved by means of the multipoint covalent attachment on the Sepabeads support.     

Immobilization on octyl-agarose beads and some catalytic features of commercial preparations of lipase a from Candida antarctica (Novocor ADL): Comparison with immobilized lipase B from Candida antarctica

Lipase A from Candida antarctica (CALA, commercialized as Novocor ADL) was immobilized on octyl-agarose, which is a very useful support for lipase immobilization, and coated with polyethylenimine to improve the stability. The performance was compared to that of the form B of the enzyme (CALB) immobilized on the same support, as both enzymes are among the most popular ones used in biocatalysis. CALA immobilization produced a significant increase in enzyme activity vs. p-nitrophenyl butyrate (pNPB) (by a factor of seven), and the coating with PEI did not have a significant effect on enzyme activity. CALB reduced its activity slightly after enzyme immobilization. Octyl-CALA was less stable than octyl-CALB at pH 9 and more stable at pH 5 and, more clearly, at pH 7. PEI coating only increased octyl-CALA stability at pH 9. In organic solvents, CALB had much better stability in methanol and was similarly stable in acetonitrile or dioxane. In these systems, the PEI coating of octyl-CALA permitted some stabilization. While octyl-CALA was more active vs. pNPB, octyl-CALB was much more active vs. mandelic esters or triacetin. Thus, depending on the specific reaction and the conditions, CALA or CALB may offer different advantages and drawbacks. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2735, 2019     

Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

Application of multi-component reaction for covalent immobilization of two lipases on aldehyde-functionalized magnetic nanoparticles; production of biodiesel from waste cooking oil

Lipase from Rhizomucor miehei (RML) and Thermomyces lanuginosa lipase (TLL) were immobilized on silica core-shell magnetic nanoparticles (Fe₃O₄@SiO₂) produced by coating Fe₃O₄ core with silica shell. The nanoparticles were functionalized with aldehyde groups followed by immobilization of RML and TLL by using a multi-component reaction in an extremely mild condition. Rapid immobilization of both enzymes (1.5−12 h) with high immobilization yields (81–100%) was observed. The maximum loading capacity of the support was determined to be 81 mg for RML and 97 mg for TLL. The thermal stability of the immobilized derivatives of RML and TLL were greatly improved by retaining 54 and 97 % of their initial activities at 65 °C, respectively. The immobilized preparations were used to produce biodiesel by transesterification of waste cooking oil. In an optimization study, Response Surface Methodology (RSM) and a central composite rotatable design (CCRD) were used to study the effect of amount of biocatalyst, temperature, reaction time, water adsorbent (wt.%) and ratio of t-butanol to oil (wt.%) on the yield of biodiesel production. Biodiesel production yield by immobilized TLL reached 93.1 % under optimal conditions while the maximum yield for RML was 57.5 %. Both immobilized derivatives showed high reusability after 5 cycles of the reaction.     

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

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

Enhancing the catalytic properties of porcine pancreatic lipase by immobilization on SBA-15 modified by functionalized ionic liquid

The mesoporous silica SBA-15 was modified by carboxyl-functionalized ionic liquid (COOH-IL-SBA). The prepared support was used to immobilize porcine pancreatic lipase (PPL) by physical adsorption (PPL-COOH-IL-SBA) and covalent attachment (PPL-CON-IL-SBA). Enzymatic properties of the immobilized PPL were investigated in the triacetin hydrolysis reaction. It was found that carboxyl functionalized ionic liquid modification of the support surface was an effective method to improve the properties of immobilized PPL. Incorporating into the functionalized SBA-15 made PPL more resistant to temperature and pH changes, compared with PPL immobilized on parent SBA-15 (PPL-SBA). Especially, after the covalent attachment to a functionalized support, the stability of PPL was improved obviously, which retained 81.25% and 52.50% of the original activity after incubation for 20 days and four times recycling, respectively, whereas PPL-SBA exhibited only 58.80% and 27.78% of the original activity under the same conditions. In addition, physical and chemical properties of the supports and immobilized PPL were characterized by small-angle X-ray powder diffraction (SAXRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), nitrogen adsorption, nuclear magnetic resonance (NMR) and thermogravimetry (TG). The images and data confirmed chemical modification in SBA-15 and PPL immobilization on the tested support.     

An efficient immobilizing technique of penicillin acylase with combining mesocellular silica foams support and <Emphasis Type="Italic">p</Emphasis>-benzoquinone cross linker

To improve the performance of covalently immobilized penicillin acylase (PA), the immobilization was carried out in mesocellular silica foams (MCFs) using p-benzoquinone as cross linker. The characterizations of the immobilized enzyme were studied carefully. The results showed that the relative activity of the immobilized PA was increased to 145% of that of free enzyme. The activity was 3.7 folds of that of PA on the silica nanoparticles. The enzyme in MCFs presented a turnover equal to that of free enzyme. It was also found that the optimum pH of the immobilized PA shifted to pH 7.5 and the optimum reaction temperature rose from 45 to 50 degrees C. Furthermore, the stability of PA was ameliorated greatly after immobilization. Fourier transform infrared spectroscopy showed no major secondary structural change for PA confined in MCFs. The proposed covalent immobilizing technique would rank among the potential strategies for efficient immobilization of PA.     

Simple and efficient immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads

Two commercial porous styrene-divinylbenzene beads (Diaion HP20LX and MCI GEL CHP20P) have been evaluated as supports to immobilize lipase B from Candida antarctica (CALB). MCI GEL CHP20P rapidly immobilized the enzyme, permitting a very high loading capacity: around 110 mg CALB/wet g of support compared to the 50 mg obtained using decaoctyl Sepabeads. Although enzyme specificity of the enzyme immobilized on different supports was quite altered by the support used in the immobilization, specific activity of the enzyme immobilized on MCI GEL CHP20P was always higher than those found using decaoctyl Sepabeads for all assayed substrates. Thus, a CALB biocatalyst having 3-8 folds (depending on the substrate) higher activity/wet gram of support than the commercial Novozym 435 was obtained. Half-live of CAL-Diaion HP20LX at 60 °C was 2-3 higher than the one of Novozym 435, it was 30-40 higher in the presence of 50% acetonitrile and it was around 100 folds greater in the presence of 10 M hydrogen peroxide.Results indicate that styrene-divinylbenzene supports may be promising alternatives as supports to immobilize CALB.     

Immobilization and stabilization of recombinant multimeric uridine and purine nucleoside phosphorylases from Bacillus subtilis

We selected the PnpI/PupG (PNP) with specificity for ribo- and deoxyriboguanosine and ribo- and deoxyriboinosine and the Up/Pdp (UP) with specificity for uridine, thymidine, and deoxyuridine from the purine and pyrimidine salvage pathway of the Gram-positive bacterium Bacillus subtilis. Then, an extensive study of the UP (uridine phosphorylase) and PNP (purine nucleoside phosphorylase) immobilization and stabilization was carried out: optimal UP preparation was achieved by immobilization onto Sepabeads coated with poly(ethyleneimine) and finally cross-linked with aldehyde dextran (UP-Sep-PEI-Dx); optimal immobilized PNP was prepared onto glyoxyl-agarose. Both derivatives were highly stable and active even under drastic experimental conditions (pH 10, 45 degrees C) unlike the free enzymes which were promptly inactivated. The derivatives prepared were successfully used in the synthesis of 2'-deoxyguanosine by enzymatic transglycosylation in aqueous solution between 2'-deoxyuridine and guanine.