Multipoint Covalent Attachment Research Articles

Rational design of biocatalysts based on covalent immobilization of acylase enzymes

Acylases catalyze the hydrolysis of amide bonds. Penicillin G acylase (PGA) is used for the semi-synthesis of penicillins and cephalosporins. Although protein immobilization increases enzyme stability, the design of immobilized systems is difficult and usually it is empirically performed. We describe a novel application of our strategy for the Rational Design of Immobilized Derivatives (RDID) to produce optimized acylase-based immobilized biocatalysts for enzymatic bioconversion. We studied the covalent immobilization of the porcine kidney aminoacylase-1 onto aldehyde-based supports. Predictions of the RDID1.0 software and the experimental results led to the selection of glyoxyl-Sepharose CL 4B support and pH 10.0. One of the predicted clusters of reactive amino groups generates an enzyme-support configuration with highly accessible active sites, contributing with 82% of the biocatalyst’s total activity. For Escherichia coli PGA, the predictions and experimental results show similar maximal amounts of immobilized protein and activity at pH 8.0 and 10.0 on glyoxyl-Sepharose CL 10B. However, thermal stability of the immobilized derivative is higher at pH 10.0 due to an elevated probability of multipoint covalent attachment. In this case, two clusters of amino groups are predicted to be relevant for PGA immobilization in catalytically competent configurations at pH 10.0, showing accessible active sites and contributing with 36% and 44% of the total activity, respectively. Our results support the usefulness of the RDID strategy to model different protein engineering approaches (site-directed mutagenesis or obtainment of fusion proteins) and select the most promising ones, saving time and laboratory work, since the in silico-designed modified proteins could have higher probabilities of success on bioconversion processes.

Glyoxyl-ficin: An example where a more intense multipoint covalent attachment may decrease enzyme stability.

The immobilization of ficin extract (sulfidryl proteases) on glyoxyl agarose beads stabilized it, but there is a clear stability maximum after 3 h of immobilization, being the 24 h biocatalyst less stable when thermally inactivated in presence of air. The incubation of the thermally inactivated biocatalysts in the presence of dithiothreitol permitted to recover almost total activity of the biocatalysts prepared for 24 h of immobilization, while the enzyme immobilized for 3 h recovered less activity. This suggested that the main cause of the faster enzyme inactivation of the 24 h biocatalyst was the Cys oxidation. This was confirmed by inactivating the Cys blocked biocatalysts by using 2,2′-dipyridyldisulfide. Again, a higher percentage of the initial activity (when unblocked by incubation in mercaptoethanol the thermally inactivated biocatalysts) was observed for the 24 h biocatalyst. The use of helium atmosphere enabled to greatly improve the enzyme stability of all immobilized biocatalysts, confirming the highest stability of the biocatalyst immobilized for 24 h. Results suggested that this biocatalyst was in fact the most rigid one, but that the distortion induced by the immobilization produced a higher exposition of the catalytic Cys, and this way, its oxidation becomes faster in the presence of oxygen.

Multipoint Immobilization at Inert Center of Papain on Homo-Functional Diazo-Activated Silica Support: A Way of Restoring “Above Room-Temperature” Bio-Catalytic Sustainability

Although enzymes play a significant role in industrial applications, their potential usage at high-level efficiency, particularly above room temperature, has not yet been fully harnessed. It brings above room-temperature catalytic sustainability of an immobilized (imm.) bio-catalyst as a long pending issue to improve enzyme stability, activity, specificity, or selectivity, particularly the enantio-selectivity over the native-enzymes. At this juncture, in a robust methodology, a heterogeneous solid phase bio-catalyst, {Si(OSi)4(H2O)1.03}n=328{OSi(CH3)2-NH-C6H4-N═N}4{papain}(H2O)251, has efficiently been prepared by immobilizing papain on homo-functionalized SG (silica-gel) via multipoint covalent attachment. The bio-catalyst is easy to be recovered and reused multiple times. The homo-functional -N═N+, which appears on the SG-surface, makes the multipoint diazo-links with the inert center of the tyrosine-moiety to couple the enzyme where all the amino, thiol, phenol, and so forth, groups of the protein, including those that belong to the active-site, remain intact. The immobilized enzyme (13.9 μmol g-1) swims in pore-water within the pore-channel, remains stable up to 70 ± 5 °C, and exhibits wider temperature adaptability in performing its hydrolyzing activities. The relative activity, 78 ± 2% at 27 °C, remains quantitative for 60 days and can be reused for 60 cycles with 53% activity at room-temperature. The thermal (relative activity: 87%; incubated at 70 ± 5 °C for 24 h) and mechanical (relative activity: 92%; incubated at 2500 rpm for 2 h at 27 °C) stability was outstanding.

Laccase multi-point covalent immobilization: characterization, kinetics, and its hydrophobicity applications.

Laccase from Myceliophthora thermophila was immobilized using one-point and multi-point covalent attachment on both a native and a modified new commercial epoxy carrier (Immobead 150P). After 10 cycles of operation at pH 3.0 and temperature 70°C, the multi-point covalently immobilized laccase on the modified Immobead 150P performed best in terms of immobilization characteristics, retaining 95% of its initial activity. Thermodynamic parameters of thermal inactivation emphasized the positive impact of the immobilization procedure. At 50°C, the immobilized and free enzyme activity levels dropped by 27 and 73%, respectively, after 48h of incubation. The immobilized enzyme enhanced its stability in alkaline conditions, resuming 95% of its original activity after 3h at pH 9.0. Immobilization reduced substrate affinity because the free laccase's Km value was lower than that of the immobilized laccase. Finally, the application of immobilized laccase in an innovative wood treatment process was tested by grafting lauryl gallate (LG) in order to provide hydrophobic properties to the wood. The results showed a relative water contact angle of 85.7% for treated wood, whereas the control showed only 26.6%, after 4min of contact between water and beechwood surface. KEY POINTS: • Multi-point covalent immobilization of a commercial laccase on a commercial support. • Enzymatic parameters generally improved by immobilization process. • New application of immobilized laccase: enzymatic-assisted wood hydrophobization.

Efficient Synthesis of (S)-1-Boc-3-aminopiperidine in a Continuous Flow System Using ω-Transaminase-Immobilized Amino-Ethylenediamine-Modified Epoxide Supports

We developed a continuous flow system using ω-transaminase covalently immobilized on an epoxy resin modified with ethylenediamine (EDA) to synthesize (S)-1-Boc-3-aminopiperidine, which is a key intermediate for several novel inhibitors. A second functional group (−NH3+) was introduced to rapidly adsorb ω-transaminase, which was further rigidified by multipoint covalent attachment to the modified resin with high binding efficiency (>95%) and activity recovery (75%). The resulting immobilized ω-transaminase was stable with 90% residual activity after 15 reuse cycles. We synthesized (S)-1-Boc-3-aminopiperidine using this continuous flow system, and 95% conversion was obtained within 10 min of residence. The system was continuously operated for 24 h and achieved a space–time yield of 930.73 g·L–1·day–1, indicating outstanding reaction efficiency. This efficient continuous bioprocess has potential for large-scale production of (S)-1-Boc-3-aminopiperidine.

Effect of amine length in the interference of the multipoint covalent immobilization of enzymes on glyoxyl agarose beads

Trypsin, chymotrypsin, penicillin G acylase and ficin extract have been stabilized by immobilization on glyoxyl agarose, adding different aliphatic compounds bearing a primary amine group during the immobilization: ethyl amine, butyl amine, hexyl amine (at concentrations ranging from 0 to 20 mM) and octyl amine (from 0 to 10 mM) to analyze their effects on the immobilized enzyme stability. As expected, the presence of amines reduced the intensity of the enzyme-support multipoint covalent attachment, and therefore the enzyme stability. However, it is clear that this effect is higher using octyl amine for all enzymes (in some cases the enzyme immobilized in the presence of 10 mM octyl amine was almost inactivated while the reference kept over 50 % of the initial activity). This way, it seems that the most important effect of the presence of aminated compounds came from the generation of steric hindrances to the enzyme/support multi-reaction promoted by the ammines that are interacting with the aldehyde groups. In some instances, just 1 mM of aminated compounds is enough to greatly decrease enzyme stability. The results suggested that, if the composition of the enzyme extract is unknown, to eliminate small aminated compounds may be necessary to maximize the enzyme-support reaction.

Capture of enzyme aggregates by covalent immobilization on solid supports. Relevant stabilization of enzymes by aggregation

In this paper, a novel procedure for the immobilization and stabilization of enzymes is proposed: the multipoint covalent attachment of bi-molecular enzyme aggregates. This immobilization protocol allows the “capture” and fixation of the enzyme aggregate on the support surface. In addition to stabilization by multipoint attachment, enzyme aggregation promotes very interesting stabilizing effects. In the presence of low concentrations of polyethylene glycol (30 %) the dimeric amine oxidase from Pisum sativum forms soluble bi-molecular aggregates. Enzyme aggregates were analyzed by Dynamic Light Scattering and by full chemical loading of a mesoporous support (10 % agarose gels activated with glyoxyl groups). The soluble aggregate was immobilized by multipoint attachment on glyoxyl- agarose at pH 8.5 though the four amino termini of the two dimeric molecules (Lys residues are not reactive at this pH). The immobilized aggregated structure cannot undergo any movement (translational or rotational) after multipoint attachment and the aggregate is “fixed” on the support surface even after the removal of PEG. The immobilized aggregate was further incubated at pH 10 in order to allow the Lys residues to react with the glyoxyl groups on the support. Enzyme aggregation has an important effect on enzyme stabilization: the aggregated derivative was 40 fold more stable than a similar derivative of the isolated enzyme and 200 fold more than native enzymes in experiments of thermal inactivation.

Optimization of immobilization conditions of Bacillus atrophaeus FSHM2 lipase on maleic copolymer coated amine-modified graphene oxide nanosheets and its application for valeric acid esterification

The thermoalkalophilic lipase from Bacillus atrophaeus (BaL) was immobilized onto amine-functionalized graphene oxide nanosheets coated with the poly (maleic anhydride-alt-1-octadecene) copolymer (GO-NH2-PMAO) and activated with glutaraldehyde as spacer arm through interfacial activation and subsequent multipoint covalent attachment. Experimental design method was applied for optimization of immobilization conditions including GO-NH2-PMAO concentration, buffer concentration, pH, sonication time, enzyme concentration, glutaraldehyde concentration, time, and temperature. The optimum specific activity of the immobilized BaL (105.95 ± 2.37 U/mg) reached at 5 mg/mL for GO-NH2-PMAO, 25 mM of buffer, pH 6.0, 60 min sonication time, 100 mM glutaraldehyde, 60 U/mL of enzyme, and 4 h of immobilization time at 25 °C, which was very close to the predicted amount (106.08 ± 1.42 U/mg). Maximum immobilization yield (81.35%) and efficiency (277.63%) were determined in optimal immobilization conditions. The obtained results clearly indicated that the immobilized BaL exhibited better stability at extreme temperature and pH than the free BaL. At temperature of 90 °C and pH 11, more than 90% of the initial activity of the immobilized BaL was retained. Furthermore, the immobilized BaL retained about 90% of its initial activity after 10 days of storage and 6 cycles of application. The esterification studies showed that maximum bioconversion of valeric acid to pentyl valerate using the free BaL (34.5%) and the immobilized BaL (96.3%) occurred in the xylene medium after 48 h of incubation at 60 °C. Therefore, the BaL immobilized on GO-NH2-PMAO was introduced as an effective biocatalyst to synthesize green apple flavour ester.

Co-Immobilization and Co-Localization of Oxidases and Catalases: Catalase from Bordetella Pertussis Fused with the Zbasic Domain

Oxidases catalyze selective oxidations by using molecular oxygen as an oxidizing agent. This process promotes the release of hydrogen peroxide, an undesirable byproduct. The instantaneous elimination of hydrogen peroxide can be achieved by co-immobilization and co-localization of the oxidase and an auxiliary catalase inside the porous structure of solid support. In this paper, we proposed that catalase from Bordetella pertussis fused with a small domain (Zbasic) as an excellent auxiliary enzyme. The enzyme had a specific activity of 23 U/mg, and this was almost six-fold higher than the one of the commercially available catalases from bovine liver. The Zbasic domain was fused to the four amino termini of this tetrameric enzyme. Two domains were close in one hemisphere of the enzyme molecule, and the other two were close in the opposite hemisphere. In this way, each hemisphere contained 24 residues with a positive charge that was very useful for the purification of the enzyme via cationic exchange chromatography. In addition to this, each hemisphere contained 10 Lys residues that were very useful for a rapid and intense multipoint covalent attachment on highly activated glyoxyl supports. In fact, 190 mg of the enzyme was immobilized on one gram of glyoxyl-10% agarose gel. The ratio catalase/oxidase able to instantaneously remove more than 93% of the released hydrogen peroxide was around 5–6 mg of catalase per mg of oxidase. Thirty milligrams of amine oxidase and 160 mg of catalase were co-immobilized and co-localized per gram of glyoxyl-agarose 10BCL (10% beads cross-linked) support. This biocatalyst eliminated biogenic amines (putrescine) 80-fold faster than a biocatalyst of the same oxidase co-localized with the commercial catalase from bovine liver.

Improvement of enzymatic performance of Asclepias curassavica L. proteases by immobilization. Application to the synthesis of an antihypertensive peptide

The aim of this work was to study different immobilization strategies on silica supports in order to obtain robust biocatalysts from latex proteases of Asclepias curassavica L., a South American native plant. Immobilized enzyme performance was evaluated under harsh reaction conditions such as the synthesis of the antihypertensive peptide N-α-CBZ-Val-Gly-OH.Proteases from A. curassavica, named asclepain, were immobilized (0.51–5.56 mg of protein/ g of support) in non-functionalized silica (S), in glyoxyl-silica (GS) and in octyl-glyoxyl-silica (OGS), by adsorption, and multi-point covalent attachment on mono and hetero-functional supports, respectively, under previously determined optimal immobilization conditions. Immobilization yields were expressed as activity yield (Ya) and protein yield (Yp).Asclepain-OGS showed the highest Ya (178 ± 1.62 %) meaning an expressed activity 1.8 times higher than the offered activity, while Yp was 75 ± 0.4 %. Ya for asclepain-S and -GS were 64 ± 1.45 % and 16 ± 0.37 %, respectively. Best results were attributed to the ability of OGS support to guide the enzyme before covalent attachment, increasing its reactivity. Asclepain-OGS led to product yield of 95.5 ± 0.14 %, five times higher than soluble asclepain in the synthesis of N-α-CBZ-Val-Gly-OH, after 3 h in 30 % methanol in 0.1 M Tris-HCl buffer pH 8.

A mild intensity of the enzyme-support multi-point attachment promotes the optimal stabilization of mesophilic multimeric enzymes: Amine oxidase from Pisum sativum

Stabilization of dimeric enzymes requires the stabilization of the quaternary structure as well as the 3D one. Both subunits may be easily immobilized on a highly activated support. Additional stabilization of the 3D structure may be achieved via multipoint covalent attachment (MCA) on highly activated supports. In the case of monomeric enzymes or thermophilic dimeric ones, the optimal stabilization is obtained via the most intense MCA and it is associated to a small loss of catalytic activity. However, in the case of mesophilic enzymes, a very intense MCA of both subunits may promote negative effects, e.g., associated to distortions of the assembly between subunits and a subsequent very important loss of catalytic activity.A dimeric mesophilic amine oxidase from P.sativum was stabilized by MCA on glyoxyl-agarose. Both subunits were covalently immobilized on the support through the region with the highest density in Lys residues. In addition to that, an interesting activity/stabilization binomial was obtained after only 3 h of enzyme-support multiinteraction (50 % of activity/350 fold stabilization). However, after 24 h of enzyme-support multi-interaction this binomial activity-stabilization decreased down to 30/150. A moderate multiinteraction seems to be the optimal strategy for immobilization-stabilization of mesophilic dimeric enzymes and it promotes moderate losses of activity and interesting stabilizations against the combined effect of heat, acid pH and ethanol. The control of the intensity of enzyme-support multi-interactions becomes now strictly necessary.

Immobilization and stabilization of d-hydantoinase from Vigna angularis and its use in the production of N-carbamoyl-d-phenylglycine. Improvement of the reaction yield by allowing chemical racemization of the substrate

This work reports the immobilization of a multimeric d-hydantoinase (DHTase) from Vigna angularis (E.C. 3.5.2.2.) on agarose beads activated with glyoxyl groups aiming to improve its stability via multipoint covalent attachment. The final reduction with sodium borohydride resulted in a drop in enzyme activity that could be decreased by adding Zn2+ or Mg2+. The optimal preparation with high activity (58 % recovered activity) and stability (around 86-fold more stable than the free enzyme) was obtained by DHTase immobilization on glyoxyl agarose for 24 h at 25 °C and pH 10.05, and a borohydride reduction step in the presence of 10 mM Zn2+ (DHTase-Glx). The enzyme was almost fully immobilized on glyoxyl agarose (19.8 mg/g of support) when offering 20 mg/g. This immobilized biocatalyst was used to catalyze the hydrolysis of d,l-phenylhydantoin under substrate racemization conditions, which produced 99 % of N-carbamoyl-d-phenylglycine after 9 h reaction.

Multi-Point Covalent Immobilization of Enzymes on Glyoxyl Agarose with Minimal Physico-Chemical Modification: Stabilization of Industrial Enzymes.

Stabilization of enzymes via immobilization techniques is a valuable approach in order to convert a necessary protocol (immobilization) into a very interesting tool to improve key enzyme properties (stabilization). Multipoint covalent attachment of each immobilized enzyme molecule may promote a very interesting stabilizing effect. The relative distances among all enzyme residues involved in immobilization have to remain unaltered during any conformational change induced by any distorting agent. Amino groups are very interesting nucleophiles placed on protein surfaces. The immobilization of enzyme through the region having the highest amount of amino groups (Lys residues) is key for a successful stabilization. Glyoxyl groups are small aliphatic aldehydes that form very unstable Schiff's bases with amino groups, and they do not seem to be useful for enzyme immobilization at neutral pH. However, under alkaline conditions, glyoxyl supports are able to immobilize enzymes via a first multipoint covalent immobilization through the region having the highest amount of lysine groups. Activation of supports with a high surface density of glyoxyl groups and the performance of very intense enzyme-support multipoint covalent attachments are here described.

Multi-Point Covalent Immobilization of Enzymes on Supports Activated with Epoxy Groups: Stabilization of Industrial Enzymes.

Commercial epoxy supports may be very useful tools to stabilize proteins via multipoint covalent attachment if the immobilization is properly designed. In this chapter, a protocol to take full advantage of the support's possibilities is described. The basics of the protocol are as follows: (1) the enzymes are hydrophobically adsorbed on the supports at high ionic strength. (2) There is an "intermolecular" covalent reaction between the adsorbed protein and the supports. (3) The immobilized protein is incubated at alkaline pH to increase the multipoint covalent attachment, thereby stabilizing the enzyme. (4) The hydrophobic surface of the support is hydrophylized by reaction of the remaining groups with amino acids in order to reduce the unfavorable enzyme-support hydrophobic interactions. This strategy has produced a significant increase in the stability of penicillin G acylase compared with the stability achieved using conventional protocols.

Functionalization of Electrospun Poly(Acrylonitrile-co-Styrene/Pyrrole) Copolymer Nanofibers for Using as a High-performance Carrier for Laccase Immobilization

Enzyme immobilization onto nanomaterials is a strategy to overcome the difficulties of the free enzymes applications where the immobilization process affords excellent reusability and operational stability against temperature and pH, and high surface area for the catalytic reaction. In this work, poly(Acrylonitrile-co-Styrene/Pyrrole), (poly(AN-co-ST/ Py)), copolymer nanofibers were successfully fabricated using the electrospinning technique. Laccase enzyme from Trametes versicolor was immobilized onto functionalized poly(AN-co-ST/Py) nanofibers by covalent attachment using polyethylenediamine (PEI) via physical adsorption and multipoint covalent attachment to the functional groups onto the polymeric surface. Since the relative activity of the immobilized Laccase depends on the amount of the coupled PEI onto the polymeric nanofibers, a colorimetric assay via copper ions complex formation was applied for the estimation of the process yield. Accordingly, the various factors affecting the PEI coupling process such as concentration, time, pH and temperature were investigated; Besides their influence on the morphology of the resultant nanofibers. Furthermore, thermal and pH stability, storage stability, and reusability were evaluated and compared. The optimum reaction temperature and pH for the immobilized enzyme were 70 °C and 6.0, respectively. The scanning electron microscopy (SEM) images showed that the modified nanofibers with PEI have kept its nanofibers structure and uniform morphology with an average diameter of approximately 559.2 nm. Within 35 days, the immobilization process reduces the activity loss from 59 % to 29 %, while thermal incubation at 50 °C for 2 hrs causes activity loss for the free and immobilized enzymes by 65 % and 30 % respectively. Therefore, the prepared copolymer nanofibers proposed in this work showed promising potential for applications to enzyme immobilization.

Chitosan activated with divinyl sulfone: a new heterofunctional support for enzyme immobilization. Application in the immobilization of lipase B from Candida antarctica.

A novel heterofunctional support for enzyme immobilization, chitosan-divinyl sulfone, was assessed in this study. The activation of chitosan with DVS was carried out at three different pHs (10.0, 12.5 and 14.0) and a Candida antarctica Lipase B (CALB) was selected as the model enzyme. After immobilization, the biocatalysts were incubated under alkaline conditions in a buffer to facilitate the multipoint covalent attachment, followed by incubation in ethylenediamine (EDA) aiming at blocking the remaining reactive groups. The highest thermal stability was obtained when pH 10.0 was used during support activation. These results were shown to be better than those obtained when using glutaraldehyde as the support-activating reagent. Subsequently, the immobilization pH was investigated (5.0, 7.0 and 10.0) prior to alkaline incubation, with the highest enzyme stability levels found at pH 10.0. Finally, the selected biocatalyst was used in the hydrolysis of ethyl hexanoate and presented an activity of 14,520.37 U/g of immobilized lipase at pH 5.0. These results show that chitosan activated with divinyl sulfone is a very promising support for enzyme immobilization and the proposed protocol is able to successfully improve enzyme stability.

Developing of an efficient membrane based downstream processing for oncolytic measles viruses

Xylooligosaccharides display interesting prebiotic effects on human health. The endoxylanase Xys1Δ, from Streptomyces halstedii JM8, was immobilized and stabilized on glyoxyl-agarose beads by multipoint covalent attachment using a novel strategy based on surface coating with a multilayer of polymers. The optimal modification consisted of surface coating with a bilayer formed by a layer of derived dextran polymers and a layer of polyethylenimine. The optimized biocatalyst was 550-fold more stable than one-point covalent immobilized Xys1Δ (at 70 °C, pH 7). This biocatalyst was tested for the production of xylooligosaccharides from soluble xylans from various sources. Hydrolysis of beechwood, wheat straw and corncob xylans was 93% in 4 h, 44% in 5 h and 100% in 1 h, respectively. Maximum values of xylooligosaccharides were found for beechwood at 20.6 mg/mL, wheat at 12.5 mg/mL and corncob at 30.4 mg/mL. The optimized biocatalyst was reused for 15 reaction cycles without affecting its catalytic activity.

Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent

Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.

Preparation of a robust immobilized biocatalyst of β-1,4-endoxylanase by surface coating with polymers for production of xylooligosaccharides from different xylan sources

Xylooligosaccharides display interesting prebiotic effects on human health. The endoxylanase Xys1Δ, from Streptomyces halstedii JM8, was immobilized and stabilized on glyoxyl-agarose beads by multipoint covalent attachment using a novel strategy based on surface coating with a multilayer of polymers. The optimal modification consisted of surface coating with a bilayer formed by a layer of derived dextran polymers and a layer of polyethylenimine. The optimized biocatalyst was 550-fold more stable than one-point covalent immobilized Xys1Δ (at 70 °C, pH 7). This biocatalyst was tested for the production of xylooligosaccharides from soluble xylans from various sources. Hydrolysis of beechwood, wheat straw and corncob xylans was 93% in 4 h, 44% in 5 h and 100% in 1 h, respectively. Maximum values of xylooligosaccharides were found for beechwood at 20.6 mg/mL, wheat at 12.5 mg/mL and corncob at 30.4 mg/mL. The optimized biocatalyst was reused for 15 reaction cycles without affecting its catalytic activity.

Different strategies to immobilize lipase from Geotrichum candidum : Kinetic and thermodynamic studies

Abstract The use of different strategies to immobilize lipase from Geotrichum candidum (GCLI) produced through submerged fermentation was evaluated in order to obtain a biocatalyst with a high stability and activity. Crude and purified GCLI were immobilized by different protocols such as interfacial activation, ionic adsorption and multipoint covalent attachment. The biocatalyst prepared using purified GCLI immobilized on MANAE-agarose exhibited the highest hydrolytic activity (45.40 ± 0.19 IU/g of support) among all the supports used, therefore it was selected for kinetic and thermodynamic studies and the influence of the pH and the protein loading was investigated. The maximum adsorption capacity of 4.51 ± 0.21 mg/g of support was observed at pH 7.0 and 25 °C with a hydrolytic activity of 81.10 ± 2.11 IU/g. GCLI immobilization on MANAE-agarose also reduced the apparent activation energy by 50% compared to the soluble enzyme and increased the amount of product formed in hydrolysis reactions, which was verified by calculating the apparent kinetic parameters Vmax (46.30 IU/mL) and Km (261.77 mmol/L). All thermodynamic apparent parameters determined from thermal stability assays showed that immobilized GCLI allowed a great increase in thermostability. Also, the prepared biocatalyst was able to hydrolyze methyl phenylacetate better than methyl mandelate.