Activity Recovery Yield Research Articles

The synergism of lytic polysaccharide monooxygenases with lichenase and their co-immobilization on silica nanospheres for green conversion of lichen biomass

Enzyme-assisted valorization of lichenan represents a green and sustainable alternative to the conventional chemical industry. The recently discovered lytic polysaccharide monooxygenases (LPMOs) are essential components of state-of-the-art enzyme cocktails for lichenin bioconversion. The LPMOs named SpyTag fused LPMOs (AST) from Chaetomium globosum was functionally expressed in E. coli and exhibited 1.25-fold synergism with lichenase, whereas AST alone produced no detectable reducing sugars. HPLC results further confirm that AST does not alter the endogenous hydrolysis mode of lichenase but rather enhances its hydrolysis efficiency by disrupting the long chain of lichenan and releasing more reducing ends. To the best of our knowledge, this was the first report on the synergistic effect of LPMOs and lichenase, which may have great synergistic potential in the conversion of lichen biomass. Furthermore, a novel strategy for the covalently immobilizing AST and lichenase on silica nanoparticles (SNPs) from the cell lysate in a single step was proposed, which exhibited high activity recovery (82.9%) and high immobilization yield (94.8%). After 12 independent runs, about 67.4 % of the initial activity of the immobilized enzymes was retained. The resulted biocatalyst systems exhibited the green and sustainable strategy in the bioconversion of lichen biomass as well as other diverse polysaccharides.

Pilot-Scale Production of Chito-Oligosaccharides Using an Innovative Recombinant Chitosanase Preparation Approach.

For pilot-scale production of chito-oligosaccharides, it must be cost-effective to prepare designable recombinant chitosanase. Herein, an efficient method for preparing recombinant Bacillus chitosanase from Escherichia coli by elimination of undesirable substances as a precipitate is proposed. After an optimized culture with IPTG (Isopropyl β-d-1-thiogalactopyranoside) induction, the harvested cells were resuspended, disrupted by sonication, divided by selective precipitation, and stored using the same solution conditions. Several factors involved in these procedures, including ion types, ionic concentration, pH, and bacterial cell density, were examined. The optimal conditions were inferred to be pH = 4.5, 300 mM sodium dihydrogen phosphate, and cell density below 1011 cells/mL. Finally, recombinant chitosanase was purified to >70% homogeneity with an activity recovery and enzyme yield of 90% and 106 mg/L, respectively. When 10 L of 5% chitosan was hydrolyzed with 2500 units of chitosanase at ambient temperature for 72 h, hydrolyzed products having molar masses of 833 ± 222 g/mol with multiple degrees of polymerization (chito-dimer to tetramer) were obtained. This work provided an economical and eco-friendly preparation of recombinant chitosanase to scale up the hydrolysis of chitosan towards tailored oligosaccharides in the near future.

Virus-templated magnetic composite hydrogels for surface immobilization of mimic-free-lipase.

Surface immobilization of enzymes on magnetic-recoverable carriers is of great interest and importance for the biocatalysis of relatively large molecules. In this work, the nanosized amino-rich filamentous M13 virus, a versatile biological scaffold, was applied as the unique soft backbone for lipase immobilization. Based on the structure and capsid proteins of M13 phages, the magnetic-recoverable mimic-free-lipases (MFLs) composed of the M13 hydrogels and magnetic particles were developed in two designs. In the first design, nanosized wild M13 phages were crosslinked into a phage hydrogel through the N-terminals of pVIII peptides while NH2-Fe3O4 magnetic nanoparticles (MNPs) were attached to the M13 virus through glutaraldehyde, forming the M13-(NH2-Fe3O4) magnetic phage hydrogel. In the second design, special M13 with Fe3O4 affinity pIII-peptide (FAP-M13) was biopanned for strongly binding towards bare Fe3O4 with the "hook"-like pIII-peptide (N-LPLSTQH-C). TEM observation confirmed the direct grasp of FAP-M13 on bare Fe3O4, forming the magnetic (FAP-M13)-Fe3O4 virus hydrogel. Lipases were uniformly anchored on the phage surface of nanoscale by crosslinking with the N-terminals of pVIII peptides, and then lipase@M13-(NH2-Fe3O4) and lipase@(FAP-M13)-Fe3O4 MFLs were constructed. For both MFLs, high activity recovery yield (>95%) and efficient magnetic separation were characterized. Significantly reduced MNP-usage-amount and enhanced lipase-loading-amount both by about 40 folds were obtained, compared with the conventional NH2-Fe3O4 carriers. The quantified Km and Vmax/Km values were almost equal to those of the free lipases, verifying free-enzyme-mimicking features of the MFLs. High pH-tolerance, wide temperature adaptability, enhanced thermal stability and stable magnetic separation capability of both MFLs were also observed. In particular, the (FAP-M13)-Fe3O4 magnetic virus hydrogel simply using bare Fe3O4 MNPs would be more convenient and economical in the scaled-up biocatalysis.

Refolding with Simultaneous Purification of Recombinant Serratia marcescens Lipase by One-Step Ultrasonication Process.

A new lipase from Serratia marcescens SRICI-01 (Trx-SmL) was successfully overexpressed in Escherichia coli with thioredoxin (Trx) fusion tag. Intriguingly, the concentration of potassium phosphate buffer (KPB) showed significant impact on the aggregation state of Trx-SmL during ultrasonic disruption. The proportion of inclusion bodies increased dramatically with the increase of KPB concentration from almost completely soluble in 10mM KPB to insoluble in 200mM KPB. Based on this new finding, a novel method for refolding and purification of recombinant Trx-SmL was developed by one-step ultrasonication. The Trx-SmL was firstly precipitated in 200mM KPB, washed for three times, and subsequently subjected to ultrasonic process in 10mM KPB where refolding and purification occurred simultaneously. This established method was proved to be a straightforward, economical, and efficient purification approach to facilely obtain recombinant Trx-SmL protein with high purity (> 90%) and activity recovery yield (> 80%) from cell lysates. The application potential of the purified fusion Trx-SmL was further demonstrated by kinetic bioresolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] producing optically pure (-)-MPGM, a key intermediate for diltiazem, with an overall yield of 41.5% and ee of 99%.

Enhanced Activity of Immobilized Lipase by Phosphonium-Based Ionic Liquids Used in the Support Preparation and Immobilization Process

Biocatalysis has significant advantages over chemocatalysis in the context of sustainability since environmentally compatible catalysts (enzymes) and mild reaction conditions are used. However, enzymes are labile macromolecules, and strategies such as the addition of cosolvents or additives or their immobilization in solid supports are the target of intensive investigation to improve the catalytic performance and recyclability potential. In this work, we investigated the use of phosphonium-based ionic liquids (ILs) on the activity of immobilized Burkholderia cepacia lipase (BCL) by two approaches: (i) use of ILs to prepare silica used as support and (ii) use of ILs during the enzyme immobilization process. Several phosphonium-based ILs were investigated, allowing us to address the cation alkyl side chain and anion nature effects. The enzymatic performance depends on the IL employed to prepare silica, with a positive effect observed when employing ILs comprising cations with longer alkyl side chains and more hydrophobic anions. The best identified IL, namely, [P666(14)][NTf2], allows for a relative activity of 209.8% and immobilization yield of 77.3% and is capable of being recycled eight times (keeping more than 50% of the enzyme initial activity). When ILs are added during the BCL immobilization process, similar negative and beneficial effects are observed. With IL [P666(14)][NTf2], the immobilized biocatalyst has a relative activity of 322.7%, a total activity recovery yield of 91.1%, and can be recycled 17 times (down to 50% of the enzyme initial activity). Finally, both approaches were combined; i.e., IL [P666(14)][NTf2] was used both in the material preparation and immobilization process of the enzyme. This strategy allows for an increase in the relative activity up to 231%, an immobilization yield of 98%, and an increase of 9% in the enzyme relative activity. Although BCL activity is not significantly enhanced by this strategy, the combined use of the IL in silica preparation and during the enzyme immobilization process increased the recyclability potential of the immobilized biocatalyst material, capable of being recycled 26 times, while keeping more than 50% of the enzyme initial activity (equivalent to a half-life of 13 h). The results obtained in this work open the path to the efficient use of ILs, and particularly of the less explored phosphonium-based ILs, in the biocatalysis field.

Foam fractionation for enhancing silica gel adsorption of urokinase from human urine

AbstractIn this work, foam fractionation (FF), a cost‐effective method, was used to enhance the silica gel adsorption (SGA) of urokinase from human urine. The analysis of how FF enhanced the adsorption/desorption of urokinase onto silica gel was conducted. The results show that FF increased both the adsorption rate of urokinase onto silica gel and the activity concentration of urokinase adsorbed at silica gel by pre‐enriching urokinase from urine. It also reduced the activity loss of urokinase in the desorption process. Then, a coupled technology of FF and SGA was developed. The effects of pH, superficial air flow rate, beta‐cyclodextrin concentration, and amount of silica gel added on activity recovery yield and purification fold of urokinase were studied. At pH 7.5, superficial air flow rate 1.70 mm/s, beta‐cyclodextrin concentration 0.2 g/L, and amount of silica gel added 0.9%, the activity recovery and purification fold of urokinase reached 89.5% and 56.8 in the coupled technology, which were 25.3% and 79.2% higher than those obtained by SGA alone, respectively. Furthermore, the amount of silica gel added in the coupled technology was 40.0% lower than that in SGA alone. Therefore, FF effectively enhanced the SGA of urokinase from human urine.

Enhancement of n-3 polyunsaturated fatty acid glycerides in Sardine oil by a bioimprinted cross-linked Candida rugosa lipase.

Considering the advantages of bioimprinting and carrier free immobilization, cross-linked enzyme aggregates (CLEA) were prepared by using bioimprinted Candida rugosa lipase (CRL) with Bovine serum albumin (BSA), Polyethyleneimine and glutaraldehyde. Effect of various factors such as CRL-Oleic acid ratio, CRL-BSA ratio, CRL- Polyethyleneimine ratio, glutaraldehyde loading, cross-linking time etc., on lipase activity recovery and aggregate yield were studied and optimized. This immobilized lipase (CRL-CLEA) was used for the selective hydrolysis of ester linkages of non-PUFA glycerides, with an aim to concentrate EPA and DHA glycerides in the Sardine oil. Imprinting with oleic acid in the presence of ethanol and Tween 60, and further immobilization with co-aggregates and cross-linking agent showed 10.4 times higher degree of hydrolysis compared to free enzyme. As result, 2.83-fold increase of n-3 PUFA content in deacidified oil was obtained by using CRL-CLEA. The resultant oil had negligible di- and triglycerides content, proving higher efficiency in hydrolysing ester bonds of fatty acids, other than n-3 PUFA. Reusability studies showed CRL-CLEA could be reused up to 5 runs without a substantial reduction in its performance. Improvement in degree of hydrolysis, thermostability, efficiency of hydrolysis and reusability were achieved due to bioimprinting and subsequent immobilization of CRL in the form of CLEA.

Immobilization of laccase onto porous polyvinyl alcohol/halloysite hybrid beads for dye removal.

Laccase was immobilized in polyvinyl alcohol beads containing halloysite nanotubes (PVA/HNTs) to improve the stability and reusability of enzyme. The porous structure of PVA/HNTs beads facilitates the entrapment of enzyme and prevents the leaching of immobilized laccase as well. Halloysite nanotubes act as bridge to connect the adjacent pores, facilitating the electron transfer and enhancing the mechanical properties. PVA/HNTs beads have high laccase immobilization capacity (237.02 mg/g) and activity recovery yield (79.15%), indicating it can be used as potential support for laccase immobilization. Compared with free laccase, the immobilized laccase on hybrid beads exhibits enhanced pH tolerance (even at pH 8.0), good thermal stability (57.5% of the initial activity can be maintained at 75 °C), and excellent storage stability (81.17% of enzyme activity could be retained after storage at 4 °C for 5 weeks compared with that for free enzyme of 60%). Also, the removal efficiency for reactive blue can reach as high as 93.41% in the presence of redox mediator 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate), in which adsorption and degradation exist simultaneously. The remarkable pH tolerance, thermal and storage stability, and reuse ability imply potential application of porous PVA/HNTs immobilized enzyme in environmental fields.

Bio-based degradation of emerging endocrine-disrupting and dye-based pollutants using cross-linked enzyme aggregates.

In this study, manganese peroxidase (MnP) from an indigenous white-rot fungus Ganoderma lucidum IBL-05 was insolubilized in the form of cross-linked enzyme aggregates (CLEAs) using various aggregating agents, i.e., acetone, ammonium sulfate, ethanol, 2-propanol, and tert-butanol, followed by glutaraldehyde (GA) cross-linking. The precipitant type, MnP, and GA concentrations affected the CLEAs activity recovery and aggregation yield. Among precipitants used, acetone appeared to be the most efficient aggregation agent, providing the highest activity recovery and aggregation yield of 31.26 and 73.46%, respectively. Optimal cross-linking was noticed using 2.0% (v/v) GA and 8:1 (v/v) MnP to GA ratio at 3.0h cross-linking time under continuous agitation at 4°C. The highest recovered activity and aggregation yield were determined to be 47.57 and 81.26%, respectively. The MnP-CLEAs, thus synthesized, were tested to investigate their bio-catalytic capacity for removing two known endocrine-disrupting chemicals (EDCs), e.g., nonylphenol and triclosan in a packed bed reactor system. The insolubilized MnP efficiently catalyzed the biodegradation of both EDCs, transforming over 80% in the presence of MnP-based system. A maximal of 100% decolorization was recorded for Sitara textile (SIT-based) effluent, followed by 95.5% for Crescent textile (CRT-based) effluent, 88.0% for K&N textile (KIT-based) effluent, and 84.2% for Nishat textile (NIT-based) effluent.

Effect of silica coating on Fe3O4 magnetic nanoparticles for lipase immobilization and their application for biodiesel production

The scope of this study was to prepare different organosilane-modified Fe3O4@SiO2 core magnetic nanocomposites with immobilized lipase and explore their potential application in biodiesel field. Fe3O4 magnetic nanoparticles prepared by co-precipitation method were coated with various ratios of SiO2 as per Stöber method and further functionalized by different organosilane compounds APTES (3-aminopropyltriethoxysilane) and MPTMS (3-mercaptopropyltrimethoxysilane). Functionalized Fe3O4@SiO2 magnetic nanoparticles were immobilized with free lipase NS81006 by glutaraldehyde cross-linking reagent. The functional groups, structure, morphology and magnetic susceptibility of synthesized and modified Fe3O4@SiO2 magnetic nanoparticles were characterized by FTIR, XRD, SEM, TEM, and VSM techniques. The immobilization efficiency and activity recovery were reduced by increasing the ratio of silica coating on Fe3O4. Maximum activity recovery (84% by APTES, 83% by MPTMS) and biodiesel yield (>90%) were obtained by lipase immobilized on Fe3O4@SiO2 support when the Fe3O4 and SiO2 (TEOS) ratio was low (1:0.25).

Protic ionic liquids influence on immobilization of LipaseBurkholderia cepaciaon hybrid supports

BACKGROUND This work aims to assess the use of protic ionic liquids (PIL), propanoate, butanoate and pentanoate of N-methylmonoetanolamine (C3, C4 and C5), in the preparation of hybrid supports made of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) and silica for the immobilization of lipase of Burkholderia cepacia (BCL) by covalent binding. For immobilized biocatalysts (IBs), the performance was determined for immobilization and characterization (pH, temperature, stability thermal, operational and in solvents), with the best conditions being defined. For the supports and efficient IB, the morphological and physicochemical characterization (MEV, BET, FTIR and TGA) was evaluated. RESULTS For preparation of the hybrid support, the experiment using 3 g of PHBV presented a higher percentage of mass, around 50% recovery of the support in the granulometry used. The IBs total activity recovery yield (Ya) in hybrid supports modified with ionic liquids IB-hybrid-C3 (103%), IB-hybrid-C4 (120%) and IB-hybrid-C5 (133%) were above the results reported for the control (90%), with IB-hybrid-C5 being the most efficient, and presenting better thermal and operational stability. CONCLUSION Burkholderia cepacia lipase was immobilized successfully in a hybrid support (PHBV and silica) modified with protic ionic liquid by covalent binding, resulting in an improvement in the morphological and physicochemical characteristics, and consequently with greater Ya and good thermal and operational stability. © 2016 Society of Chemical Industry

Evaluation of divinylsulfone activated agarose to immobilize lipases and to tune their catalytic properties

Divinylsulfone (DVS) activated agarose beads have been used to immobilize several lipases, those from Pseudomonas flourescens, Rhizomucor miehei, and Thermomyces lanuginosus (TLL), as well as the artificial chimeric phospholipase Lecitase Ultra. The best results in terms of activity recovery and immobilization yield were achieved using TLL. This enzyme could be immobilized at pH from 5 (with poor yield) to pH 10 (with 100% yield). The incubation of the immobilized enzymes for 72h at pH 10 before the blocking step (using ethylenediamine) improved the enzyme stability whatever the immobilization pH value, but the stabilization achieved in each case depended on the immobilization pH value, and also on the inactivation conditions.The enzyme activities versus different substrates were very dependent on the immobilization protocol and the conditions of activity determination. That way, TLL immobilized at pH 5 on DVS activated support was the most active versus methyl mandelate, even more active than TLL immobilized on octyl-agarose (between 3 and 7 fold factors depending on the pH of measure). Using ethyl hexanoate the most active preparation was the octyl-TLL preparation. The most active among the enzymes immobilized in DVS-activated supports was that just immobilized at pH 5 if the activity was determined at pH 7 or 8.5, while at pH 5 the most active was that enzyme but after incubation at pH 10.The results show that this support may be very useful for tuning lipase properties just by altering the first immobilization pH value, and that the further incubation at pH 10 improved enzyme stability, and in some cases, even increased activity.

Preparation and characterization of cross-linked enzyme aggregates (CLEAs) of Brassica rapa peroxidase

Peroxidase from white turnip (Brassica rapa) was immobilized as cross-linked enzyme aggregate (CLEA) via solvent precipitation and glutaraldehyde cross-linking. The nature of the precipitation solvent, enzyme and glutaraldehyde concentrations affected the formation and the activity of the enzyme aggregate. The aggregates resulting from cross-linking with glutaraldehyde produced insoluble catalytically active cross-linked enzyme aggregates. Acetone was selected as the more efficient solvent, giving the highest activity recovery (28%) and aggregation yield (78%). Immobilization was conducted for 3 h under agitation at 4 °C. Optimal conditions were determined as 2% (v/v) glutaraldehyde and 9:1 (v/v) enzyme to glutaraldehyde ratio. The highest activity recovery and aggregation yield were 42.5% and 82% respectively. CLEAs with enhanced thermal and acidic condition stabilities were formed when compared to the free enzyme. The cross-linked enzyme aggregates exhibited optimal pH and temperature in the range 6–7 °C and 40 °C, respectively. CLEAs of Brassica rapa peroxidase exhibited good stability in nonpolar organic solvents and they could be stored in pH 7 buffer with no activity loss at 4 °C for more than three months.

Immobilization of Candida rugosa lipase onto an eco-friendly support in the presence of ionic liquid.

Candida rugosa lipase (CRL) was immobilized on an eco-friendly support poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV), by physical adsorption, using different ionic liquids (ILs) as immobilization additives. This was to investigate the influence of cationic core ([C4mpy]Cl, [C4min]Cl), of anions ([C4min]Cl, [C4min]N(CN)2, [C4min]Tf2N), and of cation chain length ([C2min]Tf2N, [C4min]Tf2N) in the immobilization process. The immobilized biocatalysts (IB) were characterized with respect to the morphological, physico-chemical properties, total activity recovery yield (Ya), and biochemical properties of more efficient IB were evaluated. Initially, it was found that the change of cationic core did not influence in Ya compared to the control. With change of anions, it was seen that the best result was obtained for the more hydrophobic anion (Tf2N), and finally increasing the cation chain length increased Ya. IB most efficient with [C4min]Tf2N obtained 78% of Ya, more than twice the control value (30%) and a considerable enhancement of operational stability compared with the control.

Screening of immobilization method in aerogel matrix in the presence of protic ionic liquid

Aerogel and xerogel are formed via hydrolysis and polycondensation reactions of silica precursors, such as tetraethylorthosilicate (TEOS), always careful not to cause collapse, reduction in surface area and pore size. Several studies shown the use sol-gel entrapment possesses a number of desirable attributes, as the enzyme is physically entrapped in a rigid glass framework that permitted stabilization of the enzymes, terciary structure owing to the tight gel network. In this study, have focused on the screening of immobilization method in aerogel matrix in the presence of protic ionic liquid (PIL) with evaluation catalytic efficiency and operational stability. The novel mesoporous silica supports (aerogels) obtained by the sol-gel technique was used to immobilize commercial Burkholderia cepacia (BC) lipase by physical adsorption, covalent binding and encapsulation in the absence and presence de protic ionic liquid (N-methylmonoethanolamine pentanoate - C5). Catalytic efficiency was determined in the analyses of hydrolytic activities were carried out on the lipase loading solution and immobilized enzyme to determine the total activity recovery yield, Ya (%). For physical adsorption the recovery of activity with PIL was 82.95% (ADSLI) and in the absence 70.31% (ADS). Operational stability of the enzyme has also been examined and obtained similar values a half-life of 0.73 h to ADSLI and 0.88 h for ADS occurs with 2 batches. And other immobilized technique, covalent binding the results showed that in aerogels supports in the presence of C5 obtained 69.13% (CBLI) of total activity recovery yield and 39.11% (CB). Under such conditions, the operational stability tests indicated that a small enzyme deactivation occurs after 15 batches, revealing a biocatalyst half-life of 7.5 h for CBLI. For encapsulation, the recovery of activity with PIL (ENLI) was 45% and 37% in the absence PIL (EN). ENLI exhibited a half life of more than 23 batches with 70% of its activity remaining, with around 11.5 h. And EN the half-life was 6.5 hours, totaling 13 batches. Therefore, ADSLI presented good efficiency with total activity recovery yield but for operational stability ENLI showed the best between immobilization methods studied. Hence, it is rather appropriate for application in hydrolytic processes.

Horseradish peroxidase encapsulation in alginate microspheres in the presence of imidazolium ionic liquids

Biocatalysis with free enzymes may not be economically viable due to the complexity of their recovery in the reaction medium. With the objective of enabling the total activity recovery yield of biocatalyst and to improve the operational characteristics, have been applied enzyme immobilization techniques. The main interest in the enzyme immobilization is to obtain a catalyst whose activity and stability is not affected during the process when compared to the free enzyme. The objective of this work was to immobilize horseradish peroxidase (HRP) by encapsulation method in alginate microspheres in the presence of imidazolium ionic liquid (ILs). The enzyme encapsulation was carried with the enzyme incorporation in an aqueous solution of sodium alginate to 0.05 % (w / v). The influence of the enzyme loading was studied in the range of 0.0406-0.65 mg HRP / g alginate. Alginate microspheres were obtained by dripping in calcium chloride solution (1M) according to literature [1]. The HRP immobilization in the presence of ILs was conducted using the enzyme loading more appropriate, incorporating in the immobilization medium 1% (w/v) of IL. The ILs studied as additives were: [C4mim]TF2N; [C4mim]BF4; [C4mim]HSO4; [C4mim]Ac e [C4mim]PF6. The activity of free and immobilized enzyme was verified by colorimetric method based on the change of absorbance at 470 nm due to the formation of product in guaiacol oxidation for three minutes. The results obtained for biocatalysts immobilized by encapsulation suggest that the use of IL as an additive in the immobilization process causes a significant increase in the total activity recovery yield (RA) HRP (increase from 5.2 to 23.5 %). The results for the HRP immobilization showed that RA was 23.5% and 20.40% for the LIs [C4mim] HSO4 and [C4mim]TF2N, respectively. [C4mim]Ac provided RA of 13.3 %, while the biocatalyst with [C4mim] PF6 showed RA of 17.3 %. The [C4mim] BF4 showed the lowest performance as an additive among the ILs studied. According to Diego et al.(2009) [2] there is not have a rule to predict the behavior of immobilized enzymes in the presence of ILs. The results obtained for biocatalysts immobilized by encapsulation in alginate microspheres show that the use of ionic liquids in this process had a positive effect on the activity of the immobilized biocatalyst. Additional studies in the structural characteristic of the immobilized biocatalyst are being conducted to evaluate the influence of ILs in the supporting structure.

Use of ionic liquids as additives for the immobilization of lipase from Bacillus sp.

AbstractBACKGROUNDIonic liquids (ILs) have been used as additives to immobilise lipase from a new source of Bacillus sp. (ITP‐001) by the physical adsorption method supporting green poly(3‐hydroxybutyrate‐co‐hydroxyvalerate) (PHBV) in order to evaluate the influence of the cationic core ([C4mpy]Cl, [C4min]Cl), of anions ([C4min]Cl, [C4min]N(CN)2, [C4min]Tf2N) and the cation chain length ([C2min]Tf2N, [C4min]Tf2N)) in the immobilization process. The immobilized biocatalysts (IB) were characterized with regard to morphological and physico‐chemical properties, and total activity recovery yield (Ya) and biochemical properties of more efficient IB were evaluated.RESULTTotal activity recovery yield (Ya) for the immobilized biocatalysts employing LI as additives always resulted in higher values compared with the control. The most efficient was using the more hydrophobic IL [C4min]Tf2N, which represents a 2‐fold greater increase in the recovery of enzymatic activity. It retained more than 73% of its original activity after 20 reuses compared with the control, which, after ten cycles, retained only 33% of its original activity.CONCLUSIONThe anions and the cation chain length of ionic liquids have a strong influence on the immobilization process. The results for biocatalyst immobilized with [C4min]Tf2N showed an increase in total activity recovery yield and considerable improvement in operational stability. © 2014 Society of Chemical Industry

Use of polyethylene glycol in the process of sol–gel encapsulation of Burkholderia cepacia lipase

Lipases from Burkholderia cepacia were encapsulated using polyethylene glycol (PEG, Mw 1500) at various concentrations (0.5–3.0 %, w/v) as an additive during the sol–gel immobilisation process. Matrixes immobilized in the presence and absences of additives were characterized by thermal analysis [thermogravimetric (TG) and differential scanning calorimetry (DSC)], scanning electron microscopy (SEM), enzymatic activity, and total activity recovery yield (Ya). The addition of PEG increased the activity values, with Ya just above 1.0 % (w/v) in the presence of PEG. The additional of 1.0 % (w/v) PEG increased enzyme activity from 33.98 to 89.91 U g−1 and the values of recovery yield were 43.0–91.4 %, compared to values of the samples without PEG. PEG enhanced the thermal stability of the matrix structure in the temperature range 50–200 °C, as confirmed by TG and DSC analyses. This was influenced by the presence of water bound to the matrix. The SEM micrographs clearly showed an increase in the number of deposits on the material surface, producing matrices with greater porosity.

IMMOBILIZATION OF LIPASE BY ENCAPSULATION IN SILICA AEROGEL

Lipase from Burkholderia cepacia was immobilized in a silica matrix and dried in high pressure carbon dioxide media (aerogel). The protic ionic liquid (PIL) was used in the immobilization process by encapsulation. The objective of this work was to evaluate the influence of the drying technique using supercritical carbon dioxide in biocatalysts obtained through the sol-gel technique by evaluating temperature and pressure and, after selecting the best drying conditions, to investigate the application of the technique for the biocatalyst using ionic liquid as an additive in the immobilization process. The results for immobilized biocatalysts showed that the best conditions of pressure and temperature were 100 bar and 25 oC, respectively, giving a total activity recovery yield of 37.27% without PIL (EN) and 44.23% with PIL (ENLI). The operational stability of the biocatalysts showed a half-life of 11.4 h for ENLI and 6 h for EN. Therefore, solvent extraction using supercritical CO2, besides shortening drying time, offers little resistance to the immobilization of lipases, since their macropores provide ample room for their molecules. The use of the ionic liquid as an additive in the process studied for the immobilization of enzymes produced attractive yields for immobilization and therefore has potential for industrial applications in the hydrolysis of vegetable oils.

Refolding of recombinant N-acetyl- d-glucosamine 2-epimerase by a fed-batch process

N-Acetyl- d-glucosamine 2-epimerase is one of the key enzymes for the enzymatic synthesis of N-acetylneuraminic acid, a sialic acid and a critical precursor for the synthesis of some antiviral agents. Overexpression of the recombinant epimerase in Escherichia coli led to the formation of protein inclusion bodies. Refolding of guanidine HCl-solubilized protein by direct dilution resulted in the formation of soluble oligomers, mediated probably by hydrophobic interactions. The extent of aggregation of protein subunits into inactive oligomers could be efficiently reduced by employing fed-batch refolding process, in which the solubilized proteins were added continuously at a pre-determined rate. The yields of soluble proteins decreased with the feeding rates. The addition of glutathione into refolding buffer at certain stage of the refolding process could enhance the yield of soluble proteins more than two-fold, possibly by resolving the inadvertently formed disulfide bridges among the protein subunits that contain 10 cysteine residues each. Folding aids such as l-arginine and glycerol were found effective in increasing the yield of soluble proteins and the specific activity of the refolded proteins. Under the optimal condition, a specific activity of 0.47 IU/mg was obtained with an activity recovery yield of ca. 30%. The specific activity of the refolded proteins was significantly lower than that of the native protein, 1.23 IU/mg, indicating that more information concerning the 3D structure of native N-acetyl- d-glucosamine 2-epimerase and the role of its cofactor, ATP, for catalytic activity is needed for the development of a more efficient refolding process.