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Vinyl sulfone-activated silica for efficient covalent immobilization of alkaline unstable enzymes: Application to levansucrase for fructooligosaccharide synthesis
RSC Advances
Abstract
Most methodologies for covalent immobilization of enzymes usually take place at high pH values to enhance the nucleophilicity of protein reactive residues; however, many enzymes inactivate during the immobilization process due to their intrinsic instability at alkaline pH values. Vinyl sulfone (VS)-activated carriers may react with several protein side-chains at neutral pHs. In this work, levansucrase-an alkaline unstable enzyme of technological interest because it forms fructooligosaccharides (FOS) and levan from sucrose-was covalently attached to VS-activated silica at pH 7.0 in a short time (5 h). Theoretical immobilization yields were close to 95% but the apparent activity did not surpass 25%, probably due to random attachment with unproductive orientations and rigidification of the enzyme structure. Due to diffusional hindrance and/or local microenvironmental effects caused by the silica surface, the immobilized levansucrase was unable to produce levan but synthesized a similar amount of FOS than the free enzyme [95 g L�1 in 28 h, with a major contribution of FOS of the b(2 / 1) type]. The VS-activated biocatalysts showed a notable operational stability in batch reactors.
... The change of the IFOs pattern produced by R483A-LrInu CLEAs suggested that they may provide a microenvironment for enzyme molecules, which allowed only specific acceptors to diffuse into the enzyme active site, increasing product specificity. This finding was reported for other immobilized fructosyltransferases, such as levansucrase immobilized on vinyl sulfone-activated silica, which selectively produced levan-type fructooligosaccharides (LFOSs), while soluble levansucrase produced both LFOSs and levan [40]. ...
... The change of the IFOs pattern produced by R483A-LrInu CLEAs suggested that they may provide a microenvironment for enzyme molecules, which allowed only specific acceptors to diffuse into the enzyme active site, increasing product specificity. This finding was reported for other immobilized fructosyltransferases, such as levansucrase immobilized on vinyl sulfoneactivated silica, which selectively produced levan-type fructooligosaccharides (LFOSs), while soluble levansucrase produced both LFOSs and levan [40]. High performance liquid chromatography with pulsed amperometric detector (HPAEC-PAD) was also performed to confirm the findings of TLC analysis. ...
... The reduction of CLEA activity in early cycles might result from the more compact nature of CLEAs or desorption of non-covalently bound enzyme molecules after recycling of the biocatalyst. This phenomenon was also found in other covalent-immobilized enzymes, such as levansucrase immobilized on vinyl sulfone-activated silica [40] and inulosucrase immobilized on core-shell chitosan beads [25]. ...
Fructooligosaccharides are well-known carbohydrate molecules that exhibit good probiotic activity and are widely used as sweeteners. Inulin-type fructooligosaccharides (IFOs) can be synthesized from sucrose using inulosucrase. In this study, cross-linked enzyme aggregates (CLEAs) of Lactobacillus reuteri 121 inulosucrase (R483A-LrInu) were prepared and used as a biocatalyst for IFOs production. Under optimum conditions, R483A-LrInu CLEAs retained 42% of original inulosucrase activity. Biochemical characterization demonstrated that the optimum pH of inulosucrase changed from 5 to 4 after immobilization, while the optimum temperature was unchanged. Furthermore, the pH stability and thermostability of the R483A-LrInu CLEAs was significantly improved. IFOs product characterization indicated that the product specificity of the enzyme was impacted by CLEA generation, producing a narrower range of IFOs than the soluble enzyme. In addition, the R483A-LrInu CLEAs showed operational stability in the batch synthesis of IFOs.
... According to the literature [2,4,42], the DVS compound likely facilitated both surface activation of the enzyme and covalent bonding with more functional groups than the glyoxyl compound, resulting in a higher immobilization percentage. Additionally, DVS-activated supports exhibit reactivity across a broad spectrum of pH values, as demonstrated by Santos-Moriano et al. in 2016 [43]. Moreover, these supports feature a more extended spacer arm compared to glyoxyl, which enhances the interaction with protein groups. ...
Lipases have catalytic capacity in various processes such as hydrolysis. Those derived from plant sources, such as linseed, offer an economical alternative. The immobilization process facilitates the recovery and reuse of lipase, providing advantages such as resistance to high temperatures and difficulties in recovering and reusing free lipases, which makes product separation difficult. This study presents the immobilization of lipases extracted from flax seeds on octylfunctional hydrophobic supports. Additionally, the thermal stability of the derived products was evaluated in comparison with freely soluble lipase. The lipase exhibited a strong affinity for the evaluated heterofunctional hydrophobic supports, with DVS-activated octylagarose emerging as the most efficient method for immobilization, thus increasing catalytic activity upon resuspension. Furthermore, the octylagarose derivative demonstrated a notable increase in thermal stability. The main results of the study include that the soluble enzyme showed greater activity after 24 h, regardless of the temperature evaluated. The benzamide extract showed greater thermal stability, and all supports evaluated showed greater activity than the soluble enzyme after immobilization. Notably, lipase immobilized on octyl glyoxyl agarose showed the highest activity, demonstrated stability for 840 h at 60 °C, and had a half-life of 1242 h. Furthermore, the lipase immobilized in octyl glyoxyl agarose showed a stabilization factor approximately nine times greater than the free enzyme. These results suggest that immobilization, probably achieved through interfacial activation and multipoint covalent bonds, prevented the release of the enzyme into the medium with increasing temperature. This study thus highlights the significant potential of immobilizing flaxseed-derived lipase.
Graphical Abstract
... Tiss®-IMAC-Fe and Tiss®-NH 2 membranes had lower, yet the same immobilization yields, while Tiss®-Link resulted in the lowest immobilization yield for N-His 6 -ATA-wt. Tiss®-Link with vinyl sulfone groups on its surface facilitated covalent binding [36], while Tiss®-NH 2 membrane enabled electrostatic interaction with negatively charged amino acid residues on the enzyme surface due to its amine groups [25]. In contrast, Tiss®-IMAC membranes have metal ions (Cu 2+ or Fe 3+ ) embedded on polymeric nanofibers and support coordinative binding of His 6 tag [38], Further in operando testing of the immobilized enzyme was performed at various flow rates of inlet substrate solution yielding residence times from 0.5 min to 4 min. ...
... Although bare silica supports have been used for the immobilization enzymes [31,32], the low thermal and operational stabilities may be observed due to the enzyme desorption. To obtain robust biocatalysts, the surface of silica supports is generally modified [33][34][35] and this have been achieved using trimetoxysilane molecules bearing various functional groups, such as primary amino [12], glycidoxypropyltrimethoxylsilane [36], carboxylic acids [37], while activation with vinyl sulfone [38] or glutaraldehyde [39] has been also assayed. ...
The immobilization of xylanase from Trichoderma longibrachiatum via ion exchange on 3-propylsulfonic acid (Xyl/Si-SO3) and 3-aminopropyl (Xyl/Si-NH3) functionalized silica gel and covalently on 3-carboxypropyl (Xyl/Si−COOH) and silica gel functionalized with 3-aminopropyl and modified with glutaraldehyde (Xyl/Si-Glu) were performed and the prepared biocatalysts were used for clarification of orange juice. The supports and immobilized biocatalysts were characterized by scanning electron microscopy, and thermal gravimetric analysis. Free and all immobilized xylanase samples showed maximal catalytic activity at pH 6.0. The free xylanase and Xyl/Si-NH3 had a maximal catalytic activity at 50 °C, while Xyl/Si-Glu, Xyl/Si−COOH and Xyl/Si-SO3 exhibited their maximal catalytic activities at 60 °C. The Xyl/Si-Glu showed 4.2-fold higher thermal stability than the free xylanase at 50 °C and pH 6.0. The clarity of orange juice was increased by 74.7, 73.6, 59.3, 60.3, and 78.8 % respectively by using free xylanase, Xyl/Si-Glu, Xyl/Si−COOH, Xyl/Si-SO3 and Xyl/Si-NH3 at 50 °C after 180 min treatment time. The reusability studies showed that Xyl/Si-Glu, Xyl/Si−COOH, Xyl/Si-SO3 and Xyl/Si-NH3 retained 77, 71, 11 and 16 %, respectively, of their initial activities after 10 reuses. These results show that Xyl/Si-Glu and Xyl/Si−COOH are better potential candidates than Xyl/Si-SO3 and Xyl/Si-NH3 in fruit juice clarifying processes.
... Supports activated with divinyl sulfone have been used for a relatively long time for immobilization of different compounds and biomacromolecules (Arana-Peña et al., 2020a;Begara-Morales et al., 2013;Bryjak et al., 2008;Li et al., 2019;Medina-Castillo et al., 2012;Ortega-Muñoz et al., 2010;Santos-Moriano et al., 2016) (Fig. 34). However, only very recently they have been proposed as a suitable protocol for enzyme stabilization via multipoint covalent attachment (dos Santos et al., 2015a(dos Santos et al., , 2015b(dos Santos et al., , 2015c(dos Santos et al., , 2015d, with stabilization values sometime surpassing those obtained using glyoxyl supports (e.g., using chymotrypsin, 4-5 fold more stable than glyoxyl preparations (dos Santos et al., 2015a), that was 60,000 folds more stable than the free enzyme (Guisán et al., 1991)). ...
The use of enzymes in industrial processes requires the improvement of their features in many instances. Enzyme immobilization, a requirement to facilitate the recovery and reuse of these water-soluble catalysts, is one of the tools that researchers may utilize to improve many of their properties. This review is focused on how enzyme immobilization may improve enzyme stability. Starting from the stabilization effects that an enzyme may experience by the mere fact of being inside a solid particle, we detail other possibilities to stabilize enzymes: generation of favorable enzyme environments, prevention of enzyme subunit dissociation in multimeric enzymes, generation of more stable enzyme conformations, or enzyme rigidification via multipoint covalent attachment. In this last point, we will discuss the features of an “ideal” immobilization protocol to maximize the intensity of the enzyme-support interactions. The most interesting active groups in the support (glutaraldehyde, epoxide, glyoxyl and vinyl sulfone) will be also presented, discussing their main properties and uses. Some instances in which the number of enzyme-support bonds is not directly related to a higher stabilization will be also presented. Finally, the possibility of coupling site-directed mutagenesis or chemical modification to get a more intense multipoint covalent immobilization will be discussed.
... To reach this, the support, the reactive groups in enzyme and support and the immobilization protocol must be carefully selected and designed (Barbosa et al., 2013; J. C. S. D. Dos Santos et al., 2015aSantos et al., , 2015bSantos et al., , 2015cSantos et al., , 2015d. Among the most efficient active groups on the support to get this multipoint covalent attachment, glutaraldehyde Migneault et al., 2004), epoxide (Hilterhaus et al., 2008;Katchalski-Katzir and Kraemer, 2000;Mateo et al., 2007a), vinyl sulfone (Dos Santos et al., 2015aSantos et al., , 2015bSantos et al., , 2015cSantos et al., , 2015dOrtega-Muñoz et al., 2010;Santos-Moriano et al., 2016) and glyoxyl (Mateo et al., 2006) stand up. ...
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.
... Immobilization of levansucrases on some carriers resemble the in vivo microenvironment in which cell-wall anchored proteins are imbibed, thus providing information on how levan is produced in Nature (Hill, Karboune, & Mateo, 2017). In some cases protein immobilization results in modification of levan size and distribution (Chambert & Petitglatron, 1993;Esawy, Mahmoud, & Fattah, 2008;Santos-Moriano et al., 2016), and often improves the transfructosylation activity (Chiang, Wang, Chen, & Chao, 2009;Hill, Karboune, & Mateo, 2016). A Bs-SacB hybrid construction in which additional binding domains (GW repeats and/or a transitional domain) from Inulosucrase, (a fructansucrase from Leuconostoc citreum) were fused to Bs-SacB, resulted in an enzyme with a much higher transferase activity. ...
The physicochemical properties and biological activity of levan, a generic term given to oligo- and polysaccharides consisting of fructose units linked predominantly by β(2-6)bonds, are attributable to both its size and structural complexity. Branching in β(2-1)contributes to diversify levan structures and properties. There is a broad spectrum of applications for levan and accordingly it has been the subject of several comprehensive reviews. A thorough analysis focused on the product specificity of enzymes from the Glycoside-Hydrolase family 68 that synthesize levan is however missing. We analyze here traditional and novel strategies to manipulate bacterial levansucrases in favor of the generation of low- or high-molecular weight levan, including site directed mutagenesis and chemical engineering. A comparison of highly variable structural elements of levansucrases is presented in the context of their capacity to synthesize saccharides of different sizes, employing the levansucrases from Bacillus subtilis and Bacillus megaterium as references.
... This is the case of the vinylsulfone group (dos .6.2.2.3.2.1. Vinylsulfone-acyl groups supports Supports activated with divinylsulfone have shown to be reactive under a wide range of pH values (immobilizing proteins even at pH 5) (dos Santos-Moriano et al., 2016). Moreover, they can react with more groups than glyoxyl (moving from just primary amino groups to also include in the enzyme-support reaction thiol, imidazole, hydroxyl and even secondary amino groups) (Bale Oenick et al., 1990;Bryjak et al., 2008;dos Santos et al., 2015b;Labus et al., 2011;Lopez-Jaramillo et al., 2012;Medina-Castillo et al., 2012;Morales-Sanfrutos et al., 2010;Ortega-Muñoz et al., 2010;Prikryl et al., 2012). ...
Lipases are the most widely used enzymes in biocatalysis, and the most utilized method for enzyme immobilization is using hydrophobic supports at low ionic strength. This method allows the one step immobilization, purification, stabilization, and hyperactivation of lipases, and that is the main cause of their popularity. This review focuses on these lipase immobilization supports. First, the advantages of these supports for lipase immobilization
will be presented and the likeliest immobilization mechanism (interfacial activation on the support surface) will be revised. Then, its main shortcoming will be discussed: enzyme desorption under certain conditions (such as high temperature, presence of cosolvents or
detergent molecules). Methods to overcome this problem include physical or chemical crosslinking of the immobilized enzyme molecules or using heterofunctional supports. Thus, supports containing hydrophobic acyl chain plus epoxy, glutaraldehyde, ionic,
vinylsulfone or glyoxyl groups have been designed. This prevents enzyme desorption and improved enzyme stability, but it may have some limitations, that will be discussed and some additional solutions will be proposed (e.g., chemical amination of the enzyme to have
a full covalent enzyme-support reaction). These immobilized lipases may be subject to unfolding and refolding strategies to reactivate inactivated enzymes. Finally, these biocatalysts have been used in new strategies for enzyme coimmobilization, where the most stable enzyme could be reutilized after desorption of the least stable one after its inactivation.
... Attempts have been made to address the third premise by immobilizing AaeUPO [12,13], but without achieving adequate control over the immobilized enzyme. Indeed, random interactions with activated supports typically produce heterogeneous populations with different carrier arrangements and enzyme orientations, which are not particularly reproducible [14][15][16][17]. When dealing with selective transformations, it is necessary to precisely orient the enzyme during immobilization, such as in the construction of nanobiodevices and for flow biocatalysis [18]. ...
Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme’s activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.
... The strategies for enzyme immobilization are commonly classified into three groups [18]: support binding (by adsorption or covalent linkages), entrapment and cross-linking. For reactions involving the transformation of carbohydrates, covalent binding is preferred over adsorption to avoid enzyme leakage [19], but most of the commercial activated carriers are expensive [20][21][22]. Cross-linking gives rise to biocatalysts with highly concentrated enzyme activity, significant stability and low production costs due to the absence of carrier, although the recovery of activity is commonly low [14,15]. ...
The β-fructofuranosidase (Xd-INV) from the basidiomycota yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) is unique in its ability to synthesize neo-fructooligosaccharides (neo-FOS). In order to facilitate its industrial application, the recombinant enzyme expressed in Pichia pastoris (pXd-INV) was immobilized by entrapment in polyvinyl alcohol (PVA) hydrogels. The encapsulation efficiency exceeded 80%. The PVA lenticular particles of immobilized pXd-INV were stable up to approximately 40 °C. Using 600 g/L sucrose, the immobilized biocatalyst synthesized 18.9% (w/w) FOS (59.1 g/L of neokestose, 30.2 g/L of 1-kestose, 11.6 g/L of neonystose and 12.6 g/L of blastose). The operational stability of PVA-immobilized biocatalyst was assayed in a batch reactor at 30 °C. The enzyme preserved its initial activity during at least 7 cycles of 26 h.
... Glutaraldehyde [13], epoxy [14][15][16][17] and glyoxyl [18] supports have been utilized to immobilize-stabilize enzymes via multipoint covalent attachment. The activation of supports with divinylsulfone (DVS) to immobilize proteins and enzymes have been described for a long time [19][20][21][22][23][24][25][26][27]. However, it has been only recently when DVS activated supports have been described as very suitable supports to produce an extremely intense multipoint covalent attachment using proper immobilization protocols (incubation at alkaline pH value, blocking with suitable nucleophiles), improving the results achieved using glyoxyl supports because vinylsulfone can react with primary amino groups and also with imidazole, thiol or phenol groups. ...
The paper shows the preparation of a new heterofunctional agarose support: amino-vinylsulfone. This has been employed to immobilize the interesting enzyme β-galactosidase from Aspergillus oryzae. The enzyme cannot be immobilized on just vinylsulfone activated support a pH values ranging from 5.0 to 9.0. Neither the enzyme was immobilized using 200. mM of NaCl on amino-vinylsulfone support. However, the enzyme was readily immobilized at moderate ion strength at pH values from 5.0 to 9.0 via ion exchange on amino-vinylsulfone support, and later some covalent enzyme-support bonds could be formed, more rapidly at alkaline pH value. After optimization of immobilization pH, incubation pH and time, and blocking reagent, several immobilized biocatalysts on amino-vinylsulfone support having 50-80% of the initial activity and a stabilization factor of around 8-15 were prepared, depending on the exact immobilization conditions.
... The binding chemistry between the amino groups of the protein and the glyoxyl moieties of the support is rather simple and gives rise to robust linkages between the enzyme and the carrier [25][26][27]. However, the immobilization must be performed at alkaline pH in order to deprotonate amino groups and thus enhance their nucleophilicity [28]. For that reason, the stability of B. circulans β-galactosidase at pH 10.0 was assessed in order to determine the optimal immobilization conditions for this enzyme. ...
The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal) agarose beads and assayed for the continuous production of galactooligosaccharides (GOS) in a packed-bed reactor (PBR). The immobilization was fast (1 h) and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-D-galactopyranoside (ONPG). The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45 °C in a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and 45 °C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased with time; however, the maximum GOS concentration (24.2 g/L) was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-Glc. The PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases.
... Lipase immobilization on activated support with divinylsulfone (DVS). LIPB was immobilized on activated SBA-15 with Divinylsulfone (DVS), producing the biocatalyst SBA-15-APTES-DVS-LIPB, namely biocatalyst 2. Fig. 2 shows the schematic representation of activated support with divinylsulfone and the reaction between DVS reactive groups from activated supports with proteins, a similar mechanism is shown in literature [28,44,45]. ...
A recombinant Candida antarctica lipase B expressed in Pichia pastoris (LIPB) was immobilized on pore-expanded SBA-15 previously modified 3-amino-propyltriethoxysilane (APTES) and activated with two bifunctional reagents, glutaraldehyde (GA) or divinylsulfone (DVS), producing the biocatalysts: SBA-15-APTES-GA-LIPB and SBA-15-APTES-DVS-LIPB, respectively. After LIPB immobilization, both preparations were then modified with glutaraldehyde, producing the biocatalysts: SBA-15-APTES-GA-LIPB-GA, SBA-15-APTES-DVS-LIPB-DVS. Alternatively, LIPB was immobilized on SBA-15-APTES-DVS at pH 10.2 and the biocatalyst was named SBA-15-APTES-DVS-LIPB-pH10. The different biocatalysts were assayed to check the effect of the immobilization strategies on the stability and in the substrate specificity during the kinetic resolution of (R,S)-Phenylethyl acetate. The thermal stability of some new preparations were higher than LIPB adsorbed on SBA-15 (SBA-15-LIPB) and LIPB immobilized on Glyoxyl-agarose. High conversions in the enzymatic kinetic resolution were obtained (43–50%) for all biocatalysts studied. Regarding activity and stability, the SBA-15-APTES-DVS-LIPB-pH10 was the most successful strategy, since, in first cycle, the maximum conversion was obtained (50%), and the biocatalyst remained active and enantioselective even after five successive cycles.
This study explores various methods for the covalent immobilization of cysteine proteases (ficin, papain, and bromelain). Covalent immobilization involves the formation of covalent bonds between the enzyme and a carrier or between enzyme molecules themselves without a carrier using a crosslinking agent. This process enhances the stability of the enzyme and allows for the creation of preparations with specific and controlled properties. The objective of this study is to evaluate the impact of covalent immobilization under different conditions on the proteolytic activity of the enzymes. The most favorable results were achieved by immobilizing ficin and bromelain through covalent bonding to medium and high molecular weight chitosans, using 5 and 3.33% glutaraldehyde solutions, respectively. For papain, 5 and 6.67% glutaraldehyde solutions proved to be more effective as crosslinking agents. These findings indicate that covalent immobilization can enhance the performance of these enzymes as biocatalysts, with potential applications in various biotechnological fields.
Immobilization of lipases by physical adsorption improves their stability, recovery, and reusability in biotechnological processes. The present review provides an advanced bibliometric analysis and a comprehensive overview of research progress in this field. By searching Web of Science, 39,575 publications were analyzed, and 325 relevant articles were selected. Key journals, countries, institutions, and authors were identified. The most cited articles focus on biofuel production and industrial applications. The analysis revealed four research themes with a focus on the production of biofuel. The physical adsorption method is effective when the appropriate support is used. Despite a decrease in patent applications, industrial interest remains high. Future studies should focus on optimizing support materials and exploring new applications of this technique. The present review provides a detailed understanding of the immobilization of lipases by physical adsorption.
This review emphasizes the crucial role of enzyme immobilization technology in advancing the production of two main biofuels, ethanol and biodiesel, with a specific focus on the Cross-linked Enzyme Aggregates (CLEAs) strategy. This method of immobilization has gained attention due to its simplicity and affordability, as it does not initially require a solid support. CLEAs synthesis protocol includes two steps: enzyme precipitation and cross-linking of aggregates using bifunctional agents. We conducted a thorough search for papers detailing the synthesis of CLEAs utilizing amylases, cellulases, and hemicellulases. These key enzymes are involved in breaking down starch or lignocellulosic materials to produce ethanol, both in first and second-generation processes. CLEAs of lipases were included as these enzymes play a crucial role in the enzymatic process of biodiesel production. However, when dealing with large or diverse substrates such as lignocellulosic materials for ethanol production and oils/fats for biodiesel production, the use of individual enzymes may not be the most efficient method. Instead, a system that utilizes a blend of enzymes may prove to be more effective. To innovate in the production of biofuels (ethanol and biodiesel), enzyme co-immobilization using different enzyme species to produce Combi-CLEAs is a promising trend.
Efficient and convenient access to short to medium chain length levan-type fructooligosaccharides (LFOS) is needed in order realise the nutritional potential of this class of bioactive oligosaccharides. While LFOS are synthesised by fructansucrase enzymes, these reactions are routinely associated with high molecular weight fructan polymer formation. Recent studies have shown that FOS production can be enhanced by the combination of levansucrase and inulosucrase in a one-pot reaction. In the present study, the novel mixed enzyme cross-linked enzyme aggregates (combi-CLEAs) based on levansucrase (Lev) and N543A variant inulosucrase (Inu) were prepared by ammonium sulfate precipitation, followed by glutaraldehyde cross-linking. The effect of Lev and Inu ratio on the activity of combi-CLEAs was explored. The results showed that >70% of total sucrase activity was recovered after immobilization and that the combi-CLEAs produced high amounts of LFOS (degree of polymerisation 3 to 21), while high molecular weight polysaccharide production was much reduced. Biochemical characterisation indicated that the optimum pH and temperature of combi-CLEAs (pH 5.5 and 50ᵒC, respectively) were comparable to those of free enzyme; however, the stability of the enzyme was improved. In addition, these combi-CLEAs have operational stability for several reaction cycles, which makes them very attractive for biotechnology applications.
[77-77-0]
C4H6O2S
(MW 118.17)
InChI = 1S/C4H6O2S/c1-3-7(5,6)4-2/h3-4H,1-2H2
InChIKey = AFOSIXZFDONLBT-UHFFFAOYSA-N
(bifunctional Michael acceptor that gives six-membered ring heterocycles with primary amines¹ or analogous reagents²; furnishes γ-diketones in the reaction with aldehydes and a thiazolium salt (Stetter reaction)³; reasonably powerful dienophile⁴)
• Physical Data: bp 233–234 °C; bp 90–92 °C/8mmHg; mp ca. −26 °C; d 1.177 g cm⁻³.
• Form Supplied in: colorless liquid; commonly stabilized with hydroquinone; widely available.
• Preparative Methods: by dehydration of bis-β-hydroxyethyl sulfone with phosphoric acid at 280 °C or by pyrolysis of bis-β-acetoxyethyl sulfone at 500 °C.⁵
• Handling, Storage, and Precautions: toxic, corrosive liquid; vesicant and mild lachrymator; to be handled with care; use in a fume hood.
The coimmobilization of lipases from Rhizomucor miehei (RML) and Candida antarctica (CALB) has been intended using agarose beads activated with divinyl sulfone. CALB could be immobilized on this support, while RML was not. However, RML was ionically exchanged on this support blocked with ethylendiamine. Therefore, both enzymes could be coimmobilized on the same particle, CALB covalently using the vinyl sulfone groups, and RML via anionic exchange on the aminated blocked support. However, immobilized RML was far less stable than immobilized CALB. To avoid the discarding of CALB (that maintained 90% of the initial activity after RML inactivation), a strategy was developed. Inactivated RML was desorbed from the support using ammonium sulfate and 1% Triton X-100 at pH 7.0. That way, 5 cycles of RML thermal inactivation, discharge of the inactivated enzyme and re-immobilization of a fresh sample of RML could be performed. In the last cycle, immobilized CALB activity was still over 90% of the initial one. Thus, the strategy permits that enzymes can be coimmobilized on vinyl sulfone supports even if one of them cannot be immobilized on it, and also permits the reuse of the most stable enzyme (if it is irreversibly attached to the support).
In the present study, the cross-linked enzyme aggregates (CLEAs) of Y246S variant levansucrase from Bacillus licheniformis RN-01, known as oligosaccharide-producing levansucrase (OPL) was prepared for application in levan-type fructooligosaccharide (LFOS) synthesis in sucrose and fruit juices. The OPL-CLEAs with the highest immobilization yield (74.8% ± 8.9%) were prepared using 40 g/100 mL ammonium sulfate, 15 U/mL OPL, and 2 mg/mL bovine serum albumin in a 4 h cross-linking reaction. Compared with those of the free enzyme, the obtained OPL-CLEAs had a broader pH range for catalysis (pH 3–12) and displayed a higher optimum temperature. Moreover, the stability of OPL was significantly improved after immobilization, indicated by an increase in melting temperature and operational duration. Product analysis showed that the OPL-CLEAs could synthesize a similar LFOS profile as free enzymes, with a yield of up to 51%. Additionally, OPL-CLEAs could retain more than 50% of the initial activity after six cycles of reuse. Finally, the potential application of OPL-CLEAs in fruit juices was demonstrated by transforming up to 65%–75% of total sucrose in moderately acidic fruit juices. This study suggests the high potential for application of OPL-CLEAs in food industries.
Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.
The reliability and stability of array slides are a big concern for array vendors and end users. Herein, we report on a new type of array slide with high reactivity toward DNA probes and low side reaction. A one-step surface reaction via the Michael addition involved in preparing array slides was developed and characterized by x-ray photoelectron spectroscopy, contact angle, and fluorescence labeling. The effects of array fabrication and storage conditions, i.e., spotting solution pH, high humidity, and long-term storage on the reactivity of the slides were examined. The fabricated DNA arrays could realize good hybridization efficiency (38.2% for slides with 0.88 pmol/cm²), low limit of detection (4 × 10⁻¹⁴M), as well as high mismatch selectivity.
A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry (named SiO2-DVS-GSH). The successful synthesis of the SiO2-DVS-GSH material was confirmed by FT-IR, elemental analysis, and zeta potential analysis. The effects of water content, pH value, and salt concentration in the mobile phase on the model compound (uracil, uridine, cytosine, cytidine, guanosine, xanthosine, orotic acid) retention was studied, and a hydrophilic interaction liquid chromatography (HILIC) retention feature together with electrostatic interaction of the SiO2-DVS-GSH material was observed. The prepared stationary phase was further applied for the separation of oligosaccharide. In addition, the SiO2-DVS-GSH material displayed remarkable selectivity and specificity for the sialylated N-glycopeptides’ enrichment from bovine fetuin tryptic digests, even at a mass ratio of 1:1000 (w/w) to bovine serum albumin (BSA, non-glycosylated protein), showing superior performance compared to commercial ZIC-HILIC material. Moreover, the SiO2-DVS-GSH material behaved well in the N-glycopeptides’ enrichment from human serum, demonstrating its promising potential for glycoproteomics of complex biological samples.
A glutathione (GSH)-functionalized silica material was prepared using divinyl sulfone activation chemistry, and it shows remarkable selectivity and specificity for the sialylated N-glycopeptides’ enrichment.
This paper demonstrated the catalytic oxa-Michael reaction of inorganic silanol groups with vinyl sulfones, which facilitates an efficient strategy for functionalization of the silica surface. The strategy was applied on materials ranging from nanoscale to macroscale silica, and the surface functionalization was achieved in hours using organo-catalysts at mild temperature. The formation of Si-O-C bonds on the surface was characterized by solid-state 13C CP-MAS NMR, FTIR and XPS. Our strategy showed several advantages over traditional methods, and the resulting Si-O-C bond exhibited distinct behaviors towards different solvents. Organic solvents would stabilize the functionalized silica materials, while aqueous solutions would result in degradation affected by both solution and surface factors. Using divinyl sulfone as a crosslinker, a variety of molecules can be immobilized and sequentially released in a controllable manner, which would benefit a broad range of applications from sensing to drug and catalyst carriers.
With the aim to overcome the limitations of hydrogel chitosan beads (HGBs), various types of chitosan, core–shell chitosan beads (CSBs), and dried chitosan beads (DBs) were synthesized. Physical and chemical properties were compared with those of HGBs. CSBs were proved to be an effective support because they displayed higher stability and capacity over the HGBs, and thus, were selected for enzyme immobilization. Recombinant inulosucrase (INU) from Lactobacillus reuteri 121 was immobilized on CSBs using glutaraldehyde as a cross-linker. Immobilized biocatalysts (INU-CSBs) were then used for the synthesis of inulin-type fructooligosaccharide (IFOS). Biochemical characterization revealed that the optimum pH of both INU-CSBs and free enzyme was unaltered at 5.5 whereas the optimum temperature of INU-CSBs shifted from 50 °C to 60 °C. Moreover, pH stability and thermostability of INU-CSBs significantly improved. For batch synthesis of IFOS, INU-CSBs retained approximately 45% of their initial catalytic activity after being reused for 12 cycles. IFOS was also continuously synthesized in a fixed-bed bioreactor for a reaction duration of at least 30 h. The high efficiency of INU-CSBs makes them very attractive for industrial applications.
The efforts to enhance the stability of enzymes and improve their activity have generated considerable interest because of their wide applications in bioenergy conversion, proteomics research, and bioassays. This study reports a promising strategy for enzyme immobilization based on toehold-mediated DNA strand displacement on functionalized magnetic nanoparticles for the first time. The strategy provided a convenient approach to achieve sequential displacement and immobilization of different enzymes, using alkaline phosphatase (ALP), horseradish peroxidase (HRP), and trypsin as different model enzymes. Taking trypsin as an example, the enzyme immobilization procedure by DNA strand displacement exhibited high reversibility and reproducibility, which could retrieve more than 87% of the enzymatic activity after consecutive hybridization and dehybridization cycles. The thermal stability of the immobilized trypsin was significantly enhanced up to 3.1- and 2.3-fold greater than free enzyme after 45 min incubation at 60°C and 70°C, respectively, and the immobilized enzyme preserved promising enzymatic activity of more than 87% after 10 cycles. Notably, the immobilized enzyme exhibited excellent long-term incubation stability and storage stability as compared with free enzyme, and showed up to 11-fold higher stability than free enzyme towards different solvents. Significantly improved digestion efficiency of myoglobin, glycated hemoglobin, and cytochrome C was achieved with this immobilized enzyme within 10 min, and the obtained sequence coverages were 1.5-, 1.3-, and 1.6-fold higher than conventional in-solution digestion for 12 h. Thus, the developed strategy exhibited a promising alternative platform with high magnetic responsiveness and significantly enhanced properties for the immobilization of important enzymes and their broad applications.
Among several commercial enzymes screened for chitosanolytic activity, Neutrase 0.8L (a protease from Bacillus amyloliquefaciens) was selected in order to obtain a product enriched in deacetylated chitooligosaccharides (COS). The hydrolysis of different chitosans with this enzyme was followed by size exclusion chromatography (SEC-ELSD), mass spectrometry (ESI-Q-TOF), and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Neutrase 0.8L converted 10 g/L of various chitosans into mostly deacetylated oligosaccharides, yielding approximately 2.5 g/L of chitobiose, 4.5 g/L of chitotriose, and 3 g/L of chitotetraose. We found out that the neutral protease was not responsible for the chitosanolytic activity in the extract, while it could participate in the deacetylating process. The synthesized COS were tested in vitro for their neuroprotective (toward human SH-S5Y5 neurons) and anti-inflammatory (in RAW macrophages) activities, and compared with other functional ingredients, namely fructooligosaccharides.
Layered double hydroxide (LDH) nanoparticles were prepared and used as solid support for superoxide dismutase (SOD) enzyme. Structural features were studied by XRD, spectroscopic methods (IR, UV-Vis and fluorescence) and TEM, while colloidal stability of the obtained materials was investigated by electrophoresis and light scattering in aqueous dispersions. The SOD quantitatively adsorbed on the LDH by electrostatic and hydrophobic interactions and kept its structural integrity upon immobilization. The composite material showed moderate resistance against salt-induced aggregation in dispersions, therefore, heparin polyelectrolyte was used to improve the colloidal stability of the system. Heparin of highly negative line charge density strongly adsorbed on the oppositely charged hybrid particles leading to charge neutralization and overcharging at appropriate polyelectrolyte loading. Full coverage of the composite platelets with heparin resulted in highly stable dispersions, which contained only primary particles even at elevated ionic strengths. Our results indicate that the developed bionanocomposite of considerable enzymatic function is a suitable candidate for applications, wherever stable dispersions of antioxidant activity are required for instance in biomedical treatments or in chemical manufacturing processes.
This study aimed to prepare robust immobilized formate dehydrogenase (FDH) preparations which can be used as effective biocatalysts along with functional oxidoreductases, in which in situ regeneration of NADH is required. For this purpose, Candida methylica FDH was covalently immobilized onto Immobead 150 support (FDHI150), Immobead 150 support modified with ethylenediamine and then activated with glutaraldehyde (FDHIGLU), and Immobead 150 support functionalized with aldehyde groups (FDHIALD). The highest immobilization yield and activity yield were obtained as 90% and 132%, respectively when Immobead 150 functionalized with aldehyde groups was used as support. The half-life times (t(1/2)) of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated as 10.6, 28.9, 22.4 and 38.5 h, respectively at 35 degrees C. FDHI150, FDHIGLU and FDHIALD retained 69, 38 and 51% of their initial activities, respectively after 10 reuses. The results show that the FDHI150, FDHIGLU and FDHIALD offer feasible potentials for in situ regeneration of NADH.
Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final levan profile in reactions with sucrose as substrate.
A simple and inexpensive methodology, based on the use of micro-centrifuge filter tubes, is proposed for establishing the best enzyme immobilization conditions.
The immobilized biocatalyst is located inside the filter holder during the whole protocol, thus facilitating the incubations, filtrations and washings. This procedure minimizes the amount of enzyme and solid carrier needed, and allows exploring different immobilization parameters (pH, buffer concentration, enzyme/carrier ratio, incubation time, etc.) in a fast manner. The handling of immobilized enzymes using micro-centrifuge filter tubes can also be applied to assess the apparent activity of the biocatalysts, as well as their reuse in successive batch reaction cycles. The usefulness of the proposed methodology is shown by the determination of the optimum pH for the immobilization of an inulinase (Fructozyme L) on two anion-exchange polymethacrylate resins (Sepabeads EC-EA and Sepabeads EC-HA).
The micro-scale procedure described here will help to overcome the lack of guidelines that usually govern the selection of an immobilization method, thus favouring the development of stable and robust immobilized enzymes that can withstand harsh operating conditions in industry.
The combination of silica as support and vinyl sulfone as reactive group led to a pre-activated material that readily reacts to form covalent bonds by Michael-type addition with both amine and thiol groups naturally occurring in biomolecules in mild conditions compatible with the biological nature of the enzymes. A simple two step synthetic strategy was designed to access this functionalized hybrid material. Two types of vinyl sulfone silicas (N-type and S-type) differing in the chemical nature of the linkers between the silica particle and the reactive vinyl sulfone group were prepared by implementation of this strategy. The capabilities of those vinyl sulfone silicas were evaluated with the model enzymes invertase, lactase and lysozyme. Both S-type and N-type vinyl sulfone silicas coupled efficiently with the model enzymes even at 4 degrees C by simple combination of the species and the immobilized enzymes retained the enzymatic activity. The linker showed to play a major role in the non covalent interactions between the enzymes and the silicas. In terms of capacity, the S-type material is the best option although its poor flow rate when packed in columns invalidates its applications for low pressure liquid chromatography. The capabilities of the N-type material were successfully put to the test as a pre-packed column for the immobilization of invertase and further demonstrated with two real cases of relevance in proteomics: (i) purification of glutathione-S-transferase (GST) and (ii) identification of proteins that interact with thioredoxin h2 from Pisum sativum.
Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.
In the last decade, new trends for enzyme attachment to solid carriers have emerged in an attempt to rationalize the classical methods for enzyme immobilization. In silico analysis is becoming a powerful tool to predict the orientation of enzymes covalently attached to the carrier or the protein regions involved in adsorption to the support. Significantly, an array of algorithms has been established for the rational design of immobilized derivatives, which comprises both the protein size and the textural properties of the support. Ordered mesoporous materials open a challenging pathway to tailor immobilized enzymes with high volumetric activity and minimum lixiviation. In addition, fluorescence confocal microscopy is being successfully employed to understand the diffusional restrictions and the distribution of biomolecules within the support.
The easy functionalization of tags and solid supports with the vinyl sulfone function is a valuable tool in omic sciences that allows their coupling with the amine and thiol groups present in the proteogenic residues of proteins, in mild and green conditions compatible with their biological function.
The use of olive oil mill wastewaters (OMW) as organic fertilizer is limited by its phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus that is able to decrease the chemical oxygen demand (COD) and colour of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g-1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0-10.0, whereas the immobilized laccase kept the activity in the pH range 3.0-10.0. The free enzyme was quickly inactivated at temperatures above 50 oC, whereas the immobilized enzyme was very stable up to 70 oC. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds. We thank Dr. J. Martinez and T. de la Rubia (University of Granada, Spain) for giving us lyophilized OMW. We thank Thomas Boller (Degussa, Darmstadt, Germany) for supplying Eupergit C samples and for technical help. The authors thank the financial support received from the Spanish Projects BIO2003-00621, VEM2004-08559 and CAM S-0505/AMB0100. Peer reviewed
Fructansucrases, members of glycoside hydrolase family 68, catalyze both sucrose hydrolysis and the polymerization of fructose to beta-d-fructofuranose polymers. The resulting fructan polymers are distinguished by the nature of the glycosidic bond: inulin (beta-(2-1)-fructofuranose) and levan (beta-(2-6)-fructofuranose). In this study we demonstrate that Zymomonas mobilis levansucrase exists in two active forms, depending on the pH and ionic strength. At pH values above 7.0, the enzyme is mainly a dimer, whereas at pH values below 6.0, the protein forms well ordered microfibrils that precipitate out of the solution. These two forms are readily interchangeable simply by changing the pH. Surprisingly the manner in which the enzyme is arranged strongly affects its product specificity and kinetic properties. At pH values above 7.0, the activity of the enzyme as a dimer is mainly sucrose hydrolysis and the synthesis of short fructosaccharides (degree of polymerization, 3). At pH values below 6.0, in its microfibril form, the enzyme catalyzes almost exclusively the synthesis of levan (a degree of polymerization greater than 20,000). This difference in product specificity appears to depend on the form of the enzyme, dimer versus microfibril, and not directly on the pH. Images made by negative stain transmission electron microscopy reveal that the enzyme forms a very ordered structure of long fibrils that appear to be composed of repeating rings of six to eight protein units. A single amino acid replacement of H296R abolished the ability of the enzyme to form microfibrils with organized fibril networks and to synthesize levan at pH 6.0.
This work addresses the effect of sucrose concentration and temperature on the three activities displayed by the levansucrase from Zymomonas mobilis: formation of levan (polymerization), production of short-chain fructooligosaccharides (FOS), and sucrose hydrolysis. Of the conditions tested, levan formation reached the highest value at 4 °C and 100 g/L sucrose. The increase of temperature (40 °C) and sucrose concentration (600 g/L) caused a significant decrease of the levan concentration and a higher production of FOS. However, an increase of the temperature also caused an enhancement of the undesired hydrolytic activity. Several inulin-type FOS (1-kestose, nystose, 1F-fructosylnystose), neoFOS (blastose, neokestose, neonystose) and levan-type FOS (6-kestose, 6,6-nystose) were synthesized by levansucrase. The latter compound was purified and characterized by mass spectrometry and 2D NMR. Using 600 g/L sucrose at 40 °C, the maximum yield of FOS was reached at 85% sucrose conversion; at this point, the reaction mixture contained (in weight basis) 31% glucose, 14% fructose, 15% sucrose and 40% FOS (including a small contribution of levan).
BACKGROUND
Due to their exceptional mechanical, electrical and chemical properties, carbon nanotubes (CNT) have been used in nanotechnology field to create new nanostructures. Consequently, there is an increasing interest in understanding and controlling the interactions of this nanomaterial with biological molecules, such as enzymes. In this context, peroxidases have been immobilized on CNT for various potential applications, such as sensing, drug delivery and biocatalysis. There are but a few studies about the influence of the nanoscale environment on the function of these enzymes.RESULTSOxygen functional groups are introduced by the oxidation of multi-walled carbon nanotubes (MWCNTs) in an initial step and then selectively removed by a thermal treatment at well defined temperatures. The immobilization efficiency and catalytic activity of peroxidase were analyzed as function of the pH. Pristine MWCNTs show excellent immobilization capacity (100%) and high enzyme activity, but low thermal stability (at 40 °C) owing to mostly hydrophobic interaction between peroxidase and the support. MWCNTs oxidized with HNO3 and at posteriori heated at 400 °C, mostly presenting hydroxyl surface groups, provided the best compromise between peroxidase activity and thermal stability, which has been attributed to the formation of hydrogen bonds between the enzyme and the support. The storage stability of peroxidase immobilized on that support was 4.5 times higher than for the free peroxidase.CONCLUSIONMWCNTs present high affinity to adsorb peroxidase, which makes them excellent supports for the immobilization and stabilization of this enzyme, which constitutes a great advantage for industrial applications.
Levan is a homopolymer of fructose naturally obtained from both plants and microorganisms. Microbial levans are more advantageous, economical and industrially feasible with numerous applications. Bacterial levans are much larger than those produced by plants with multiple branches and molecular weights ranging from 2 to 100 million Da. However levans from plants generally have molecular weights ranging from about 2000 to 33,000Da. Microbial levans have wide range of applications in food, medicine, pharmaceutical, cosmetic and commercial industrial sectors. With excellent polymeric medicinal properties and ease of production, microbial levan appear as a valuable and versatile biopolymer of the future. The present article summarizes and discusses the most essential properties of bioactive microbial levan and recent developments in its production, characterization and the emerging applications in health and industry.
Copyright © 2014 Elsevier Ltd. All rights reserved.
Different filamentous fungi isolated from molasses and jams (kiwi and fig) were screened for fruc-tooligosaccharides (FOS) producing activity. Two strains, identified as Penicilium sizovae (CK1) and Cladosporium cladosporioides (CF(2)15), were selected on the basis of the FOS yield and kestose/nystose ratio. In both strains the activity was mostly mycelium-bound. Starting from 600 g/L of sucrose, maximum FOS yield was 184 and 339 g/L for P. sizovae and C. cladosporioides, respectively. Interestingly, the highest FOS concentration with C cladosporioides was reached at 93% sucrose conversion, which indicated a notable transglycosylation to hydrolysis ratio. The main FOS in the reaction mixtures were identified by HPAEC-PAD chromatography. C cladosporioides synthesized mainly 1-kestose (158 g/L), nystose (97 g/L), F-1-fructosylnystose (19 g/L), 6-kestose (12 g/L), neokestose (10 g/L) and a disaccharide (34 g/L) that after its purification and NIVIR analysis was identified as blastose [Fru-beta(2 -> 6)-Glc]. P. sizovae was very selective for the formation of F-1-FOS (in particular 1-kestose) with minor contribution of neoFOS and negligible of levan-type FOS.
Currently, prebiotics are all carbohydrates of relatively short chain length. An important group is the fructooligosaccharides, which are a special kind of prebiotics associated to their selective stimulation of the activity of certain groups of colonic bacteria that have a positive and beneficial effect on intestinal microbiota, reducing incidence of gastrointestinal infections, respiratory and also possessing a recognized bifidogenic effect. Traditionally, these prebiotic compounds have been obtained through extraction processes from some plants, as well as through enzymatic hydrolysis of sucrose. However, different fermentative methods have also been proposed for the production of fructooligosaccharides, such as solid-state fermentation utilizing various agroindustrial by-products. By optimizing the culture parameters, fructooligosaccharides yields and productivity can be improved. The use of immobilized enzymes and cells has also been proposed as being an effective and economic method for large-scale production of fructooligosaccharides. This paper is an overview on the results of recent studies on fructooligosacharides biosynthesis, physicochemical properties, sources, biotechnological production and applications.
Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immo-bilized enzyme systems allows an easy recovery of both enzymes and products, multiple re-use of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This paper is a review of the recent literatures on enzyme immobilization by various techniques, the need for immobilization and different applications in industry, covering the last two decades. The most recent papers, patents, and reviews on immobilization strategies and application are reviewed.
A modification of the classical calcium alginate enzyme entrapment technique is described aiming to overcome some of the limitations of the former gel-based biocatalysts. Dried alginate entrapped enzymes (DALGEEs) were obtained dehydrating calcium alginate gel beads containing entrapped enzymes. A fructosyltransferase from Aspergillus aculeatus, present in Pectinex Ultra SP-L, was entrapped using this technique. The resulting DALGEEs were successfully tested both operating batchwise and in a continuous fixed-bed reactor for fructooligosaccharides (FOS) synthesis from sucrose. Interestingly, DALGEEs did not re-swell upon incubation in concentrated (600 g/L) sucrose solutions, probably due to the lowered water activity (aw) of such media. Confocal laser scanning microscopy of DALGEEs revealed that the enzyme molecules accumulated preferably in the shell of the particles. DALGEEs showed an approximately 30-fold higher volumetric activity (300 U/mL) compared with the calcium alginate gel beads. Moreover, a significant enhancement (40-fold) of the space-time-yield of fixed-bed bioreactors was observed when using DALGEEs as biocatalyst compared with gel beads (4030 g/day L of FOS vs. 103 g/day L). The operational stability of fixed-bed reactors packed with DALGEEs was extraordinary, providing a nearly constant FOS composition of the outlet during at least 700 h. It was also noticeable their resistance against microbial attack, even after long periods of storage at room temperature. The DALGEEs immobilisation strategy may also be useful for other biotransformations, in particular when they take place in low aw media.
Immobilized enzymes are becoming increasingly popular as reusable, selective analytical chemical reagents in solid-phase flow-through reactors, as membranes in sensors and as films in dry reagent kits. Classification must encompass the properties of the original enzyme, the type of support used and the methods of support activation and enzyme attachment. Important characteristics of an immobilized enzyme viz a viz the enzyme in solution, e.g., apparent activity, stability and lifetime must also be reported.
Levansucrase from Zymomonas mobilis expressed in Escherichia coli was immobilized onto the surface of magnetite. Optimum conditions for the immobilization were, pH 4.0; immobilization reaction time of 30 min and 8 U of enzyme per gram of matrix. Immobilization yield was 75% under optimized conditions. The maximum production yield of levan by the immobilized levansucrase was about 42% on a fructose released basis with sucrose as substrate at 100 g/l. Thermal stability of the levansucrase increased by immobilization procedure. Furthermore, immobilized enzyme showed the less polymerizing activity in levan formation, producing the low-molecular weight (MW) levan. The molecular weight of levan can be controlled by using the immobilized levansucrase in high yield. Immobilized levansucrase retained 61% of the original activity after five repeated uses.
Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 C.
The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.
Carbohydrate-mediated molecular recognition is involved in many biological aspects such as cellular adhesion, immune response, blood coagulation, inflammation, and infection. Considering the crucial importance of such biological events in which proteins are normally involved, synthetic saccharide-based systems have emerged as powerful tools for the understanding of protein-carbohydrate interactions. As a new approach to create saccharide-based systems, a set of representative monosaccharides (D-mannose, D-glucose, N-acetyl-D-glucosamine, and L-fucose) and disaccharides (lactose, maltose, and melibiose) were derivatized at their anomeric carbon with a vinyl sulfone group spanned by an ethylthio linker. This vinyl sulfone functionalization is demonstrated to be a general strategy for the covalent linkage of a saccharide in mild conditions via Michael-type additions with the amine and thiol groups from functionalized supports and those naturally present in biomolecules. The introduction of the ethylthio linker between the biorecognizable element (i.e., saccharide) and the reactive group (i.e., vinyl sulfone) was found to preserve the functionality of the former. The capability of the vinyl sulfone saccharides for the study of lectin-carbohydrate interactions was demonstrated by (i) immobilizing them on both amine-functionalized supports (glass slides and microwell plates) and polylysine-coated glass slides to create sugar arrays that selectively bind lectins (ii) coupling to model proteins to yield neoglycoproteins that are recognized by lectins and (iii) using vinyl sulfone saccharides as tags to allow the detection of the labeled biomolecule by HRP-lectins. The above results were further put tothe test with a real case: detection of carbohydrate binding proteins present in rice ( Oryza sativa ).
Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.
Functionalization of carbon nanotubes (CNTs) with proteins is often a key step in their biological applications, particularly in biosensing. One popular method has used the cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to covalently conjugate proteins onto carboxylated CNTs. In this article, we critically assess the evidence presented in these conjugation studies in the literature. As CNTs have a natural affinity for diverse proteins through hydrophobic and electrostatic interactions, it is therefore important to differentiate protein covalent attachment from adsorption in the immobilization mechanism. Unfortunately, many studies of conjugating proteins onto CNTs using EDC lacked essential controls to eliminate the possibility of protein adsorption. In studies where the attachment was claimed to be covalent, discrepancies existed and the observed immobilization appeared to be due to adsorption. So far, bond analysis has been lacking to ascertain the nature of the attachment using EDC. We recommend that this approach of covalent immobilization of proteins on CNTs be re-evaluated and treated with caution.
The catalytic properties of levansucrase bound to hydroxyapatite were studied as a possible model for enzyme behaviour when associated in vivo to matrices such as the cell wall of bacteria or tooth surfaces. The activity of the immobilised enzyme was mainly directed towards its polymerase activity. The yield of levan reached 85%. The kcat of the enzyme for sucrose transformation was increased and the Km for this substrate was unmodified. These properties allow the design of a system for the large-scale production of high-molecular-weight branched-chain levan in vitro in high yield.
A new levan fructotransferase (LFTase) isolated from Arthrobacter oxydans J17-21 was characterized for the production of difructose dianhydride IV (DFA IV). LFTase was purified to apparent homogeneity by Q-Sepharose ion exchange chromatography, Mono-Q HR 5/5 column chromatography, and gel permeation chromatography. The enzyme had an apparent molecular mass of 54000 Da. The optimum pH for the enzyme-catalyzed reaction was pH 6.5, and the optimum temperature was observed at 45 degrees C. The LFTase was activated by the presence of CaCl(2) and EDTA-2Na but inhibited strongly by MnCl(2) and CuSO(4) at 1 mM and completely by FeSO(4) and Ag(2)SO(4) at 1 mM. A bacterial levan from Zymomonas mobilis was incubated with an LFTase; final conversion yield from the levan to DFA IV was 35%. Neither inulin, levanbiose, sucrose, dextran, nor starch was hydrolyzed by LFTase. DFA IV was very stable at acidic pH and high temperature, thus indicating that DFA IV may be suitable for the food industry and related areas.
A fructosyltransferase present in Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus, was purified to 107-fold and further characterised. The enzyme was a dimeric glycoprotein (20% (w/w) carbohydrate content) with a molecular mass of around 135 kDa for the dimer. Optimal activity/stability was found in the pH range 5.0-7.0 and at 60 degrees C. It was stable or slightly activated (upto 1.4-fold) in the presence of reducing agents, such as dithiothreitol and 2-mercaptoethanol, and detergents, such as sodium dodecylsulphate and Tween 80. The enzyme was able to transfer fructosyl groups from sucrose as donor producing the corresponding series of fructooligosaccharides: 1-kestose, nystose and fructosylnystose. Using sucrose as substrate, the k(cat) and K(m) values for transfructosylating activity were 1.62+/-0.09 x 10(4)s(-1) and 0.53+/-0.05 M, whereas for hydrolytic activity the corresponding values were 775+/-25s(-1) and 27+/-3 mM. At elevated sucrose concentrations, the fructosyltransferase from A. aculeatus showed a high transferase/hydrolase ratio that confers it a great potential for the industrial production of prebiotic fructooligosaccharides.
The main point was the search for a proper carrier and the kind of carrier activation for trypsin (EC 3.4.21.4) immobilization. The acrylic and cellulose-based carriers were specially prepared in that they possessed the most often used anchor groups: -OH, -NH(2), DEAE and/or -COOH. The immobilization procedures were selected to apply mainly to protein amine groups and appropriate anchor groups on the carrier. As activity tests low (N-benzoyl-dl-arginine-p-nitroanilide, BAPNA) and high (casein) molecular weight substrates were used. It was found, as a rule, that trypsin bound to -COOH groups with the help of carbodiimide was less active and that the amount of bound protein and measured activity (BAPNA) are considerably higher when protein is immobilized via divinyl sulfone. Both rules were observed irrespective of the nature of the polymer matrix. Both types of carriers were found suitable for trypsin immobilization and they were far better than the corresponding Eupergit C-bound enzyme preparations. Taking into account storage stability and activity for both substrates, the divinylsulfone linkage formed between unmodified Granocel and trypsin was the most effective method for the enzyme immobilization. For this preparation, BAPNA and casein conversion, thermal stability at 60 degrees C and estimated kinetic parameters were compared with those obtained for the native enzyme. It was shown that mass transport limitations could be effectively eliminated by suitable conditions and immobilized trypsin was considerably more stable. The values k(cat)/K(m) indicated that the immobilized enzyme was even better as amidase activity was regarded and its potential for protein hydrolysis was only less than twice.
beta-Fructofuranosidases are powerful tools in industrial biotechnology. We have characterized an extracellular beta-fructofuranosidase from the yeast Schwanniomyces occidentalis. The enzyme shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose and raffinose, with different catalytic efficiencies (k(cat)/K(m)). Although the main reaction catalysed by this enzyme is sucrose hydrolysis, it also produces two fructooligosaccharides (FOS) by transfructosylation. A combination of (1)H, (13)C and 2D-NMR techniques shows that the major product is the prebiotic trisaccharide 6-kestose. The 6-kestose yield obtained with this beta-fructofuranosidase is, to our concern, higher than those reported with other 6-kestose-producing enzymes, both at the kinetic maximum (76gl(-1)) and at reaction equilibrium (44gl(-1)). The total FOS production in the kinetic maximum was 101gl(-1), which corresponded to 16.4% (w/w) referred to the total carbohydrates in the reaction mixture.
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Production of identified FOS by vinyl sulfone-immobilized levansucrase Reaction conditions: 5 U mL À1 LEV-VS, 600 g L À1 sucrose, 50 mM sodium acetate buffer pH 5
- Fig
Fig. 7 (A) Production of identified FOS by vinyl sulfone-immobilized
levansucrase. Reaction conditions: 5 U mL À1 LEV-VS, 600 g L À1
sucrose, 50 mM sodium acetate buffer pH 5.4, 40 C. (B) FOS quantification of LEV and LEV-VS reactions at 28 h.
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