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Continuous production of fructooligosaccharides and invert sugar by chitosan immobilized enzymes: Comparison between in fluidized and packed bed reactors
Abstract
In this work, β-fructofuranosidase and β-fructosyltransferase were covalently immobilized on chitosan spheres, using glutaraldehyde as a coupling agent, in order to produce invert sugar and fructooligosaccharides (FOS). Maxinvert L was used to make β-fructofuranosidase biocatalyst yielding 7000 HU/g. A partial purified β-fructosyltransferase from Viscozyme L was used to prepare the other biocatalyst yielding 2100 TU/g. The production of invert sugar and FOS was evaluated using different continuous enzymatic reactors: two packed bed reactors (PBR) and two fluidized bed reactors (FBR). The invert sugar production achieved a yield of 98% (grams of product per grams of initial sucrose) in the PBR and 94% in the FBR, whereas FOS production achieved a yield of 59% in the PBR and 54% in the FBR. It was also observed in both cases that varying the flow rate it is possible to modulate the FOS composition in terms of nystose and kestose concentrations. The operational stability of FOS produced in the PBR was evaluated for 40 days showing no reductions in yields.
... 8,10 Nevertheless, enzymatic reactions in PBRs require stable biocatalysts and reaction conditions that will minimize the limitations of mass transfer and allow fast and efficient access of the substrate molecules to the enzyme active sites. 8,10,11 Several studies have reported that immobilized extracellular FTases and whole cells with transfructosylation activity present a higher operational stability than the soluble enzymes. 3,9,[12][13][14] Whole cells are prominent among these biocatalysts because they * Correspondence to: RF provide the reaction medium with enzymes immobilized in their own biomass of origin, and hence, do not require purification and immobilization steps. ...
... 3,6,8,[17][18][19][20][21] Furthermore, some of these cells have presented a high potential for FOS production in continuous reactors. 5,8,10,11 The productivity of FOS in a PBR was reported to be 1.7 times higher than that in a batch reactor under the same reaction conditions using immobilized whole cells. 10 Particularly, the cells of the fungus A. oryzae IPT-301 have presented one of the greatest transfructosylation activities among numerous strains of filamentous fungi. ...
... The values of the parameters V max and K m were equal to 634 ± 85 U g −1 and 157 ± 68 g L −1 , respectively. The effective contact between the substrate and the enzyme in the PBR was determined through the enzymatic productivity, which was calculated by Eqn (11). The enzyme productivity obtained was 522 U g −1 min −1 . ...
BACKGROUND
Fructooligosaccharides (FOS) are prebiotic sugars used in the production of functional foods. These sugars can be produced by the transfructosylation reaction of sucrose catalyzed by fructosyltransferase enzymes (FTase, E.C.2.4.1.9). The aim of this work was to evaluate the use of cells of Aspergillus oryzae IPT‐301 as biocatalysts for the sucrose transfructosylation reaction in a packed bed reactor (PBR).
RESULTS
The morphology of the cells was observed by scanning electron microscopy, and BET specific surface areas were examined by nitrogen physisorption The highest transfructosylation activity (660 U g⁻¹) of the cells was obtained at 25 min of reaction by adopting a reaction temperature of 50 °C, sucrose concentration of 470 g L⁻¹, and volumetric flow of 15 mL min⁻¹. The rise in volumetric flow provided an increase in the transfructosylation activity, but also a faster deactivation of the biocatalyst. Enzyme kinetics was described using the Michaelis–Menten model, with a Vmax of 632.8 U g⁻¹ and a km of 160.7 g L⁻¹. The cells showed constant enzymatic activity for 12 h of reaction in the PBR operated with discontinuous flow at flow rates of 5 mL min⁻¹ and 11.5 mL min⁻¹, and in the PBR operated with continuous flow at a flow rate of 5 mL min⁻¹. The transition from diffusional to kinetic regimes was observed starting from 11.5 mL min⁻¹.
CONCLUSION
The results obtained suggest a high potential of application of the whole A. oryzae IPT‐301 cells for continuous FOS production in PBR. © 2022 Society of Chemical Industry (SCI).
... Enzyme beads are less susceptible to breaking from mechanical shear stress and the ratio between the substrate and the enzyme is much lower in a PBR than in conventional batch reactors, and in addition their results in the reaction performance being higher (Oliveira, Dias, Silva, & Porto, 2018;Sen, Nath, & Bhattacharjee, 2017). PBRs are widely used in several industrial applications including the invert sugar production (Koli & Gaikar, 2017;Lorenzoni et al., 2015). However, these studies were performed using commercial enzyme preparations from yeasts. ...
... The hydrolysis remained efficient up to 100 min (70.22%) (Fig. 6). Lorenzoni et al. (2015) reported a similar qualitative behavior for a maximum production in PBR using the same flow rate and the commercial preparation Maxinvert L. In Table 2 Kinetic and thermodynamic parameters for thermal denaturation of FFase from Aspergillus tamarii URM4634 immobilized on chitosan beads. comparison with other processes, Mafra et al. (2018) reported a conversion of sucrose of 75% and 95% for batch and fed-batch processes, respectively, using a commercial invertase immobilized by Crosslinking Enzyme Aggregates (CLEAs). ...
... However, the authors obtained yields in longer time intervals, 4 and 16.7 h for batch and fed-batch processes, respectively, which are long-time intervals compared with the present results. Most studies on sucrose hydrolysis in enzymatic reactors have been conducted using commercial enzyme preparations, mainly from yeasts (Koli & Gaikar, 2017;Lorenzoni et al., 2015). Studies on other microbial sources, such as filamentous fungi, are still scarce. ...
β-fructofuranosidase (FFase) from Aspergillus tamarii URM4634 was immobilized covalently in chitosan beads. It was characterized biochemically, studied in terms of kinetic and thermodynamic parameters, and applied on conversion of sucrose for invert sugar production in a packed bed reactor (PBR). The optimum reactional conditions were determined and obtained at pH 5.0 and 60 °C. FFase was thermostable at 50–55°C. At 50°C, the enzyme shows longer half-life (t1/2) (594.13 min) and a higher D-value (1,973.64 min). This indicates that immobilized FFase was stable at temperature commonly used in invert sugar production. The following thermodynamic parameters were obtained: activation energy (E*d = 301.57 kJ mol⁻¹), enthalpy (298.76 ≤ ΔH*d ≤ 298.89 kJ mol⁻¹), entropy (579.88 ≤ ΔS*d ≤ 589.27 J K⁻¹ mol⁻¹) and Gibbs free energy (100.29 ≤ ΔG*d ≤ 108.47 kJ mol⁻¹). The high E*d, ΔH*d and ΔG*d values confirmed FFase thermostability. The high and positive values for ΔS*d indicate an increase in disorder due opening of the enzyme structure. The sucrose hydrolysis in PBR showed a maximum invert sugar yield (96.0%) at 15 min of operation. The hydrolysis process remained efficient up to 100 min (70.22%). The results obtained in the present study provide a good indication that immobilized FFase on chitosan beads in PBR is efficient to invert sugar production for food industry.
... Enzyme beads are less susceptible to breaking from mechanical shear stress and the ratio between the substrate and the enzyme is much lower in a PBR than in conventional batch reactors, and in addition their results in the reaction performance being higher (Oliveira, Dias, Silva, & Porto, 2018;Sen, Nath, & Bhattacharjee, 2017). PBRs are widely used in several industrial applications including the invert sugar production (Koli & Gaikar, 2017;Lorenzoni et al., 2015). However, these studies were performed using commercial enzyme preparations from yeasts. ...
... The hydrolysis remained efficient up to 100 min (70.22%) (Fig. 6). Lorenzoni et al. (2015) reported a similar qualitative behavior for a maximum production in PBR using the same flow rate and the commercial preparation Maxinvert L. In Table 2 Kinetic and thermodynamic parameters for thermal denaturation of FFase from Aspergillus tamarii URM4634 immobilized on chitosan beads. comparison with other processes, Mafra et al. (2018) reported a conversion of sucrose of 75% and 95% for batch and fed-batch processes, respectively, using a commercial invertase immobilized by Crosslinking Enzyme Aggregates (CLEAs). ...
... However, the authors obtained yields in longer time intervals, 4 and 16.7 h for batch and fed-batch processes, respectively, which are long-time intervals compared with the present results. Most studies on sucrose hydrolysis in enzymatic reactors have been conducted using commercial enzyme preparations, mainly from yeasts (Koli & Gaikar, 2017;Lorenzoni et al., 2015). Studies on other microbial sources, such as filamentous fungi, are still scarce. ...
Pectinex Ultra SP-L, a food-grade enzymatic preparation with high transfructosylating activity, was covalently immobilized on chitosan beads. The highest immobilization yield (95.9%) was obtained using 4.0% glutaraldehyde for 60 min and 80 rpm at 25 ± 1 °C. The immobilized biocatalyst showed good operational stability, being able to retain, after the third cycle of reuse, no less than 100.0 and 73.9% of starting hydrolytic and transfructosylating activities, respectively. Results of residual activity experiments allowed estimating, for irreversible thermal inactivation of both free and immobilized enzyme, the activation energy (E*d = 234.3 and 242.2 kJ·mol⁻¹), enthalpy (234.4≤ΔH*d≤234.2 kJ·mol⁻¹ and 239.4≤ΔH*d≤239.3 kJ·mol⁻¹), entropy (381.2≤ΔS*d≤379.8 J·mol⁻¹·K⁻¹ and 390.4≤ΔS*d≤389.7 J·mol⁻¹·K⁻¹) and Gibbs free energy (109.3≤ΔG*d≤103.9 kJ·mol⁻¹ and 111.3≤ΔG*d≤103.6 kJ·mol⁻¹). The use of the immobilized enzyme in a packed bed reactor allowed obtaining, after 100 min, a fructo-oligosaccharide mixture containing more than 25 g L⁻¹ 1-kestose.
... There are a large number of bioreactors with different process engineering properties being their choice decisive to achieve high ScFOS yields and productivity [147]. Lorenzoni et al. [90] compared packed bed reactors and fluidised bed reactors for inverted sugar hydrolysis and ScFOS production. Focussing on the ScFOS production, a yield in the ScFOS production of 59% was obtained with the packed bed reactors and 54% for the fluidised bed reactors [90]. ...
... Lorenzoni et al. [90] compared packed bed reactors and fluidised bed reactors for inverted sugar hydrolysis and ScFOS production. Focussing on the ScFOS production, a yield in the ScFOS production of 59% was obtained with the packed bed reactors and 54% for the fluidised bed reactors [90]. Accordingly, the use of packed bed bioreactors is more profitable for ScFOS synthesis than fluidised bed reactors. ...
... Nevertheless, the differences of packed bed bioreactors with the fluidised bed reactors are not too large being then considered the latter bioreactor type an alternative. In addition, Lorenzoni et al. [90] concluded that the packed bed bioreactor is better than the fluidised bed reactor due to its simpler technology and lower requirements of working volumes, which makes a more profitable ScFOS production. ...
Short-chain fructooligosaccharides (ScFOS) are a group of linear fructose oligomers that include 1-kestose, 1-nystose and 1-β-fructofuranosylnystose. ScFOS, which naturally occur at low levels in different plant products, are of high interest as food ingredients because of their prebiotic character, organoleptic characteristics and technological properties. Two different industrial processes are used to achieve large-scale ScFOS production: inulin hydrolysis (enzymatic or chemical hydrolysis) or sucrose biotransformation by transfructosylation (enzymatic synthesis) using specific enzymes like fructosyltransferases and fructofuranosidases. Enzymatic ScFOS synthesis seems to be more advantageous than inulin hydrolysis since it is less expensive, and leads to lower molecular weight FOS. The biotechnological process described to carry out this catalysis includes the production of transfructosylation enzymes, separation, enzyme immobilisation and finally the ScFOS production and purification. Such ScFOS production processes may be conducted under submerged or solid-state fermentation under discontinuous or continuous conditions. Several methodologies with different economic/environmental costs and production yields have been described to carry out these ScFOS production stages, although industrial scale-up needs to be optimised. This review tries to address a revision about enzymatic ScFOS production methods and its scale-up to industrial levels.
... A imobilização de enzimas para a produção de FOS é desejável, pois apresenta uma série de vantagens sobre o uso de enzimas solúveis, como o aumento da estabilidade da enzima, a recuperação facilitada do produto e a reutilização do biocatalisador, reduzindo o custo total do processo . A imobilização da FTase para a síntese de FOS foi descrita por Detofol et al. (2015) e Lorenzoni et al.(2015). No primeiro estudo a FTase de Rhodotorula sp foi imobilizada por adsorção e reticulação sobre um suporte sólido-16 Saccharomyces cerevisiae JZH, JZHΔS, JZH-ssInuC, JZHΔS-SSC e JZHΔS-TSC 17 Glicose, frutose, sacarose e moléculas de inulina ácido (minério de nióbio), no segundo a enzima foi imobilizada covalentemente em partículas de quitosano utilizando glutaraldeído como agente de acoplamento (DETOFOL et al., 2015;LORENZONI et al, 2015). ...
... A imobilização da FTase para a síntese de FOS foi descrita por Detofol et al. (2015) e Lorenzoni et al.(2015). No primeiro estudo a FTase de Rhodotorula sp foi imobilizada por adsorção e reticulação sobre um suporte sólido-16 Saccharomyces cerevisiae JZH, JZHΔS, JZH-ssInuC, JZHΔS-SSC e JZHΔS-TSC 17 Glicose, frutose, sacarose e moléculas de inulina ácido (minério de nióbio), no segundo a enzima foi imobilizada covalentemente em partículas de quitosano utilizando glutaraldeído como agente de acoplamento (DETOFOL et al., 2015;LORENZONI et al, 2015). ...
... Os reatores de leito fixo apresentaram rendimentos mais elevados para síntese FOS, 59%, a um fluxo de 0,08 ml/min. As concentrações de GF 3 e GF 2 foram 218 ±2 g/L e 133 ±1 g/L, respectivamente (LORENZONI et al, 2015). , caracterizaram a LEV de Z. mobilis imobilizada para a síntese FOS e otimizaram a reação para uso na indústria. ...
Os frutooligossacarídeos são carboidratos muitas vezes considerados prebióticos que desempenham um papel fundamental no equilíbrio da microbiota intestinal e na saúde humana. Eles têm despertado um grande interesse pela indústria de alimentos, pois geralmente são empregados como ingredientes que alteram a designação de alimentos para alimentos funcionais. Os frutooligossacarídeos, de forma convencional, são produzidos pela reação de enzimas microbianas a partir de sacarose ou inulina como substratos. Vários estudos sobre a síntese de frutooligossacarídeos têm sido realizados, buscando a otimização dos parâmetros de produção, o desenvolvimento de processos mais eficientes, os diferentes métodos de fermentação e novas fontes microbianas produtoras de enzimas. Assim, esta revisão tem como objetivo revisar os últimos progressos na produção biotecnológica de frutooligossacarídeos.
... To enhance reusability, the expressed recombinant enzyme will be immobilized. Chitosan, a natural polymer derived from the deacetylation of chitin, is ideal for immobilization due to its renewability and low cost [9]. Chitosan could be used for immobilization with the activation step such as the addition of glutaraldehyde as the hydroxyl and amino groups of chitosan can make chemical bonds with the carboxyl group of the enzyme [10,11] thus, making chitosan has a high affinity with protein. ...
... The optimum pH was observed using different buffers such as acetate buffer (pH 4-5), phosphate buffer (pH 5-7), and tris-HCl buffer (pH [8][9]. The recombinant thermostable α-amylase exhibits its highest activity at pH 6, with a slight decrease noted at pH 7, followed by a gradual decline in activity until pH 9, where a sharp plummet occurs (Fig. 2). ...
Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable α-amylase production. Recombinant technology was used to express thermostable α-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 • C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30-90 • C) and pH range (6-8), with optimal activity at 70 • C and pH 6 with great stability at 60 • C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca 2+. The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization.
... These findings suggest that the particle-particle interactions happening in the fluidizedbed system may have led to enzyme loss or mechanical damage to the capsules, compromising their operational stability [33]. Although the fluidized-bed is less prone to column clogging, preferential flow path formation, and bed compression due to weight, the lower hydrolysis rate in this operation mode may also be attributed to the inhibitory effect of reaction products [20,34]. ...
... Despite the reduced inhibition of β-galactosidase activity by the reaction product galactose after immobilization (Fig. 3A), this monosaccharide still interfered with the yield of C@S-Gal enzymatic hydrolysis reactions. Lorenzoni et al. [34] observed that the production of fructooligosaccharides and inverted sugar was approximately 5% higher within packed-bed reactors compared to fluidized-bed ones when using β-fructofuranosidase and β-fructosyltransferase enzymes immobilized on chitosan-based supports. Furthermore, the recirculation of a fraction of the hydrolysis product to generate bed fluidization may have reduced enzyme activities due to the inhibitory effect of the reaction product (glucose). ...
The objective of this study was to develop a bioprocess for lactose hydrolysis in diverse dairy matrices, specifically skim milk and cheese whey, utilizing column reactors employing a core–shell enzymatic system featuring β-galactosidase fused to a Cellulose Binding Domain (CBD) tag (β-galactosidase-CBD). The effectiveness of reactor configurations, including ball columns and toothed columns operating in packed and fluidized-bed modes, was evaluated for catalyzing lactose hydrolysis in both skim milk and cheese whey. In a closed system, these reactors achieved lactose hydrolysis rates of approximately 50% within 5 h under all evaluated conditions. Considering the scale of the bioprocess, the developed enzymatic system was capable of continuously hydrolyzing 9.6 L of skim milk while maintaining relative hydrolysis levels of approximately 50%. The biocatalyst, created by immobilizing β-galactosidase-CBD on magnetic core-shell capsules, exhibited exceptional operational stability, and the proposed bioprocess employing these column reactors showcases the potential for scalability.
... These glutaraldehyde-amino supports have great versatility and can be used for the immobilization of several enzymes. [29][30][31][32][33][34] The amine group provides a cation group to act on ionic adsorption; also, the glutaraldehyde gives some hydrophobicity character and chemical reactivity. 26 Thus, using low ionic strength the enzyme can experience some ionic exchange with the amino groups in the support. ...
... 36,37 Thus, chitosan presents amino and hydroxyl groups in its surface, allowing reactions with crosslinking agents such genipin, 38 and glutaraldehyde. 29,31,34 The primary amino groups of chitosan presents a pKa value of 6.3. 39 Several studies have demonstrated excellent results with the immobilization of enzymes in chitosan substrates, such as β-Galactosidase, 32 cyclodextrin glycosyltransferase, 40 invertase, 29 β-fructofuranosidase, 41 glucose oxidase, 42 catalase and pepsin. ...
In this work, we studied the covalent immobilization of α‐acetolactate decarboxylase (ALDC) on glutaraldehyde activated chitosan beads for application in beers to prevent off‐flavor. The immobilized preparation was characterized regarding yield, efficiency, activity recovery, thermal and pH stabilities, and operational stability in beer. The concentration of activating agent was evaluated and at the best condition an immobilization yield of 84% and activity recovery of 9.1% were verified. Optimal pH was 6.0 for free and immobilized enzymes, however, the immobilized enzyme showed higher relative activity at the tested pH, with emphasis on the pH range from 5.0 to 6.5. Besides, immobilized enzyme showed stabilization factor of almost sevenfold at 60°C compared to free form. The immobilized biocatalyst was tested in repeated batches in beer for α‐acetolactate conversion to acetoin, and after 12 cycles the enzyme presented 80% of its initial activity. To date, this is the most stable immobilized preparation of ALDC reported.
... The literature increasingly presents diverse promising applications of gel particles that eventually scale up to be implemented in fluidized beds. For example, profitable sugars could be continuously produced in LSFB columns at higher flow rates [28]. Emergent technologies for cleaner production such as aerobic cultures for effluent treatment [29] and carbon dioxide capture by immobilized carbonic anhydrase [30] can be implemented at the pilot to industrial-scale GLSFBs. ...
... Emergent technologies for cleaner production such as aerobic cultures for effluent treatment [29] and carbon dioxide capture by immobilized carbonic anhydrase [30] can be implemented at the pilot to industrial-scale GLSFBs. Although most of the processes involving immobilized enzymes are currently carried out in fixed beds, fluidized beds avoid dead zones, and thus the risk of potentially contaminating the product stream [28,31]. Additional industrially relevant advantages of fluidized beds compared to fixed bed reactors are lower pressure drop and superior heat and mass transfer characteristics [31,32]. ...
Currently, various industrial processes are carried out in fluidized bed reactors. Knowing its internal dynamics is fundamental for the intensification of these processes. This work assesses the motion of fluidized calcium alginate spheres under the influence of an upward fluid flow within a 1.2 m high and 0.1 m inner diameter acrylic column. The liquid–solid fluidized bed was compared with a gas–liquid–solid fluidized bed operation mode in terms of mixing behavior. The radioactive particle tracking technique is a proper methodology to study the internal dynamics of these kinds of equipment. Data gathered were analyzed with Shannon entropy as a dynamic mixing measure. Mixing times were found to be between 1 and 2.5 seconds for both fluidization modes. The liquid–solid fluidized bed presents a rather smooth mixing time profile along the column. On the other hand, the gas–liquid–solid fluidized bed showed high sensitivity of entropy production with height, reaching a sharp tendency break at the second quartile of the column. The Glansdorff–Prigogine stability measure can accurately capture flow regime transitions of the gas–liquid–solid fluidized bed, allowing it to be used to construct reliable operative windows for fluidization equipment.
... Immobilization enhanced considerably the thermal stability of the biocatalyst comparing to FE at 60 C, and retained its activity after 50 cycles of batch FOS synthesis. Lorenzoni et al. (2015) evaluated inverted sugar and FOS production using this biocatalyst under two PBR and two fluidized bed reactors (FBR), producing 98 and 94% (grams of invert sugar per grams of initial sucrose), respectively, and 59 and 54% (grams of FOS per grams of initial sucrose), in the PBR and FBR, respectively. In both modes of production, varying the flow rate was possible to modulate the product composition in terms of DP3 and DP4 concentrations. ...
... After 50 cycles of use, both immobilized systems maintained more than 80% its activity. Similar results were obtained by Lorenzoni et al. (2014Lorenzoni et al. ( , 2015, with chitosan particles activated with GA, that lead to not only higher thermal stability but also a retention of high enzymatic activity after 50 cycles of use in a batch production of FOS. For immobilization in chitosan, Nam et al. (2017) proposed that the occurrence of optimum pH and temperature shifts, when compared with the FE may occur due to alterations of the physical and chemical properties of the enzyme during the immobilization process. ...
The highly demanding conditions of industrial processes may lower the stability and affect the activity of enzymes used as biocatalysts. Enzyme immobilization emerged as an approach to promote stabilization and easy removal of enzymes for their reusability. The aim of this review is to go through the principal immobilization strategies addressed to achieve optimal industrial processes with special care on those reported for two types of enzymes: β-galactosidases and fructosyltransferases. The main methods used to immobilize these two enzymes are adsorption, entrapment, covalent coupling and cross-linking or aggregation (no support is used), all of them having pros and cons. Regarding the support, it should be cost-effective, assure the reusability and an easy recovery of the enzyme, increasing its stability and durability. The discussion provided showed that the type of enzyme, its origin, its purity, together with the type of immobilization method and the support will affect the performance during the enzymatic synthesis. Enzymes’ immobilization involves interdisciplinary knowledge including enzymology, nanotechnology, molecular dynamics, cellular physiology and process design. The increasing availability of facilities has opened a variety of possibilities to define strategies to optimize the activity and re-usability of β-galactosidases and fructosyltransferases, but there is still great place for innovative developments.
... Penicillium sp. and Fusarium sp (Ganaie et al., 2014a). FOS production can be evaluated by using commercial fructofuranosidase and fructosyltransferase covalently immobilized on chitosan using glutaraldehyde (Lorenzoni et al., 2015), but it is more usually performed by using immobilized cell systems which avoids laborious enzyme extraction and purification techniques. To accomplish cell immobilization system, entrapment of cells within porous matrices such as calcium alginate or chitosan gel beads are commonly evaluated approaches (Huang et al., 2016;Ganaie et al., 2014b). ...
... To accomplish cell immobilization system, entrapment of cells within porous matrices such as calcium alginate or chitosan gel beads are commonly evaluated approaches (Huang et al., 2016;Ganaie et al., 2014b). (Lorenzoni et al., 2015) operating two packed bed reactors (PBR) and two fluidized bed reactors (FBR) found FOS production achieved a 59% yield in the PBR and 54% in the FBR. They also observed that varying the flow rate it was possible to modulate the FOS composition and the operational stability in the PBR was evaluated for 40 days with no reductions in yields. ...
... Moreover, the liquid flow maintains the biocatalyst in a hydrodynamic balance between the force of gravity and drag forces derived by the upflow substrate stream (Van Zessen, Tramper, Rinzema, & Beeftink, 2005). Several studies have reported the widespread and successful application of FBRs in the food industry, for example, for apple juice clarification (Diano et al., 2008), the production of fructo-oligosaccharides and invert sugars (Lorenzoni et al., 2015), lactose hydrolysis (Roy & Gupta, 2003), the flavour enhancement of beverages (Gueguen, Chemardin, Pien, Arnaud, & Galzy, 1997) and other catalytic reactions (Saponjic et al., 2010). The benefits derive from the continuous operational mode as well as improved mass transfer (Lorenzoni et al., 2015). ...
... Several studies have reported the widespread and successful application of FBRs in the food industry, for example, for apple juice clarification (Diano et al., 2008), the production of fructo-oligosaccharides and invert sugars (Lorenzoni et al., 2015), lactose hydrolysis (Roy & Gupta, 2003), the flavour enhancement of beverages (Gueguen, Chemardin, Pien, Arnaud, & Galzy, 1997) and other catalytic reactions (Saponjic et al., 2010). The benefits derive from the continuous operational mode as well as improved mass transfer (Lorenzoni et al., 2015). ...
... The main factors influencing the void fraction were particle shape, size, and packing. [11][12][13] As for nonaqueous reactions, such as those conducted in an oil medium (e.g., transesterification and esterification), the literature contains a vast collection of immobilized biocatalysts, such as calcium oxide/nanocrystal cellulose/ polyvinyl alcohol, 14 pumice granules loaded with lithium, 15 lipases immobilized on silica-hydroxyl ethyl cellulose particles, 16 and magnetic nanosupports. 3 However, there is a lack of studies on the use of hydrogel beads in PBR in nonaqueous reactions, which commonly involve not only oil but also alcohol (oil transesterification for biodiesel production). ...
There has been a growing interest in ecofriendly enzymatic processes. However, enzyme solubility limits the application of many biocatalysts in continuous systems, requiring the development of cost‐effective strategies for enzyme immobilization. Based on this premise, this study investigated the application of lipase immobilized in starch–alginate beads for oil transesterification in a tubular reactor. An economical derivative was produced by immobilizing Eversa Transform 2.0 in 50:50 (w/w) starch–alginate beads using the jet‐cutting technique. The biocatalyst had a particle size of about 500 μm and activity of 138.67 ± 18.53 U g⁻¹. X‐ray photoelectron spectroscopy showed nitrogen content ranging from 6.38% to 7.29%, with uniform distribution of lipase throughout the beads. Nitrogen isotherms were characteristic of mesoporous materials, with an average pore diameter of 48.09 Å and low surface area (0.69 m² g⁻¹). A face‐centered central composite design was used to study soybean oil transesterification. In the best four runs, the process achieved a mean triglyceride conversion of 45%. High ester productivity levels (2.05 × 10⁻²% ester g⁻¹ biocatalyst min⁻¹ or 1.5 × 10⁻⁴% ester U⁻¹ min⁻¹) were obtained. Biocatalyst reuse led to a twofold increase in ester concentration (14.57% vs 7.7%). These findings confirm the successful development of a low‐cost biocatalyst suitable for use in continuous reactions.
... As the reaction rate of immobilized enzymes does not necessarily increase linearly with increasing enzyme amount or catalyst loading (Ganguly & Nandi, 2015;Ryu et al., 2003), the higher CLEC amount applied in our PBR compared with our FBR might also play a role here. Further, the productivity of the PBR was overall higher than the productivity of the FBR, which is in agreement with other literature precedents (Bódalo-Santoyo et al., 1999;Lorenzoni et al., 2015). ...
Halohydrin dehalogenase HheG is an industrially interesting biocatalyst for the preparation of different β‐substituted alcohols starting from bulky internal epoxides. We previously demonstrated that the immobilization of different HheG variants in the form of cross‐linked enzyme crystals (CLECs) yielded stable and reusable enzyme immobilizes with increased resistance regarding temperature, pH, and the presence of organic solvents. Now, to further establish their preparative applicability, HheG D114C CLECs cross‐linked with bis‐maleimidoethane have been successfully produced on a larger scale using a stirred crystallization approach, and their application in different chemical reactor types (stirred tank reactor, fluidized bed reactor, and packed bed reactor) was systematically studied and compared for the ring opening of cyclohexene oxide with azide. This revealed the highest obtained space‐time yield of 23.9 kgproduct gCLEC⁻¹ h⁻¹ Lreactor volume⁻¹ along with the highest achieved product enantiomeric excess [64%] for application in a packed‐bed reactor. Additionally, lyophilization of those CLECs yielded a storage‐stable HheG preparation that still retained 67% of initial activity (after lyophilization) after 6 months of storage at room temperature.
... It showed increased operational stability for 40 days of continuous FOS production. The FOS yield obtained was 59% in packed bed reactor and 54% in fluidized bed reactor [100]. Mycelia bound FTase from A. aculeatus M105 was entrapped in calcium alginate beads (3%, w/v). ...
In the present study, generation of prebiotic fructooligosaccharides (FOS) using Aspergillus tamarii FTase was optimized by applying response surface methodology. Optimal FOS (251 g L⁻¹) was generated at 28.4°C, pH 7.0 and 50% (w/v) sucrose leading to 1.97‐fold yield enhancement. The m‐FTase was purified using ultrafiltration followed by HiTrap Q HP anion exchange chromatography resulting in 2.15‐fold purified FTase with 12.76 U mg–1 specific activity. Purified FTase (75 kDa) had Km and Vmax values of 1049.717 mM and 2.094 µmol min–1 mg–1, respectively. FOS incorporation led to upregulation of caspase 3, caspase 9, and Bax genes suggesting mitochondrial apoptosis activation in cancer cells. The study describes characteristics of purified FTase from A. tamarii, production optimization of FOS and unravels the role of FOS in anticancer activity against HT‐29 cells.
Practical Application
This study provides detailed insights of kinetic and thermodynamic characteristics of purified FTase, a prebiotic FOS‐generating enzyme. Moreover, the role of the apoptotic genes involved in anticancer activity, and the prebiotic potential of FOS is also investigated. These findings are important in the context of FOS applications, and the optimized production strategies make it useful for industrial application.
... Compared with PBRs, FBRs are less susceptible to column clogging, preferred path formation, and column compression [19,237]. Hidayat et al. [234], in investigating the continuous production of oleic fructose ester using C. rugosa immobilized on modified Amberlite IRA96, opted to use FBR, given that fructose forms crystals during the reaction, which may reduce substrate flow and can cause clogging in reactors such as PBRs. ...
Lipases are efficient biocatalysts with numerous applications in different industrial sectors, such as pharmaceutical, food, and fine chemistry industries. Enzyme immobilization further extends the applications of lipases by enhancing stability, selectivity, and half-life. However, obtaining high catalytic efficiency in reactions catalyzed by immobilized lipases requires optimization of reaction conditions (presence or absence of organic solvents, temperature, medium viscosity) and operational characteristics. This is a technical review focused on exploring the state-of-the-art of industrial applications of immobilized lipases in different reactor systems. Articles published between 2015 and 2020 were selected and analyzed to identify the major factors affecting the application of immobilized lipases, such as types of enzyme support, enzyme–support interactions (immobilization methods), substrate characteristics, and reactor configurations. The most common reactor configurations are discussed, as well as their advantages and disadvantages. In the current literature, studies on immobilized lipases and enzymatic reactors focus on developing strategies to minimize mass transfer limitations and eliminate the need for organic solvents.
... It showed increased operational stability for 40 days of continuous FOS production. The FOS yield obtained was 59% in packed bed reactor and 54% in fluidized bed reactor [100]. Mycelia bound FTase from A. aculeatus M105 was entrapped in calcium alginate beads (3%, w/v). ...
Background
Fructosyltransferases (FTases) are drawing increasing attention due to their application in prebiotic fructooligosaccharide (FOS) generation. FTases have been reported to occur in a variety of microorganisms but are predominantly found in filamentous fungi. These are employed at the industrial scale for generating FOS which make the key ingredient in functional food supplements and nutraceuticals due to their bifidogenic and various other health-promoting properties.
Scope and approach
This review is aimed to discuss recent developments made in the area of FTase production, characterization, and application in order to present a comprehensive account of their present status to the reader. Structural features, catalytic mechanisms, and FTase improvement strategies have also been discussed in order to provide insight into these aspects.
Key findings and conclusions
Although FTases occur in several plants and microorganisms, fungal FTases are being exploited commercially for industrial-scale FOS generation. Several fungal FTases have been characterized and heterologously expressed. However, considerable scope exists for improved production and application of FTases for cost-effective production of prebiotic FOS.
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HIGHLIGHTS
• Fructosyltrasferase (FTase) is a key enzyme in fructo-oligosaccharide (FOS) generation
• Developments in the production, properties, and functional aspects of FTases
• Molecular modification and immobilization strategies for improved FOS generation
• Fructosyltransferases are innovation hotspots in the food and nutraceutical industries
... Enzyme immobilisation may be achieved by anion exchange resins with amines as functional groups by mixing previously purified enzymes with the corresponding resin [6]. Immobilisation with chitosan is another possibility for continuous FOS production [114]. However, all these concepts have in common to be complex and laborious. ...
Fructans are carbohydrates consisting of fructose monomers linked by β-2,1- and/or β-2,6-glycosidic bonds with linear or branched structure. These carbohydrates belong to the group of prebiotic dietary fibre with health-promoting potential for humans and mammals due to their indigestibility and selective stimulation of microorganisms in the gastrointestinal tract. This makes fructans interesting mainly for healthy food as well as animal feed applications. As a consequence of a growing public awareness for animal welfare, dietary fibre and thus fructans move into the focus as a fibre-rich feeding improving not only animals’ health but also their well-being. Against this background, this paper summarises the known effects of fructans focusing on pigs and highlights the state of the art in fructan production processes from plant material as well as selected current research lines. Additionally, an attempt is made to assess the potential of European fructan production for an application as animal feed. Based on this, challenges in the field of fructan production are addressed and alternative substrates for fructans are discussed and pointed out.
... The FOSs have attracted special attention because of their prebiotic properties and also their sweet taste being very similar of sucrose [13]. These are short chains of fructose molecules which are excellent source of soluble fibres and do not add calories because they are not digestible by stomach enzyme [14,15]. Keeping the above facts in view, the present study was conducted to optimize fructooligosaccharide fortified apple-whey based herbal RTS beverage and to evaluate its quality during storage. ...
Apple based beverages are rich source of sugars and dietary fibres but deficit in proteins and some minerals like calcium. Whey- a major environmental pollutant from dairy industry is an excellent source of proteins and calcium. Non-nutritive sweeteners can be used for the development of low calorie hypoglycaemic beverages. Therefore, the present work was planned to optimize and evaluate the effect of fructooligosaccharide (FOS) incorporation on physico-chemical, nutritional and sensory characteristics of apple-whey blended ready-to-serve beverage. Herbal apple-whey blended beverage was prepared by using 75% apple juice+25% whey with 2.5% jaljeera extract and 13oB TSS. Results revealed that the beverage with 75% sweetos (mixture of fructooligosaccharide and sucralose) was found most acceptable with overall acceptability score of 8.59±0.26. Selected beverage had 12.20±0.01oBrix TSS, 0.30±0.01% acidity, 6.28±0.03% reducing sugar, 9.43±0.06% total sugars, 10.57±0.05 mg/100 g ascorbic acid, 37.84±0.03 mg/100 g total phenols, 15.64±0.02 mg/100 mL calcium, 0.28±0.03% protein and 1.59±0.03% FOS thus depicting enhanced nutritional value. Beverages were stored successfully for a period of 60 days under ambient and refrigerated conditions. However, various quality parameters of RTS beverage were retained higher under refrigerated storage conditions. Conclusively, this creates a scope for better health beverage as well as efficient utilization of whey.
... Another important step in bioreactor design is to determine the optimum enzymatic reaction system to be utilized [29,30]. As previously mentioned, FOS production, primarily 1-kestose, is significantly influenced by the initial concentration of SS because transfructosylation is favoured over hydrolysis at high sucrose concentration. ...
Fructooligosaccharides (FOS) have gained significant attention for their prebiotic properties. Given that sugarcane syrup (SS) is sucrose-rich but with other nutritional benefits, its direct transformation into FOS may add value to this product. Therefore, the aims of this study were to develop FOS conversion from SS and to define the kinetic behaviour of FOS synthesized in a 1-L specialized pH-stat bioreactor (SPSB). The SS was composed of sucrose (58.93%) with considerable antioxidant capacities and Ƴ-aminobutyric acid. The developed SPSB process consisted of three stages: evaporation of sugarcane juice into syrup (68–75 °Brix) (stage 1), optimization of the Viscozyme L and SS mixture at different reaction temperatures (47–55 °C) (stage 2), and upscaling of the optimized reaction system under defined conditions in a 1 L-SPSB system (stage 3). In the 1 L-SPSB system, the enzymatic reaction yielded 32.22% of FOS from SS after a 6 h reaction, which is comparable with a pure system containing an equivalent concentration of 10% of sucrose as initial substrate with 39.55% yield. This result demonstrated the efficient conversion of SS into FOS, supporting the utilization of sugarcane juice for its health benefits.
... In this reactor, the flow of substrate keeps the immobilized enzyme particles in a fluidized state, thus obtaining a high catalytic surface area. FBRs have widespread application in the food industry [i.e. for apple juice clarification (Diano et al., 2008), for the production of fructo-oligosaccharides and invert sugars (Lorenzoni et al., 2015), for lactose hydrolysis (Roy & Gupta, 2003), for the flavour enhancement of beverages (Gueguen, Chemardin, Pien, Arnaud, & Galzy, 1997) and for controlling malolactic fermentation in wine (Cappannella et al., 2016)]. ...
The recently growing demand of gluten-reduced beer is leading to the development of diverse approaches to be applied in brewing. The current work focuses on the development of an innovative and sustainable biocatalytic tool for the continuous production of gluten-reduced beer, based on the application of immobilized prolyl endopeptidase from Aspergillus niger (AN-PEP). This food-grade protease has been immobilized on A. niger chitosan beads and applied, for the first time, for the reduction of gluten in a commercial beer from barley malt. The immobilization procedure was optimized for maximizing the specific activity of the biocatalyst (0.016 I.U./mgBSAeq) and the best performance was reached using an immobilization solution at an initial protein concentration of 0.3 mgBSAeq/mL.
The immobilization increased the thermal stability of the protease, which showed similar catalytic properties in synthetic beer (toward the synthetic substrate Z-Gly-Pro-pNA) when it was applied at 20 °C or at 50 °C. The continuous treatment in fluidized bed reactor (FBR), containing 10 g of immobilized AN-PEP (corresponding to 0.0036 gBSAeq), was optimized varying the flow rate (Qv). The suitable conditions to achieve reduction of the intact gluten of authentic beer was Qv of 728 mL/min. The continuous treatment in FBR allowed us to reduce the initial gluten content (65 mg/kg) in the commercial beer from barley malt, reaching the concentration of 19 mg/kg after 9 h and 15 mg/kg after 10 h of treatment.
... La inmovilización de enzimas por unión covalente se ha realizado sobre: microporos de poli(2-hidroxietil metacrilato-glicidil metacrilato) (Danisman et al., 2004), lecitina obtenida de la leguminosa Cajanus cajan usando glutaraldehído (Ahmad et al., 2001), superficies de polianilina modificadas por copolimerización con ácido acrílico (Chen et al., 2000), tubos de cloruro de polivinilo (Kumar et al., 2008), microperlas de poli(estireno-2-hidroxietil metacrilato) activadas químicamente (Altinok et al., 2008), esferas de quitosano en reactores de lecho empaquetado y lecho fluidizado (Lorenzoni et al., 2015), matriz de poli(metacrilato de 3-metiltienilo)/poli(ácido 3-tiofenacético) (Dizge et al., 2008), entre otras. ...
En el estudio se desarrolló un método alternativo para la hidrólisis de sacarosa por β-D-fructofuranosidasa de Saccharomyces cerevisiae inmovilizada sobre nanopartículas magnéticas de ferrita de cobalto (NPM-CoFe2O4), una metodología que permite el reúso de la entidad biológica. Los resultados revisados en la literatura alertan sobre la modificación de la actividad de las enzimas cuando son inmovilizadas; por esta razón se cuantificaron los cambios en las propiedades catalíticas de la enzima inmovilizada para conocer la eficacia de este sistema a escala de laboratorio. Las nanopartículas magnéticas fueron sintetizadas por el método de reducción poliol y caracterizadas por Difracción de rayos X, Magnetometría de Muestra Vibrante, Microscopia Electrónica de Barrido y Microscopia Electrónica de Transmisión. Las nanopartículas se recubrieron con quitosano y se activaron con glutaraldehído, el cual acopló la β-D-fructofuranosidasa sobre las nanopartículas mediante unión covalente. La inmovilización se caracterizó por Espectroscopía Infrarroja con Transformada de Fourier, y la cantidad de enzima inmovilizada y el rendimiento de la inmovilización se determinó por el método espectrofotométrico para la cuantificación de proteína de Bradford. Se investigó el comportamiento catalítico de la enzima en función del pH y la temperatura. El pH operacional óptimo fue 0.5 más alto para la enzima inmovilizada respecto a la enzima libre. La temperatura operativa óptima fue de 50°C para la enzima libre e inmovilizada. Luego de la inmovilización Vmáx disminuyó 2.96% y Km aumentó en un factor de 1.7. Las bio-nanopartículas retuvieron un 95.89 y 91.79% de la actividad inicial, en el segundo y tercer ciclo de uso.
... The two temperatures were selected to minimize any form of protein inactivation or interactions due to many temperature variations as the enzyme preparation is not completely purified. 36 A summary of the immobilization results is displayed in Table 1. ...
Short‐chain fructooligosaccharides (scFOS) are nutraceuticals with numerous applications in the food and pharmaceutical industries. The production of scFOS using immobilized biocatalysts offers some functional and technical advantages over free enzyme counterparts. To investigate the economic potential of the immobilized enzyme system relative to the free enzyme system, a techno‐economic comparison was conducted on three methods of scFOS production (powder and syrup forms) at a capacity of 2000 t per annum (tpa) by enzymatic synthesis from sucrose: the free enzyme (FE), calcium alginate immobilized enzyme (CAIE), and amberlite IRA 900 immobilized enzyme (AIE) systems. These processes were simulated in Aspen Plus to obtain the mass and energy balances and to estimate the operating and capital costs, followed by economic evaluation and sensitivity analysis. Profitability analysis showed that all three systems are economically viable as their associated minimum selling prices (MSP) were well below the scFOS market price of 5 kg⁻¹ because the savings on cost as a result of enzyme immobilization could not offset the additional costs associated with immobilization. Sensitivity analysis demonstrated that total operating cost, fixed capital investment, and internal rate of return (% IRR) have the greatest effects on the MSP. Furthermore, the syrup form of scFOS production leads to 29% less MSP, compared to powder form. In addition, the studied plant capacities of 5000 and 1000 tpa showed 10% and 16% reductions on MSP respectively. © 2019 Society of Chemical Industry and John Wiley & Sons, Ltd
... The two temperatures were selected to minimize any form of protein inactivation or interactions due to many temperature variations as the enzyme preparation is not completely purified. 36 A summary of the immobilization results is displayed in enzyme structure during immobilization by adsorption. 37 However, the efficiency values were greater than that of the CA beads. ...
The immobilization of β‐fructofuranosidase for short‐chain fructooligosaccharide (scFOS) synthesis holds the potential for a more efficient use of the biocatalyst. However, the choice of carrier and immobilization technique is a key to achieving that efficiency. In this study, calcium alginate (CA), Amberlite IRA 900 (AI900) and Dowex Marathon MSA (DMM) were tested as supports for immobilizing a novel engineered β‐fructofuranosidase from Aspergillus japonicus for scFOS synthesis. Several immobilization parameters were estimated to ascertain the effectiveness of the carriers in immobilizing the enzyme. The performance of the immobilized biocatalysts are compared in terms of the yield of scFOS produced and reusability. The selection of carriers and reagents was motivated by the need to ensure safety of application in the production of food‐grade products. The CA and AI900 both recorded impressive immobilization yields of 82 and 62%, respectively, while the DMM recorded 47%. Enzyme immobilizations on CA, AI900 and DMM showed activity recoveries of 23, 27, and 17%, respectively. The CA, AI900 immobilized and the free enzymes recorded their highest scFOS yields of 59, 53, and 61%, respectively. The AI900 immobilized enzyme produced a consistent scFOS yield and composition for 12 batch cycles but for the CA immobilized enzyme, only 6 batch cycles gave a consistent scFOS yield. In its first record of application in scFOS production, the AI900 anion exchange resin exhibited potential as an adequate carrier for industrial application with possible savings on cost of immobilization and reduced technical difficulty.
... This produces higher yields, which suggests that such enzyme preparations can be used as viable alternatives for industrial FOS production (Nguyen, Rezessy-Szabó, Czukor, & Hoschke, 2011;Singh & Singh, 2010;Singh et al., 2016). The commercial exploitation of industrial biotransformations using enzymes is heavily dependent on their cost, however, and often this process is hampered by the lack of prolonged operational stability, and difficulties in recovering and reusing the enzymes (Lorenzoni et al., 2015). Alternatives commonly used to overcome such technical drawbacks are enzyme immobilization (which is only justified if the enzyme is expensive, or is inactivated under reaction conditions), or in situ enzyme production by selected microorganisms because of their easy cultivation and high yields, which is the best way to obtain low-cost enzymatic preparations (Cadena et al., 2010;Vega-Paulinoa & Zúniga-Hansena, 2012;Xu et al., 2016). ...
Saccharomyces paradoxus, a native microorganism of the aguamiel, was used successfully for endoinulinase synthesis for agave fructooligasaccharide (FOS) production. We optimized the fermentation parameters to maximize the enzyme synthesis, and we performed enzyme kinetics studies to achieve agave fructans hydrolysis. The results showed that under constant operating conditions (pH 7.7, 40 °C, 175 rpm of agitation, and 0.005 VVM of aeration) results in the production of an enzymatic extract with 49.57 mg/L. This enzymatic extract, when mixed with an agave fructans solution containing 37.8 g/L, allowed us to obtain products with 18% more FOS content the original concentration. The mass spectrum plot shows that the hydrolyzed product contains FOS with a degree of polymerization from 5 to 9 hexose units. These results are promising because they show FOS production from agave and confirm that importance of using native strains in the design of directed fermentation processes.
... This strategy allowed maximum amount of enzyme to be immobilized over a larger specific surface area thereby leading to increased FOS yield (Chen et al., 2014). For continuous production of FOS, comparative studies have been carried out on fluidized bed and packed bed reactors of which the latter reactor was found to be operationally more stable as well as kinetically favorable when continuously run for 40 days (Lorenzoni et al., 2015). ...
... mixture of saccharides including fructosyl-nystose (GF4), nystose (GF3), kestose (GF2), glucose (G), fructose (F), and non-reacting sucrose (GF). Recently, continuous production techniques employing packed bed reactors (Tanriseven and Aslan 2005;Ghazi et al. 2005;Chen et al. 2014), fluidized bed reactors (Lorenzoni et al. 2015), and enzyme membrane reactors with inert (Ur Rehman et al. 2016) and catalytic membranes (Nishizawa et al. 2000;Hicke et al. 2006) have received considerable attention. In contrast with a conventional batch process utilizing soluble enzymes in a stirred-tank reactor, these techniques allow the production of an enzyme-free product in a continuous manner by recovering the biocatalysts. ...
Short-chain fructo-oligosaccharides (FOS) are considered as low-calorie carbohydrates with prebiotic function. They can be produced from sucrose by fructosyltransferase activity, resulting in a mixture of saccharides with different chain lengths. Current practice for carbohydrate analysis involves the use of time-costly and off-line chromatographic procedures. This study is dedicated to the development of an artificial neural network (ANN) model for predicting carbohydrate composition from the direct measurement of UV spectra. A total of 182 samples were generated by operating an enzyme membrane reactor (EMR) under both optimal and suboptimal settings. The concentration data determined by HPLC and corresponding absorbance readings were used to train a two-layer feedforward neural network. The optimized model was then validated by using new observations that were not involved in the training. The model explained 98, 97, and 88% of the variation in the composition of the new observations regarding the main components sucrose, kestose, and glucose with a mean squared error of prediction of 6.59, 3.40, and 2.81, respectively. The results indicate that the proposed UV-ANN method has a great potential to be used for the real-time monitoring of the bioconversion.
Graphical Abstractᅟ
... This strategy allowed maximum amount of enzyme to be immobilized over a larger specific surface area thereby leading to increased FOS yield (Chen et al., 2014). For continuous production of FOS, comparative studies have been carried out on fluidized bed and packed bed reactors of which the latter reactor was found to be operationally more stable as well as kinetically favorable when continuously run for 40 days (Lorenzoni et al., 2015). ...
Sustainable agriculture is vitally important in today’s world because it offers
the potential to meet our future agricultural needs, something that conventional
agriculture will not be able to do. Global agriculture has to double food
production by 2050 in order to feed the world’s growing population and at the
same time reduce its reliance on inorganic fertilizers and pesticides. To achieve
this goal, there is an urgent need to harness the multiple beneficial interactions
that occur between plants and microorganisms. Microorganisms has vital
role in agriculture in order to promote the exchange of plant nutrients and
reduce application of chemical fertilizers as much as possible. Plant Growth-
Promoting Rhizobacteria (PGPR) is able to exert a positive effect leading
plant growth. Beneficial plant– microbe interactions in the rhizosphere can
influence plant vigor and soil fertility. The eco-friendly approaches inspire a
wide range of application of plant growth promoting rhizobacteria (PGPRs),
endo- and ectomycorrhizal fungi, cyanobacteria and many other useful
microscopic organisms led to improved nutrient uptake, plant growth and
plant tolerance to abiotic and biotic stress. The present chapter highlighted on
the beneficial influences of microorganisms on plant growth include nitrogen
fixation, acquisition and uptake of major nutrients, promotion of shoot and
root growth, disease control or suppression and improved soil structure.
Some of the commonly promoted and used beneficial microorganisms in
agriculture worldwide include Rhizobia, Bacillus, Pseudomonas, Mycorrhiza,
Azospirillum, Trichoderma, Streptomyces species and many more. This chapter presents the current perspectives on the role of beneficial microflora in
agriculture sustainability.
Keywords: Sustainable agriculture, PGPR, Rhizosphere, Microbial
interaction, Biofertilizer.
... Several material for enzyme immobilization such as bentonite [3], sheparose, gelatin and chitosan were generally used [4]. Chitosan is one of the interesting material, which a natural polymer allowed high biocompatibility, low cost, renewable, and easy to obtain from chitin by deacetylation with alkali [5]. The lower level of N-acetyl groups (<40%) provides greater solubility in pH below 6.5, nontoxic, available in different forms (powder, gel, fibers, and membranes), and easy to derivate and demonstrating high affinity protein. ...
The purpose of this study was to optimize the amylase immobilization using a chitosan bead and to characterize immobilized amylase of Bacillus thuringiensis Bacteria HCB6. This study was started of amylase production, continued by immobilization optimization including ratio of chitosan:enzymes, enzyme-matrix contact time, substrate concentration, pH effect, incubation temperature effect, reaction time, and stability of immobilized enzyme. Amylase activity assay was dinitro salicylic (DNS) method. The results showed the optimum chitosan:enzyme ratio was 2.5: 1 (v/v), immobilization contact time of 18 hours and immobilization efficiency of 87.93%. Furthermore, immobilized amylase of B. thuringiensis HCB6 showed optimum substrate concentration of 1.5%, optimum pH of 6, optimum incubation temperature of 37 ° C, and the reaction time of 30 minutes. The Michaelis-Menten constant KM value for free and immobilized amylase were 5.30% and 1.33% respectively. Immobilized amylase can be used up to five times with the remaining activity of 43.3%.
... The results of this study are anticipated to contribute to the development of alternative and low-cost CG sorbent materials for the sequestration of agrochemicals in aquatic environments. The CG materials can be further modified for use in large-scale applications such as fluidized bed reactors (Lorenzoni et al. 2015;Zhou et al. 2014a) for the sequestration of contaminants in drinking water reservoirs. ...
The sorption properties are reported for chitosan and its cross-linked forms (chitosan-glutaraldehyde; CG) with some model agrochemical sorbates [pentachlorophenol (PCP), 2,4-dichlorophenol (2,4-DCP) and 2,4-dichlorophenoxy acetic acid (2,4-D), dicamba and carbofuran]. The CG cross-linked materials were prepared at variable C:G monomer mole ratios: 1:0.5 (CG1), 1:1 (CG2), (CG3). The sorbents were characterized using diffuse reflectance infrared Fourier transform spectroscopy, thermogravimetric analysis and a dye sorption method using phenolphthalein. The sorption studies were carried out in aqueous solution at pH 9 except for dicamba and carbofuran (pH 7). The isotherm results were evaluated by the Sips, Freundlich, and Langmuir models. The Sips model provided the “best-fit” results where the sorption capacity increased as the cross-linker content of the CG materials increased. The relative uptake for chitosan and its cross-linked forms adopted the following order: PCP > 2,4-DCP > 2,4-D. In the case of dicamba and carbofuran, the former had a higher sorptive uptake. The variable uptake of the sorbates were attributed to their relative lipophilicity where the main driving force of these solid-solution systems relates to hydrophobic effects, in accordance with the tunable physicochemical properties of the chitosan sorbent materials.
Short-chain fructooligosaccharides (sc-FOS) are prebiotics beneficial to human health, which can be synthesized from raw material containing a high sucrose content. Sugarcane syrup (SS) without molasses removal contains sucrose as a major sugar and is a rich source of several bioactive compounds. The aim of this study is to investigate factors affecting sc-FOS synthesis from SS using commercial enzyme preparations containing fructosyltransferase activity as biocatalysts. sc-FOS content increased significantly as the sucrose concentration of SS in the reaction mixture increased up to 40% (w/v). Changes in carbohydrate compositions during the transfructosylating reaction of a pure sucrose solution and SS prepared from the two sugarcane cultivars Khon Kaen 3 and Suphanburi 50, catalyzed by Pectinex Ultra SP-L and Viscozyme L, showed similar profiles. Both enzymes showed a high ability to transfer fructosyl moieties to produce sc-FOS and a plateau of the total sc-FOS concentration was observed after 4 h of reaction time. For the pure sucrose solution and SS (Suphanburi 50), Viscozyme showed a superior ability to convert sucrose to Pectinex, with a higher sc-FOS yield (g FOS/100 g of initial sucrose), GF2 or 1-kestose yield (g GF2/g of initial sucrose) and GF3 or nystose yield (g GF3/g of initial sucrose). sc-FOS syrup (FOS SS) and the foam-mat-dried syrup powder prepared from SS and FOS SS, respectively, contained a high total phenolic content and possessed higher antioxidant activities than those prepared from pure sucrose, but contained lower calories.
Fructooligosaccharides (FOS) are fructose oligomers that are beneficial to human health and nutrition for being prebiotic sugars. Their production occurs by a transfructosylation reaction in sucrose molecules catalyzed by fructosyltransferase enzymes (FTase, E.C.2.4.1.9) adhered to microbial cells. The purpose of this work was to study the preparation, enzymatic activity, and stability of glutaraldehyde-crosslinked Aspergillus oryzae IPT-301 cells used as biocatalyst for the transfructosylation reaction of sucrose in a packed bed reactor (PBR), aiming at FOS production. The highest transfructosylation activity (AT) was presented by the biocatalyst prepared by crosslinking at 200 rpm and 45 min. The highest AT in the PBR were obtained at 50?C, flow rates from 3 mL min-1 to 5 mL min-1 and sucrose concentrations of 473 g L-1 and 500 g L-1. The enzymatic kinetics was described using the Michaelis-Menten model. Finally, the biocatalyst showed constant AT of approximately 75 U g-1 and 300 U g-1 for 12 h of reaction in the PBR operating in continuous and discontinuous flow, respectively. These results demonstrate a high potential of glutaraldehyde-crosslinked A. oryzae IPT-301 cells as heterogeneous biocatalysts for the continuous production of FOS in PBR reactors.
Fructooligosaccharides, a low‐calorie alternative sweetener with prebiotic effects, can be synthesized from sucrose via enzymatic reactions. Recently, methods for producing novel fructooligosaccharides using free and immobilized Bacillus licheniformis RN‐01 levansucrase were developed at the laboratory scale. However, there was a need to apply engineering knowledge in production process design and scale‐up to develop and evaluate the feasibility of this technology in the industrial scale. In this paper, three different types of reactors for fructooligosaccharides production using B. licheniformis RN‐01 levansucrase were compared in terms of technical and economic aspects. The previously collected experimental data, including enzyme activities, immobilization yields, product yields, and material balances, were used to estimate the capital and operating costs of fructooligosaccharides production using (a) immobilized enzyme in a stirred‐tank reactor, (b) immobilized enzyme in a packed‐bed reactor, and (c) free enzyme in a stirred‐tank reactor. The technical issues in reactor design and operation were also evaluated. The results showed that, in terms of minimum product selling prices, the process using immobilized enzyme had more economic advantages than the process using free enzyme because the cost savings from enzyme purchasing could compensate the additional equipment and chemical costs in the enzyme immobilization process. The results could be a guideline for selecting suitable reactor types for fructooligosaccharides production and provide directions for further reactor design and development.
The aim of this study was to continuously hydrolyze mango peel pectin, an agricultural waste, into pectic oligosaccharides (POS) with antibacterial activity. A packed-bed reactor (PBR) with immobilized polygalacturonase was designed, and the effects of the process conditions (flow rate, substrate concentration, and enzyme amount) were evaluated. The optimal conditions were obtained using response surface analysis, and a stable POS production with a yield of 94.56% was achieved. The PBR retained 82% of its initial activity after continuous operation for 72 h. The POS products exhibited good antibacterial activity against E. coli, S. aureus, B. subtilis, and S. typhimurium. The destructive effects of POS on the membrane system integrity were observed via scanning electron microscopy and the leakage of intracellular nucleic acids. Thus, the PBR is a powerful tool for continuous POS production, and hence, it can be applied as an alternative to chemical preservatives in the food industry.
The current demand for the need of sustainable processes is increasing day by day. The considerable growth in biotechnological industries is attributed to enhanced enzyme production and their stability and development in technologies for process intensification, which can pivot way towards large scale production and economical formulation. White biotechnology deals with the implementation of biotechnology at the industrial level. It is considered one of the key tools, which can be utilized to boost the bio-economy. With the increasing research in the field of biocatalysis, it is proved to be a green tool in the synthesis of fine chemicals. It is quite evident that biocatalysis has numerous advantages over chemical catalysis. Immobilization of enzymes is the key towards practical and commercial viability of the processes, which improves activity, stability, and reusability of enzymes, thereby making the biocatalytic reactions more sustainable and economical. The amalgamation of biocatalysis with process intensification techniques such as microwaves, ultrasound, hydrodynamic cavitation, and the combination of chemical and biological catalysis can be an effective way to enhance the yield and productivity of these processes. The current review summarizes and discusses the applications of immobilized enzymes used in different process intensification techniques. The techniques utilized can help in achieving better productivity and enhanced yields as compared to those with conventional techniques. These advances in bioprocesses will lead to rapid application at the industrial level for strengthening the bio-based economy.
Immobilization of biological molecules such as cells, enzymes, antibodies and other catalytically active cellular components greatly enhances their application in biocatalysis. Therefore, immobilized biomolecules have extensively been investigated for successful technological advancement. This chapter provides a comprehensive overview on the evolution of immobilization that took place over the years with the fundamental understanding on immobilization and importance of immobilization support structures called “scaffolds” followed by practical applicability reported in multidisciplinary fields like industrial bioprocessing, biomedicine, biosensing and biorecognition, as well as environmental biotechnology but partly restricted to activities in authors laboratory. In last section of the chapter, latest computational practices like biosimulation techniques have been discussed which helps in predicting the mode of steady execution and realization of biomolecule’s immobilization for viable bioprocess developments.
The feasibility of operation on a fluidized bed reactor (FBR) with inulinase imbedded in the gelatin alginate microspheres was investigated in order to improve the higher inulin conversion yields and enzyme stability than that of the previously obtained with the packed bed reactors. The operation processes were based on statistical analyses, the operational conditions of immobilized inulinase in a FBR system have been determined for immobilized enzyme load of 18 g, substrate concentration of 80 g/L, expansion ratio of 1.4 and substrate flow rate at 0.5 mL/min. According to the above-mentioned research parameters, the continuous fructose preparation with FBR system was sustainable for 10 days (240 h) and gained the productivity of 86.4 g/Ld. Compared with the previous results of the packed-bed reactor, the immobilized inulinase in the FBR system was applied in the inulin conversion, which appeared more effective. This study suggested that a system for the continuous and efficient enzymatic conversion of inulin in the FBR was founded, which could be potentially applicable for the scale-up production.
Graphic Abstract
The diagram of fluidized-bed reactor with immobilized inulinase for continuous inulin conversion
Fructo- and galacto-oligosaccharides (FOS and GOS) are non-digestible oligosaccharides with prebiotic properties that can be incorporated into a wide number of products. This review details the general outlines for the production of FOS and GOS, both by enzymatic synthesis using disaccharides or other substrates, and by hydrolysis of polysaccharides. Special emphasis is laid on technological aspects, raw materials, properties, and applications.
The bioprocessing industry is regarded as one of the fastest growing sectors with an estimated compound annual growth rate of 8.6%. The global market for biopharmaceuticals is projected to rise to a market value of USD 727.1 billion by 2025. Due to the unique nature of bioprocessing industries wherein micro-organisms are employed to manufacture the desired products, these processes are prone to additional hazards such as biological hazards and dust explosion amongst others. This necessitates the need to review the existing research in the fields of biotechnology and bioprocessing to reduce/eliminate these hazards and pave the path towards a safer bioprocessing industry. The study involves developing a framework comprising of studying the recent technologies that reduce/eliminate these hazards involved in the bioprocessing industries that include dust explosions, loss of containment of toxic chemicals, loss of containment of biohazard/active product ingredient, fire, and explosion and mapping these technologies with respect to inherent safety principles that include substitution, minimization, moderation and simplification with an overall objective of minimizing the risk associated with bioprocesses and moving towards an inherently safer bioprocessing industry.
Microbial enzymes find application in enzymatic reactions for the production of fructooligosaccharides (FOS). Demands for an alternative healthy sweetener and multifunctional FOS have prompted investigators to explore microorganisms for microbial enzyme production as well as to develop bioprocesses for the production of high-fructose syrup and oligosaccharides based on transfructosylation of sucrose or hydrolysis of inulin. Fructosyltransferases have been characterized in several molds, yeasts, and a few bacteria. Recently, there has been a growing interest in finding novel enzyme producers by using agroindustrial media in both submerged and solid-state fermentation. This review discusses current knowledge on production, properties, and applications of microbial enzymes involved in the production of fructooligosaccharides and considers recent advances in the field. It also describes a comprehensive and illustrative analysis on basic and applied aspects of fungal enzymes, different substrates of microbial growth, and fundamental biotechnological and catalytic aspects to illustrate the potential of these biocatalysts in the food and bioprocessing industries.
Invertases or β-d-fructofuranosidases are used to hydrolyze sucrose and polysaccharides, which have the same type of β-d-fructofuranosyl bond, to obtain fructose and glucose as final products. These enzymes are also able to perform this reaction in the reverse direction. The resulting mixture of the fructose and glucose is referred as “inverted sugar” because of the inversion of its optical property from the positive rotation to the negative rotation. Invertases are important in the food industry, especially in confectionery, as a catalytic agent in obtaining an artificial sweetener. Thus, it is used for the preparation of formulas that prevent crystallization of certain sweet preparations, employing in the chocolate industry. In some syrup, it is also employed to increase its sweetening properties such as manufacturing of soft caramel fillings. The most common form of this inverted sugar is honey, which is a supersaturated mixture of glucose and fructose. In addition, invertases are able to synthesize fructooligosaccharides through fructotransferase where sucrose is presented in high concentrations. The fructooligosaccharides are associated to improve human health. Invertases are produced by plants, bees, and microorganisms. But, the filamentous fungi belonging to the Aspergillus genus and yeast such as Saccharomyces cerevisiae and Candida utilis are the most prominent organisms used for invertase production. The purpose of this chapter is to compile results of recent advances on invertase including its production, application, and molecular characterization.
Fructofuranosidase (EC 3.2.1.26) from Aureobasidium sp. ATCC 20524, recovered from 5 L fermented medium, purified by two simple steps with a yield of 65 % and a purification factor of 16, was immobilized by adsorption onto titanium dioxide (FTIO). The enzyme was also covalently immobilized onto TiO 2 coated with polyethyleneimine (FTIOP) and encapsulated in gellan gum (FTIOPG). FTIO and FTIOP recorded an activity of 903 U g ⁻¹ and 9212 U g ⁻¹ , respectively. The immobilized enzyme showed high activity and stability at pH levels ranging from 4.0 to 8.0 and there were no changes in the temperature profile for either methodology when compared with free fructofuranosidase. The immobilized biocatalysts were reused 7 times for FOS production without significant activity loss, except FTIO at pH 5.0. Gellan gum was used for FTIOP encapsulation. FOS production was performed in a batch and a continuous reactor using FTIOPG as a biocatalyst. Batch conversion (g FOS /g initial sucrose ) was around 60 % for initial sucrose concentrations of 100, 300 and 600 g L ⁻¹ , at a time of maximum conversion. Fixed-bed reactor operational stability was remarkable, providing a constant FOS production in the outlet of the column during 720 h.
Dextransucrase from Leuconostoc mesenteroides B-512F was covalently immobilized on glutaraldehyde-actived chitosan particles. The best initial protein loading (400 mg/g of dried support) showed 197 U/g of catalytic activity. The optimal reaction pH and temperature of this new biocatalyst were determined to be 4.5 and 20 °C, respectively. Regarding the thermal stability, the immobilization enhanced enzyme protection against high temperatures, whereas glucose and maltose acted as stabilizers. The biocatalyst was stable under storage at 5 °C for a month. The biocatalyst presented good operational stability, retaining up to 40% of its initial activity after ten batch cycles of reaction to obtain oligosaccharides. These results suggest the use of the immobilized dextransucrase on chitosan particles as a promising novel biocatalyst to produce dextran and oligosaccharides.
Cadaverine, a five carbon diamine (1,5-diaminopentane), plays a role as a building block of polyamides and it can be made by fermentation or direct bioconversion. To improve its production by increasing reusability of immobilized enzyme and avoid separation of enzyme in bioconversion, a continuous Llysine bioconversion process for cadaverine production has been developed. Various divalent cations, alginate concentrations, cell density with alginate and flow rate of feed were examined to maximize the lysine decarboxylase activity of the whole-cell immobilized beads. Under the selected conditions,123 h of continuous cadaverine production has been performed and 5.5 L of 819 mM cadaverine were produced with 14 mL reactor resulting in 466.5 g of cadaverine. Cadaverine production was possible with small volume reactor maintaining relatively high concentration of substrate
Thermomyces lanuginosus lipase (TLL) was covalently immobilized on Immobead 150 (ImmTLL) via epoxy groups of the support and the lysine of the enzyme to be used in the synthesis of butyl butyrate. The optimization of the esterification reaction parameters was carried out by central composite design and response surface methodology, having product yield as target, with all tested variables showing to be statistically significant on the conversion. The optimal conditions for butyl butyrate synthesis were: temperature of 40 °C, substrate molar ratio of 3:1 butanol:butiric acid, 40% of enzyme amount in relation to substrate weight, and 2.5% of water (volume fraction of reaction), using hexane as solvent. Under these conditions, over 84% of conversion was obtained after 4 h of reaction in a batch reactor, with a productivity of 0.27 mmol g−1 h−1 per gram of derivative. The butyl butyrate synthesis was also evaluated in a continuous reactor, using two configurations: packed-bed reactor (PBR) and fluidized-bed reactor (FBR). The highest productivity per gram of derivative in the continuous process was 1.01 mmol g−1 h−1, reached using the PBR containing 1.5 g of ImmTLL and glass beads. The biocatalyst presented operational stability at 40 °C, maintaining 83% of its initial conversion capacity after 8 cycles of reuse in batch reactor and 63% after 30 days of continuous operation in the PBR, using a flow rate of 0.02 mL min−1. These results suggest that this immobilized system can be successfully used to produce butyl butyrate for food, cosmetics, and for pharmaceutical applications.
In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).
Invertase is an enzyme widely distributed among plants and microorganisms that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant Saccharomyces invertase at 3.3 A resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, Saccharomyces invertase displays an unusual quaternary structure. Monomers associate in two different kinds of dimers that are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constrains that limit the access to the active site of the disaccharide (sucrose) or short oligosaccharides of up to four units. Comparative analysis of GH32 enzymes shows that formation of the Saccharomyces invertase octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge on the activity of this important family of enzymes. This, in turn, gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition.
Fructooligosaccharides (FOSs) were prepared from sucrose using fungal fructosyl transferase (FTase) obtained from Aspergillus oryzae MTCC 5154. The resulting mixture consisted of glucose (28-30%), sucrose (18-20%) and fructooligosaccharides (50-54%) as indicated by HPLC analysis. Identification of oligomers present in the mixture of fructooligosaccharides was carried out using NMR spectroscopy and LC-MS. No compounds other than mono-, di-, tri-, tetra- and pentasaccharides were identified in the FOS mixture prepared using FTase. NMR and LC-MS spectra proved the absence of any toxic microbial metabolites of Aspergillus species in FOS thereby emphasizing its safe use as a food ingredient. Animal studies conducted on streptozotocin-induced diabetic rats suggested that the use of FOS as an alternative non-nutrient sweetener is without any adverse effects on various diabetes-related metabolic parameters. Despite the high free-sugar content associated with it, FOS did not further aggravate the hyperglycemia and glucosuria in diabetic animals, even at 10% levels. On the other hand, by virtue of its soluble fibre effect, it has even alleviated diabetic-related metabolic complications to a certain degree.
'Chemical Reactor Modeling' closes the gap between chemical reaction engineering and fluid mechanics and provides the basic theory for momentum, heat and mass transfer in reactive systems.
In this chapter the characteristics of fluidized gas–solid suspensions are described, and the basic designs of fluidized bed reactors are sketched. Several modeling approaches that have been applied to described these units are outlined.
A synthetic mixture of fructo-oligosaccharides (mFOS), consisting largely of nystose (GF3) and a lesser amount of 1-kestose (GF2) has been reported to be selectively utilized by bifidobacteria. In the present study, we tried to identify which fructo-oligosaccharide molecule in mFOS is really involved in the stimulation of bifidobacteria in the gut, using both the gnotobiotic murine model and in vitro culture. 1-Kestose administration to gnotobiotic mice that were associated with human fecal microbiota significantly increased the number of bifidobacteria while mFOS administration was unable to sustain bifidobacteria in these hosts. Moreover a simultaneous decrease in the number of clostridia was found in host mice administered 1-kestose but not in those administered mFOS. The acetate/propionate ratio in the feces was far higher in host mice administered 1-kestose than in those administered mFOS, suggesting the selective growth activation of bifidobacteria by 1-kestose. The culture study demonstrated that 1-kestose exerts a strong growth-stimulating activity on bifidobacteria but a negligible one on clostridia. On the other hand, nystose was able to stimulate clostridia if the clostridia were exposed to nystose for some time. These results suggest the superiority of 1-kestose to mFOS, which consists largely of nystose, in the selective stimulating activity on bifidobacteria.
Twenty-five commercial enzyme preparations for use in the food industry were assayed for transfructosylation activity. Three preparations showed high transfructosylation activity from sucrose as well as the ratio of transferase and hydrolase activities. Short-chain fructooligosaccharides (sc-FOS) were not hydrolyzed by the three enzyme preparations after a 12 h reaction time. At a 6 h reaction time, yield and volumetric productivity were in the range from 58.8 to 62.6% (g sc-FOS/100 g initial sucrose) and 52.5 to 55.9 g sc-FOS/L h, respectively. One enzyme preparation was then evaluated for sc-FOS synthesis. Thus, environmental factors influencing the reaction were studied on products. Total sc-FOS concentration was not affected by temperature, pH and enzyme concentration at the studied levels, but high concentrations of sucrose affected the sc-FOS formation. The results suggest that these enzyme preparations can be exploited as a source of food-grade fructosyltransferase, in addition to Pectinex Ultra SP-L.
Fluidized-bed reactors present a number of advantages that make them an attractive alternative in processes involving biocatalysts. However, fluidized-bed bioreactors are also realtively complex, basically for two reasons. First, their use requires the biocatalyst, commonly cells or enzymes, to be immobilized into or onto a solid support. Second, the hydrodynamic characterization is difficult, especially in those systems where three phases (gas—liquid—solid) are involved. The mathematical model of a fluidized-bed bioreactor needs to take into account those hydrodynamic aspects that will determine the flux model in the reactor. Moreover, the description of other aspects is also required: the mechanisms of transport between the different phases, the kinetic equations for the phenomena taking place in the biocatalytic particles, such as cell growth, product formation, substrate consumption, enzyme deactivation, and the mass balance equations in the reactor. In addition to these aspects, the application of fluidized-bed bioreactors to different kind of processes is also discussed. The potential of this type of bioreactor is also emphasized from the point of view of the different number of possible modifications in the design both of the bioreactor and the biocatalyst particles, in order to enhance its operation.
Glutaraldehyde is one of the most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemistry of the glutaraldehyde/amino reaction will be commented upon. This reaction is still not fully clarified, but it seems to be based on the formation of 6-membered heterocycles formed by 5 C and one O. Then, we will discuss the production of intra- and inter-molecular enzyme crosslinks (increasing enzyme rigidity or preventing subunit dissociation in multimeric enzymes). Special emphasis will be placed on the preparation of cross-linked enzyme aggregates (CLEAs), mainly in enzymes that have low density of surface reactive groups and, therefore, may be problematic to obtain a final solid catalyst. Next, we will comment on the uses of glutaraldehyde in enzymes previously immobilized on supports. First, the treatment of enzymes immobilized on supports that cannot react with glutaraldehyde (only inter and intramolecular cross-linkings will be possible) to prevent enzyme leakage and obtain some enzyme stabilization via cross-linking. Second, the cross-linking of enzymes adsorbed on aminated supports, where together with other reactions enzyme/support crosslinking is also possible; the enzyme is incorporated into the support. Finally, we will present the use of aminated supports preactivated with glutaraldehyde. Optimal glutaraldehyde modifications will be discussed in each specific case (one or two glutaraldehyde molecules for amino group in the support and/or the protein). Using preactivated supports, the heterofunctional nature of the supports will be highlighted, with the drawbacks and advantages that the heterofunctionality may have. Particular attention will be paid to the control of the first event that causes the immobilization depending on the experimental conditions to alter the enzyme orientation regarding the support surface. Thus, glutaraldehyde, an apparently old fashioned reactive, remains the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst.
The enzymatic synthesis of fructooligosaccharides (FOS) was carried out using a partially purified β-fructofuranosidase from the commercial enzyme preparation Viscozyme L. Partial purification of β-fructofuranosidase from Viscozyme L was done by batch adsorption using ion-exchange resin DEAE-Sepharose, showing a 6-fold increase in specific activity. The biocatalyst was then covalently immobilized on glutaraldehyde-activated chitosan particles. Thermal stability of the biocatalyst was evaluated at 50 °C and 60 °C, being around 100 times higher at 60 °C when compared to the free enzyme. The immobilized biocatalyst was reused 50 times for FOS production (100 min per batch at 50 °C and pH 5.5) without significant loss of activity. The average yield (grams of FOS per grams of initial sucrose) was 55%. The immobilization process combined with partial purification method resulted in a derivative with activity of 1230 Ut/g, which is among the best for FOS production.
A kinetic model based on a ping-pong mechanism was developed under the steady-state hypothesis to account for the short-chain fructooligosaccharides (sc-FOS) synthesis using the commercial cellulolytic enzyme preparation, Rohapect CM. This new mechanism takes into account the interactions between the enzyme species and potential substrates (sucrose and sc-FOS) as a single complex reaction, allowing a better understanding of the reaction kinetics.
The initial reaction rate laws appropriately describe the kinetic profiles of the examined substrates. Whereas sucrose exhibited Michaelis-Menten behavior with substrate inhibition, 1-kestose and nystose followed Michaelis-Menten and sigmoid enzyme kinetics. In addition, the enzyme was competitively inhibited by glucose and exhibited significant hydrolytic activity in the presence of nystose.
The overall model was simultaneously fitted to experimental data from three initial sucrose concentrations (0.5, 1.5 and 2.1 M) using a multi-response regression with kinetic parameters that have biochemical relevance and are independent of the enzyme concentration. According to the model, sucrose acts almost exclusively as a fructosyl donor substrate. The mathematical development described herein is expected to be suitable for modeling similar enzymatic reaction systems.
Biodiesel production by immobilized Rhizopus oryzae lipase in magnetic chitosan microspheres (MCMs) was carried out using soybean oil and methanol in a magnetically-stabilized, fluidized bed reactor (MSFBR). The maximum content of methyl ester in the reaction mixture reached 91.3 (w/v) at a fluid flow rate of 25 ml/min and a magnetic field intensity of 150 Oe. In addition, the MCMs-immobilized lipase in the reactor showed excellent reusability, retaining 82 % productivity even after six batches, which was much better than that in a conventional fluidized bed reactor. These results suggested that a MSFRB using MCMs-immobilized lipase is a promising method for biodiesel production.
Cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on glutaraldehyde-activated chitosan spheres and used in a packed bed reactor to investigate the continuous production of β-cyclodextrin (β-CD). The optimum temperatures were 75°C and 85°C at pH 6.0, respectively for free and immobilized CGTase, and the optimum pH (5.0) was the same for both at 60°C. In the reactor, the effects of flow rate and substrate concentration in the β-CD production were evaluated. The optimum substrate concentration was 4% (w/v), maximizing the β-CD production (1.32g/L) in a flow rate of 3mL/min. In addition, the biocatalyst had good operational stability at 60°C, maintaining 61% of its initial activity after 100 cycles of batch and 100% after 100h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.
From the viewpoint of process engineering in enzymatic biodiesel production, this paper provides a practical basis for development of the packed-bed reactor (PBR) on a bench scale. A key component of this work is the separation of glycerol byproduct in continuous operation. To facilitate measuring and separating the byproduct, we constructed the PBR integrated with a glycerol-separating system. Methyl ester content in the effluent increased with increasing pass number of the reaction mixture, whereas the efficiency of glycerol removal was affected significantly by the flow rate in PBR. Further optimization revealed that the PBR could be operated over a long period realizing both a high methyl ester content and an efficient glycerol removal, obtaining a product that meets the biodiesel standard for residual glycerides. To remove soluble glycerol present in the biodiesel, adsorptive purification using ion-exchange resin was applied to the PBR system. The initial glycerol content of approximately 0.053wt% was reduced to less than 0.003wt%, which was maintained for more than 140 bed volumes and repeated after regeneration of the resin using methanol. Therefore, this developed model incorporating the enzymatic PBR and glycerol-separating system is promising for practical biodiesel production, in which no wastewater treatment is necessary.
The influence of several parameters (support porosity, glutaraldehyde concentration, time of action, pH) on the activation reaction of an amine porous silica (Spherosil) with glutaraldehyde has been studied. Glutaraldehyde binding onto the support was followed by measuring the carbon content of the activated silica.We established comparisons between the quantity of glutaraldehyde retained on the support after activation and the capacity of the activated silica to bind trypsin. We have defined the optimal conditions for Spherosil activation and prepared derivatives with high enzymatic activity.Our results are in agreement with a reaction mechanism with glutaraldehyde in a polymeric form resulting from aldol condensation, rather than in a monomeric form.
β-d-Galactosidase from Kluyveromyces lactis was immobilized on glutaraldehyde-activated chitosan and used in a packed-bed reactor for the continuous hydrolysis of lactose and the synthesis of galactooligosaccharides (GOS). The biocatalyst was tested for its optima pH and temperature, thermal stability in the presence of substrate and products, and operational stability. Immobilization increased the range of operational pH and temperature, and the enzyme thermal stability was sharply increased in the presence of lactose. Almost complete lactose hydrolysis was achieved for both milk whey and lactose solution at 37°C at flow rates up to 2.6mLmin(-1). Maximal GOS concentration of 26gL(-1) was obtained at a flow rate of 3.1mLmin(-1), with a productivity of 186gL(-1)h(-1). Steady-state operation for 15 days showed the reactor stability concerning lactose hydrolysis.
In contrast to the application of soluble enzymes in industry, immobilized enzymes often offer advantages in view of stability, volume specific biocatalyst loading, recyclability as well as simplified downstream processing. In this tutorial review the focus is set on the evaluation of immobilized enzymes in respect to mass transport limitations, immobilization yield and stability, to enable industrial applications.
Crude inulinase from Aspergillus niger (A. niger) NCIM 945 was immobilized on chitosan beads using a two-step covalent immobilization approach. Immobilization conditions were optimized to achieve 86% immobilization efficiency with 83 inulinase activity units/ g of wet support. At lower pH values (pH 2.5 and 3) the relative activity of the immobilized enzyme was higher than the free enzyme. Also, thermal stability increased considerably at 50 and 60 °C after immobilization on chitosan beads. The Km values of free and immobilized inulinase were 0.25 and 0.21 mM, respectively and Vm values were close to 0.2 µM/ min/mL. In a continuous packed bed column 5% w/v inulin was completely hydrolyzed at optimized conditions of flow rate (0.1 mL/min) and incubation temperature (60 °C). This resulted in volumetric productivity of 68 g/L.h. This simple and efficient covalent immobilization approach has a potential for continuous inulin hydrolysis.
Invertase (E.C.3.2.1.26) from Saccharomyces cerevisiae was covalently immobilized on chitosan nanoparticles prepared by the ionotropic gelation method and activated with glutaraldehyde. The support was characterized and it was studied its load capacity, the influence of the presence of substrate during immobilization, and determined the biocatalyst kinetic parameters and stabilities. The light scattering analysis (LSA) and transmission electron microscopy (TEM) techniques indicated a mixture of chitosan nano and aggregated nanoparticles, providing high superficial area for enzyme immobilization. The thermal and storage stabilities, the optimal pH and temperature of the enzyme were not altered. K(m) increased 3-fold, while V(max) remained unaltered. The immobilized biocatalyst was reused for 59 batches with maximal invertase activity, the highest operational stability so far described in the literature. These results fulfill some important aspects for the enzyme immobilization: the simplicity of the protocols, the conservation of the enzyme activity, and the high operational stability.
Enzyme biocatalysis plays a very relevant role in the development of many chemical industries, e.g., energy, food or fine chemistry. To achieve this goal, enzyme immobilization is a usual pre-requisite as a solution to get reusable biocatalysts and thus decrease the price of this relatively expensive compound. However, a proper immobilization technique may permit far more than to get a reusable enzyme; it may be used to improve enzyme performance by improving some enzyme limitations: enzyme purity, stability (including the possibility of enzyme reactivation), activity, specificity, selectivity, or inhibitions. Among the diverse immobilization techniques, the use of pre-existing supports to immobilize enzymes (via covalent or physical coupling) and the immobilization without supports [enzyme crosslinked aggregates (CLEAs) or crystals (CLECs)] are the most used or promising ones. This paper intends to give the advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations. Moreover, the use of nanoparticles as immobilization supports is achieving an increasing importance, as the nanoparticles versatility increases and becomes more accessible to the researchers. We will also discuss here some of the advantages and drawbacks of these non porous supports compared to conventional porous supports. Although there are no universal optimal solutions for all cases, we will try to give some advice to select the optimal strategy for each particular enzyme and process, considering the enzyme properties, nature of the process and of the substrate. In some occasions the selection will be compulsory, for example due to the nature of the substrate. In other cases the optimal biocatalyst may depend on the company requirements (e.g., volumetric activity, enzyme stability, etc).
The effect of the support size on the properties of enzyme immobilization was investigated by using chitosan macroparticles and nanoparticles. They were prepared by precipitation and ionotropic gelation, respectively, and were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), transmission electron microscopy (TEM), light scattering analysis (LSA), and N(2) adsorption-desorption isotherms. β-Galactosidase was used as a model enzyme. It was found that the different sizes and porosities of the particles modify the enzymatic load, activity, and thermal stability of the immobilized biocatalysts. The highest activity was shown by the enzyme immobilized on nanoparticles when 204.2 mg protein·(g dry support)(-1) were attached. On the other hand, the same biocatalysts presented lower thermal stability than macroparticles. β-Galactosidase immobilized on chitosan macro and nanoparticles exhibited excellent operational stability at 37 °C, because it was still able to hydrolyze 83.2 and 75.93% of lactose, respectively, after 50 cycles of reuse.
More than 80% of the commercial value of enzymes is linked to their applications as process catalysts. Hydrolytic reactions conducted mainly with the enzyme dissolved in the aqueous medium has been the traditional way of using enzymes, this technology still representing a major share of enzyme processes. However, in recent decades the use of enzymes in organic synthesis has widened its scope of application to unprecedented levels.
Enzyme reactors can operate batch-wise or continuously; fed-batch operation has also been proposed (Kumar et al. 1996). Batch processes with the enzymes (usually hydrolases) dissolved in an aqueous reaction medium, despite its wide application have several drawbacks, since enzymes are poorly stable and hard to recover in such systems, leading to low productivity; besides, such processes are characterized by a rather low added value so that process optimization is critical for being and keeping competitive. Poor stability is usually the limiting factor in any enzyme process so that enzyme stabilization during reactor operation is a major concern (Ballesteros et al. 1998; O’Fágáin 2003) and among the many strategies for enzyme stabilization (Illanes 1999) enzyme immobilization is the most relevant (Guisán 2006). Immobilized enzymes can be used in batch processes but in this case the enzyme is recovered to be used in subsequent batches until the accumulated inactivation makes necessary to replace the spent biocatalyst. As a consequence, specific productivity (mass of product/mass of biocatalyst time of operation) is increased and bioreactor design becomes flexible to suit the particular needs of a given process.
Yeast cell wall invertase (CWI) was immobilized within calcium alginate beads. The result of entrapment was the complete immobilization of all the added CWI. Three types of biocatalysts were prepared: CWI-S, CWI-155 and CWI-157. The optimum pH values were 4.5 and 5.0 for free and immobilized invertase, respectively. The optimum temperature was 60 degrees C, for both free CWI and CWI-S immobilizate. 80 degrees C was the optimum temperature for CWI-155 whereas the optimum temperature for CWI-157 ranged from 50 degrees C to 80 degrees C. Immobilized CWI was more stable than was free CWI above optimum activity temperatures. The activation energies were 32 kJ/mol for free CWI and 45, 21 and 25 kJ/mol for CWI-S, CWI-155 and CWI-157, respectively. The Km values of free and immobilized CWI-157 were 28.4 mM and 72 mM, respectively. The V-max values were estimated as 4.5 mM/min and 0.42 mM/min, respectively. Immobilized CWI-157 was tested in a batch reactor using 70% sucrose (w/v). Complete sucrose conversion was achieved after 55 h. After 40 consecutive cycles, CWI-157 retained 90% of its activity.
Four external invertase isoforms (EINV1, EINV2, EINV3 and EINV4) from Saccharomyces cerevisiae were highly purified by isoelectric precipitation, ethanol precipitation, ion-exchange on QAE-Sephadex and gel filtration using Sephacryl S-200. Unlike previously published procedures for external invertase purification, a specially designed step elution was applied on QAE-Sephadex which enabled the separation of four isoforms. The isoforms have the same molecular mass and catalytic properties: Km for sucrose (25.6 mM), pH optimum (3.5–5.0) and temperature optimum (60 °C), but they exhibit significant difference in pI values, thermal stability and chemical reactivity. Deglycosylation studies showed that the observed differences between isoforms arise from posttranslational modifications. Results showed that external invertase is a mixture of at least four isoforms, but in order to improve the efficiency of food industry processes, only the most stable isoform (EINV1) should be purified and utilised. Substantially different chemical reactivity of the isoforms could be used to improve the yield of covalent immobilization procedures.
Poly (2-hydroxyethyl methacrylate–glycidyl methacrylate) (pHEMA–GMA) membrane was prepared by UV-initiated photopolymerization. Invertase was immobilized by the condensation reaction of the epoxy groups of glycidyl methacrylate in the membrane structure with amino groups of the enzyme. The Km values were 22 mM and 58 mM for free and immobilized enzyme, respectively. Immobilization improved the pH stability and temperature stability of the enzyme. Thermal stability was found to increase with immobilization. The half times for the activity decay at 70 °C were found to be 11 and 38 min for the free and immobilized enzyme, respectively. The immobilized enzyme activity was found to be quite stable in later experiments.
As functional materials, chitin and chitosan offer a unique set of characteristics: biocompatibility, biodegradability to harmless products, nontoxicity, physiological inertness, antibacterial properties, heavy metal ions chelation, gel forming properties and hydrophilicity, and remarkable affinity to proteins. Owing to these characteristics, chitin- and chitosan-based materials, as yet underutilized, are predicted to be widely exploited in the near future especially in environmentally benign applications in systems working in biological environments, among others as enzyme immobilization supports. This paper is a review of the literature on enzymes immobilized on chitin- and chitosan-based materials, covering the last decade. One hundred fifty-eight papers on 63 immobilized enzymes for multiplicity of applications ranging from wine, sugar and fish industry, through organic compounds removal from wastewaters to sophisticated biosensors for both in situ measurements of environmental pollutants and metabolite control in artificial organs, are reviewed.
Chitin is the most abundant natural amino polysaccharide and is estimated to be produced annually almost as much as cellulose. It has become of great interest not only as an underutilized resource, but also as a new functional material of high potential in various fields, and recent progress in chitin chemistry is quite noteworthy. The purpose of this review is to take a closer look at chitin and chitosan applications. Based on current research and existing products, some new and futuristic approaches in this fascinating area are thoroughly discussed.
A liquid-fluidized bed or packed bed with gel beads is attractive as an immobilized-cell bioreactor. The performance of such bioreactors is influenced by the physical behavior of these beads. Three different but related aspects involving the drag force between particles and liquid were studied for five types of gel beads, differing in diameter and density: (1) the terminal settling velocity of a single gel bead, (2) the pressure drop over a packed bed and (3) the voidage in a liquid-fluidized bed. Qualitatively, the same trends in these aspects were observed for gel beads as for conventional solids. Quantitatively, however, these aspects were incorrectly predicted by established models (with one exception). It was found that the drag force between gel bead and flowing liquid is smaller than that for conventional solids. As an explanation, two hypotheses are suggested. The first one attributes the drag reduction to small amounts of dissolved polymer. The second one attributes the smaller drag force to the surface nature of gel beads: gel beads contain over 95% of water and thus can be regarded as `rigid¿ water droplets. Hence, the gel bead surface might show water-like properties. As an alternative to drag-coefficient relations for conventional solids, the drag coefficient of a single gel bead in a packed or fluidized bed could successfully be described by adapting an existing relation. The success of this description facilitates a more rational design of packed and fluidized beds of gel beads.
Invertase is a commercially important enzyme used for the hydrolysis of sucrose. The hydrolysis of sucrose yields an equimolar mixture of glucose and fructose, known as invert syrup, is widely used in food and beverage industries. This enzyme is also used for the manufacture of artificial honey, plasticizing agents used in cosmetics, pharmaceutical and paper industries as well as enzyme electrodes for the detection of sucrose. Immobilization of invertase and its biotechnological applications are reviewed.
This study investigated how chain length affects fermentation properties of fructooligosaccharides (FOSs) and inulin (IN). Chain lengths of FOSs and IN vary from an average degree of polymerization (DP) of 3 to greater than 20. Three samples classified as FOSs (samples A, B, and C) and 3 samples classified as IN (samples D, E, and F) were fermented via an in vitro batch method with human fecal inoculum as the source of microbes. Samples were removed at 0, 4, 8, 12, and 24 hours for total short-chain fatty acid (SCFA), acetate, propionate, and butyrate measurement via gas chromatography. Sample chain length did not affect SCFA concentrations in a predictable manner. Sample E (90%-94% DP > 10, 6%-10% DP = 1-2), a mixture of long-chain IN and short-chain FOS, produced significantly more total SCFA and acetate than the other samples. Sample F (DP > 20), the longest-chain IN, produced the lowest concentration of butyrate at 24 hours. The rate of FOS fermentation was higher than IN fermentation during 0 to 4 hours for all SCFAs, and the rate of IN fermentation was higher than FOS fermentation during 12 to 24 hours for all SCFAs. Chain length affects in vitro fermentability, with short chains being rapidly fermented and long chains being steadily fermented. Clinical studies should follow this work to verify if these differences exist in vivo.
Baker's yeast invertase was found to catalyse transfructosylation reactions in aqueous and anhydrous organic media with sucrose as a substrate, leading to the formation of five intermediate fructans in addition to the release of D-glucose (D-Glc)and D-fructose (D-Fru). All the reaction products were separated and quantitatively estimated using high performance anion exchange-pulsed amperometric detection equipment. The unknown products were subsequently identified by linkage analysis as beta-D-Fru-(2 --> 1)-beta-D-Fru-(2 --> 1)- alpha-D-glucopyranoside (1-kestose), beta-D-Fru- (2 --> 6)-alpha-D-glucopyranoside (6-beta-fructofuranosylglucose), beta-D-Fru-(2 -->1) -beta-D-fructofuranoside (inulobiose), beta-D-Fru-(2 --> 6)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (6-kestose) and beta-D-Fru-(2 --> 6)-alpha-D-Glc-(1 --> 2)-beta-D-fructofuranoside (neokestose); and this last was eluted together with a disaccharide. The time-course of sucrose hydrolysis via fructan production in 2 ml of a 50 mM sodium acetate buffer (pH 4.5) containing 0.2 M sucrose and 25 U of invertase was different from that in 2 ml of anhydrous toluene with 1.46 M sucrose and 1,000 U of invertase as a suspended powder. Under the latter experimental conditions, invertase was found to exhibit cyclic behaviour, where sucrose was degraded and subsequently synthesised. This observation has not yet been reported, as far as we know.
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.
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