Received: 19 February 2012 Revised: 24 March 2012 Accepted: 26 March 2012 Published online in Wiley Online Library: (wileyonlinelibrary.com) DOI 10.1002/jctb.3827
Liquefied petroleum gas as solvent medium for
the treatment of immobilized inulinases
Marceli Fernandes Silva,
a
Simone Maria Golunski,
a
Diane Rigo,
a
Vin´ıcius Mossi,
a
Marco Di Luccio,
b
Marcio A. Mazutti,
c
Sibele B.C. Pergher,
d
D ´ebora Oliveira,
b
J. Vladimir Oliveira
b
and Helen Treichel
e,f
∗
Abstract
BACKGROUND: The main goal of this work is to assess the influence of pressurized liquefied petroleum gas (LPG) treatment on the enzymatic activity of immobilized inulinases. The effects of process variables were evaluated through 23experimental design.
RESULTS: Inulinase from Klyveromyces marxianus NRRL Y-7571 presented an increase of 163% in residual activity using LPG at 30 bar during 1 h exposure using a depressurization rate of 20 bar min−1. For Aspergillus niger commercial inulinase, an increment of 129% in residual activity was observed at 30 bar for 1 h treatment at the highest depressurization rate (20 bar min−1). Enzymatic activities changed significantly depending on the enzyme source and the experimental treatment conditions investigated, such as exposure time, depressurization rate and pressure.
CONCLUSION: Hence, compressed LPG appears to be a promising technique with practical relevance as a preparation step to improve enzyme activity, thus helping the development of new biotransformation processes.
c
2012 Society of Chemical Industry Keywords: inulinase; compressed fluid LPG.
INTRODUCTION
Inulin is a fructose polymer found in plants such as Jerusalem artichoke, chicory and dahlia.1,2 It has been widely investi-gated as a source for the production of ultra-high-fructose syrup (UHFS) through enzymatic hydrolysis by either the sole action of exoinulinase (exo-β-d-fructosidase, EC3.2.1.80) or the synergic action of exo- and endoinulinase (2,1-F-d-fructan fructanohydrolase,EC3.2.1.7).3,4
Inulinase can be produced by a host of microorganisms including fungi, yeast and bacteria. Among them, Aspergillus sp. (filamentous fungi) and Kluyveromyces. sp (diploid yeast) are apparently the most common choices for commercial applications.5
It has been argued that nowadays the high cost of enzyme production is one of the major obstacles to commercialization of enzyme-catalyzed processes. For this reason, recent advances in enzyme technology, such as the use of solvent-tolerant and/or immobilized inulinases, which make possible the re-utilization of the catalyst, have been made to develop cost-effective systems.6,7 In recent years, many studies regarding the utilization of alternative solvents for biocatalysis have been presented in the literature.8,9 Considerable efforts have been reported in the literature towards green chemistry reactions, with emphasis on enzymatic reactions carried out in ionic liquids10 – 13 and in sub-and supercritical fluids.14 – 16 The use of compressed fluids as solvents (normally gaseous solvents) for chemical reactions may be a promising route to completely eliminate solvent traces from reaction products. In addition, manufacturing processes
in near-critical fluids can be advantageous in terms of energy consumption, easier product recovery, adjustable solvation ability, and reduction of side reactions.
Supercritical carbon dioxide has special characteristics, such as low toxicity, working temperature compatible with the optimal temperature for enzymes and favorable transport properties that can accelerate mass-transfer-limited enzymatic reactions.14,15 Nevertheless, carbon dioxide is not the only gas whose properties
∗ Correspondence to: Helen Treichel, Chemistry and Food School, Federal
University of Rio Grande FURG P.O Box 474, CEP: 96201900, Rio Grande -RS, Brazil. E-mail: helentreichel@gmail.com
a Department of Food Engineering, URI – Campus de Erechim, Av. Sete de
Setembro, 1621, Erechim, RS, 99700-000, Brazil
b Department of Chemical and Food Engineering, Federal University of Santa
Catarina, Campus Universit´ario, Trindade, Caixa Postal 476, Florian´opolis, SC, 88040-900, Brazil
c Department of Chemical Engineering - Federal University of Santa Maria, Av.
Roraima, 1000, Santa Maria, RS, 97105-900, Brazil
d Department of Chemistry – Federal University of Rio Grande do Norte, Av.
Salgado Filho, 3000, Lagoa Nova, Natal, RN, 59078-970, Brazil
e Chemistry and Food School, Federal University of Rio Grande - FURG P.O Box
474, CEP: 96201900, Rio Grande - RS, Brazil
f Federal University of Fronteira Sul - Campus de Erechim, Av. Dom Jo˜ao Hoffmann, 313, 99700-000, Erechim, RS, Brazil
seem to be adequate for biocatalysis as the comparable dielectric constant of propane and n-butane to that of carbon dioxide support a firm belief that such pure gases and their mixtures may also be suitable as reaction media for enzyme-catalyzed bioconversions.17 – 19 In this regard, the possibility of using a cheap, widely available fluid such as liquefied petroleum gas (LPG) seems to be a promising alternative to much more costly fluids such as propane, n-butane and even carbon dioxide.
The knowledge of enzyme behavior when submitted to high-pressure fluid treatment is of primary importance, as the loss of enzyme activity may lead to undesirable poor reaction rates and low yields of target products.20,21 In fact, enzyme stability and activity may depend on the enzyme species, the characteristics of the compressed fluid, the water content of the enzyme/support and the process variables involved, which means that very distinct effects can be achieved depending on the characteristics of the system under investigation.22 – 26
Based on these aspects, the main focus of this study was to investigate the enzymatic activity of inulinases in compressed LPG using a commercial immobilized inulinase from Aspergillus niger and a home-made immobilized inulinase from Kluyveromyces
marxianus NRRL Y-7571. No experimental data about the behavior
of inulinases after treatment in compressed LPG were found in the current literature. The present report is part of a broader project and reflects our efforts to help develop new enzyme-catalyzed processes in alternative fluid media.20,21,27 – 31
EXPERIMENTAL
Chemicals and enzymesLPG was kindly donated by Petrobras and is constituted of a mixture of propane (50.3 wt%), n-butane (28.4 wt%), isobutane (13.7 wt%), ethane (wt% 4.8 wt%) and other minor constituents (methane, pentane, isopentane, etc.).
Commercial inulinase from Aspergillus niger was purchased from Sigma-Aldrich. Non-commercial inulinases were produced from Kluyveromyces marxianus NRRL Y-7571 as described below.
Inulinase production
The extract containing extracellular inulinase was obtained by solid state fermentation using sugarcane bagasse as substrate. The medium composition was optimized in previous work
by our research group as follows: 2 kg of sugarcane bagasse supplemented with pre-treated cane molasses 15 wt%, corn steep liquor (CSL) 30 wt%, and soybean bran 20 wt%. The moisture content was set to 65 wt% and autoclaved at 121◦C for 20 min. The fermentation runs were started with the inoculation of an optimized volume corresponding to a cell mass of 14 g. All experiments were carried out for 24 h.32After fermentation, the enzyme was extracted from the sugarcane bagasse by adding sodium acetate buffer 0.1 mol L−1pH 4.8 in a solid/liquid ratio of 1 : 10, following incubation at 50◦C and 150 rpm for 30 min.33 Inulinase immobilization
Inulinase from both microorganisms were immobilized according to the methodology described by Risso et al.6 Initially, a gel solution was prepared containing 16.5 g of distilled water and 0.75 g of sodium alginate, and maintained under mild heating. After complete dissolution of the alginate, 12.5 g of sucrose were added, followed by 5 mL of the solution containing the recovered inulinase, 3.5 mL of glutaraldehyde and 0.75 g of activated carbon. For sphere formation, the gel solution was pumped into a 0.2 mol L−1 calcium chloride solution in sodium acetate buffer (0.1 mol L−1and pH=4.8) containing 3.5 wt% of glutaraldehyde, and stirred slowly at 10◦C. The immobilized inulinase was maintained at 4◦C for 24 h and then washed with sodium acetate buffer (0.1 mol L−1and pH=4.8). To maintain the structure, the immobilized spheres (around 0.005 m in diameter) were immersed in a 0.2 mol L−1calcium chloride solution in sodium acetate buffer (0.1 mol L−1and pH=4.8).
High-pressure treatment of enzymes
The experiments involving the immobilized inulinases were performed in a laboratory-scale unit similar to that employed by Kuhn et al.29which consists basically of a solvent (LPG) reservoir, two thermostatic baths, a syringe pump (ISCO 260D), a stainless steel vessel (cell) with an internal volume of 3 mL, an absolute pressure transducer (Smar, LD301) equipped with a portable programmer (Smar, HT201) with a precision of ±0.37 bar, as schematically represented in Fig. 1. All lines of the experimental setup consisted of 1/16’’ OD tubing of stainless steel (HIP) and between the pump and solvent reservoir a check (one way) valve (HIP 15-41AF1-T 316SS) was positioned to avoid pressurization solvent back flow to the head of the solvent cylinder. Two
TP IP A B C D B E F G
Figure 1. Schematic diagram of the apparatus for treatment of solid enzyme with compressed solvents. A solvent reservoir; B thermostatic bath; C
additional micrometering valves (HIP 15-11AF2 316SS) completed the experimental apparatus, one located after the syringe pump, at the entrance to the high-pressure cell, to allow solvent loading, and the other just after the cell to perform solvent discharge. The high-pressure cell was submerged in the water bath and was supported by a simple device while the micrometering valves were located outside the bath.
The experimental procedure adopted for enzymes treatment in pressurized fluid consisted, first, in adjusting the thermostatic bath to 40◦C, the temperature established in the present work for all experimental runs. Then, the enzymatic preparations (0.7 g) in immobilized form were loaded into the cell. After this procedure, the system was pressurized for different exposure times, according to pre-established conditions following an experimental design, keeping a constant pressurization rate (10 bar min−1). Finally, the system was depressurized at different pre-established rates, according to the experimental design, by a programmed syringe pump piston displacement and the micrometric valve used at lower pressures, near the solvent saturation pressure. The enzymatic activity of enzymes was determined before (initial activity) and after (final activity) the treatment procedure with pressurized fluids, as previously described.
Experimental conditions
Aiming at evaluating the effects of process variables on the activities of immobilized inulinase after treatment with pressurized fluid, a 23central composite design was adopted. The experimental planning was conceived to cover, at the same time, the variable ranges commonly used for enzyme-catalyzed reactions in compressed fluids, the optimum range of activity of each enzyme and the equipment operating limits.23 – 25 The variables evaluated for immobilized inulinases were pressure (30–275 bar), depressurization rate (10–200 bar min−1) and exposure time (1–6 h). Each run of the experimental design was carried out randomly, including a central point condition performed in triplicate, for experimental error evaluation. Analysis was performed using the software Statistica6.1 (Statsoft Inc, Tulsa, OK, USA). After depressurization, the water content of the treated enzyme was measured immediately by Karl Fischer titration and the activity was measured after storage in a freezer.
Measurement of the stability of the inulinases after treatment After determination of the experimental run that led to highest residual activity, 40 g of enzyme was treated and incubated at −4◦C, for evaluation of the inulinase activity during the storage. Samples were stored for 100 days and the activity measured each 10 days, using both sucrose and inulin as substrates.
Inulinase activity assay
An aliquot of 0.5 g of the enzyme source, softened, was incubated with 4.5 mL of 2 wt/v% sucrose solution in sodium acetate buffer (0.1 mol L−1, pH 5.5) at 50◦C. Reducing sugars released were measured by the 3,5-dinitrosalicylic acid method.34 A separate blank was set up for each sample to correct the non-enzymatic release of sugars. One unit of inulinase activity was defined as the amount of enzyme necessary to hydrolyze 1µmol of sucrose per minute under the mentioned conditions (sucrose as a substrate). Results were expressed in terms of inulinase activity per gram of dry solids (U g−1ds). The residual activity was defined as the ratio between the activities after and before treatment with pressurized fluid.
Scanning electron microscopy (SEM)
Textural characterization of materials was by AUTOSORB-1 (Quantachrome Nova-2200e) in low temperature N2 adsorp-tion–desorption experiments. Before analysis, about 100 mg of each support was treated under vacuum, at 300◦C for 3 h. Measure-ments were performed at the temperature of liquid N2. Average specific area was determined by the BET method.
For scanning electron microscopy (SEM), performed with SEM SSZ 550 Shimadzu, the samples were coated with a gold film and analyzed with a JOEL – JSM 5800 at 20 kV acceleration voltage.
RESULTS AND DISCUSSION
Table 1 presents the matrix for the 23central composite design and the respective responses in terms of relative residual activity (%) of non-commercial immobilized inulinase of Kluyveromyces
marxianus NRRL Y -7571 after treatment in pressurized LPG at
40◦C. After statistical analysis of the experimental data, it is possible to observe (Fig. 2) that the interaction between pressure and time, pressure and depressurization rate and the main effect of pressure had a positive effect (P < 0.05) on the enzyme activity. The treatment shows that the depressurization rate is the factor that most affects the enzymatic residual activity. According to some authors, pressure can modify the catalytic behavior of enzymes by changing the rate-limiting step or modulating the selectivity of the enzyme.35 – 38On the other hand, the main effect of time and depressurization rate and the interaction between time and pressure had a negative effect on inulinases activity. According to Knez,37depressurization is one of the factors that most affects enzymatic activity. The pressurized fluid contacts with the tertiary structure of the enzyme in a slow way. When the system is depressurized quickly there is a fast expansion of the fluid, causing a higher flow pressure in the enzyme than in the system, which can provoke cellular fissure, causing an unfolding in the structure of the enzyme, increasing or reducing its activity and selectivity.
The values of residual activity using LPG treatment can be considered very promising, since this fluid is constituted of a mixture, is commercially used as cooking gas, and therefore has a
Table 1. Relative residual activity∗(%) of non-commercial immobi-lized inulinase of Kluyveromyces marxianus NRRL Y -7571 after treatment in pressurized LPG at 40◦C. The initial activity of the immobilized enzyme without treatment was 66 U g−1
Run P (bar) T (h) R (bar min−1) RRA∗(%) 1 −1 (30) −1 (1) −1 (20) 163.1 2 −1 (30) −1 (1) 1 (100) 131.5 3 −1 (30) 1 (6) −1 (20) 131.9 4 −1 (30) 1 (6) 1 (100) 120.0 5 1 (270) −1 (1) −1 (20) 136.0 6 1 (270) −1 (1) 1 (100) 153.2 7 1 (270) 1 (6) −1 (20) 143.1 8 1 (270) 1 (6) 1 (100) 127.8 9 0 (150) 0 (3.5) 0 (60) 146.5 10 0 (150) 0 (3.5) 0 (60) 145.1 11 0 (150) 0 (3.5) 0 (60) 144.7 P= pressure, t=exposure time and R=depressurization rate.
∗Residual activity defined as the absolute value of (final activity/initial
Figure 2. Pareto chart of effects of pressure, exposure time and
depres-surization rate on residual activities of non-commercial inulinase from
Kluyveromyces marxianus NRRL Y-7 in LPG.
much lower cost than carbon dioxide and, especially, propane and
n-butane. The LPG composition of around 50 wt% of propane and
40 wt% of n-butane+isobutane justifies the similar values found for residual enzymatic activity when compared with those verified when the enzyme was treated with pure propane and n-butane.
The increase in the potential of hydrolysis of sucrose by the inulinase, evaluated by the enzyme activity, after treatment with pressurized gases was verified; however the mechanism of this increase is not clear. Kamat et al.36suggest that enzymes exposed to supercritical fluid conditions can suffer a change in its molecular and conformational structure and the interaction of the enzyme with the solvent could cause an increase in the activity previously presented by the enzyme.
Since solvent properties affect the specific interaction with the enzymes, different effects may be obtained depending on the enzyme studied.25,38Regarding the effect of hydrostatic pressure on enzyme stability, the literature pointed out that pressure values around those used in this work have a small effect on enzyme activity.39
Table 2 presents the residual enzymatic activity values obtained after treatment of the commercial inulinase from Aspergillus niger. From Fig. 3 one can observe that the depressurization rate had a significant negative effect, however, its interaction with pressure showed a positive significant effect (P < 0.05). The results confirm that the depressurization rate is one of the factors that can promote alterations in the enzymatic cellular structure and consequently loss or gain of enzyme activity.
Matsuda et al.40suggested that large changes in the pressurized fluid density as a result of increasing pressure altered the interaction between fluid and enzyme to progressively affect the enzyme conformation and, therefore, the enantioselectivity of the reaction. Similar results were obtained at various other temperatures. A comparison of the E-values at the same pressurized fluid density but different temperatures indicated that the enantioselectivity was also temperature dependent.
Certain enzymes show a considerable apparent pressure activation.41,42 Pressure can modify the catalytic behavior of
Table 2. Relative residual activity∗(%) of the commercial immobilized inulinase from Aspergillus niger after treatment in pressurized LPG at 40degC. The initial activity of the immobilized enzyme without treatment was 100 U g−1
Run P (bar) T (h) R (bar min−1) RRA∗(%) 1 −1 (30) −1 (1) −1 (20) 129.4 2 −1 (30) −1 (1) 1 (100) 91.3 3 −1 (30) 1 (6) −1 (20) 119.5 4 −1 (30) 1 (6) 1 (100) 97.4 5 1 (270) −1 (1) −1 (20) 105.2 6 1 (270) −1 (1) 1 (100) 96.0 7 1 (270) 1 (6) −1 (20) 98.5 8 1 (270) 1 (6) 1 (100) 100.4 9 0 (150) 0 (3.5) 0 (60) 97.5 10 0 (150) 0 (3.5) 0 (60) 93.6 11 0 (150) 0 (3.5) 0 (60) 97.6 P=pressure, t=exposure time and R=depressurization rate.
∗Residual activity defined as the absolute value of (final activity/initial
activity).
Figure 3. Pareto chart of effects of pressure, exposure time and
depres-surization rate on residual activities of inulinase from Aspergillus niger in LPG.
enzyme by changing for example, the rate-limiting step46 or modulating the selectivity of the enzyme.42
If an enzyme is stable in a supercritical fluid its stability is usually not influenced by pressure for pressure ranges up to 30 MPa. On the other hand, reaction rates may be influenced by the pressure. In most cases, a pressure increase acts positively for enzymatic reactions.41Pressure-induced deactivation of enzymes takes place mostly at pressure exceeding 150 MPa. Reversible pressure denaturation mostly occurs at pressures below 300 MPa and higher pressure is needed to cause irreversible denaturation.41 Figure 4 shows the stability of the inulinase (storage at–4◦C) from Kluyveromyces marxianus NRRL Y-7571 and Aspergillus niger submitted to LPG treatment using sucrose and inulin as substrates. Inspection of the experimental data obtained in this work, as shown in Fig. 4, reveals that the enzyme from Aspergillus niger maintained its relative residual activity at 100% when inulin was used as substrate and at 52% for sucrose.
In Fig. 4 it is observed that the best values of relative residual activity for the non- commercial immobilized inulinase from
Figure 4. Stability of the inulinase (storage at−4◦C) from Kluyveromyces
marxianus NRRL Y-7571 and Aspergillus niger submitted to LPG treatment
using sucrose and inulin as substrates.
K. marxianus NRRL Y-7571 were obtained when inulin was used
as substrate (94%), however promising values were also obtained with sucrose (53%), showing that even with loss in its catalytic power the enzyme maintains its stability.
Mozhaev et al.43 proposed that pressure stabilization of chy-motrypsin against heat inactivation may arise from protein interac-tions with water. Hydration of charged groups by water molecules is favored by pressure due to solvent electrostriction. Temperature disrupts the highly ordered structure of electrostricted water and reduces the strongly destabilizing effect of pressure on ion pairs.44 Vannieuwenburgh et al.45,46 revealed that pressure treatment acts also on the velocity and the specificity of the reaction catalyzed by Aspergillus niger fructosyl-transferase.
Figures 5(a) and (b) and 6(a) and (b) show the SEM micrographs of the enzymes Aspergillus niger and Kluyveromyces marxianus
NRRL Y-7571 before and after the treatment with pressurized LPG, respectively. Figure 5(a) shows that the enzyme-support structure of the inulinase of Aspergillus niger before being submitted to the treatment with LPG is organized, however, after treatment (Fig. 4(b)) it presents a small modification in its structure that did not provoke loss in her activity. The enzyme of K. marxianus NRRL Y-7571 presents an organized enzyme-support structure before being submitted to the pressurized fluid (Fig. 5(a)), however, when it was submitted to high-pressure treatment with LPG (Fig. 5(b)) its conformation possesses a slight alteration that did not provoke activity losses. This is qualitative convincing evidence of the low effect of LPG on inulinase activity.
Early studies suggested that the activities of monomeric enzymes were stimulated, while the activities of multimeric enzymes were inhibited by application of high hydrostatic pressure.47,48,49 However, at least 15 dimeric and tetrameric enzymes have been reported to be activated by pressure.47,49
CONCLUSIONS
Based on the results obtained in this work, one may infer that the enzyme activity after treatment with pressurized LPG depends on the structural nature of the enzyme and the experimental conditions imposed, i.e. exposure time, depressurization rate and system pressure. It has been observed in experiments with immobilized inulinase that gains in enzyme activities occur under several experimental conditions, thus allowing the selection of optimal operating conditions for the advantageous application of these treated biocatalysts in many important reactions of interest to the food industry. Thus, the use of compressed fluids, such as LPG may be of technological relevance as a preceding, preparation step, to improve enzyme activity, hence helping the development of new biotransformation processes.
Figure 5. SEM of the structure of the enzyme obtained from Aspergillus niger, immobilized in sodium alginate and activated coal, before and after
treatment in pressurized LPG.
Figure 6. SEM of the structure of the enzyme obtained from Kluyveromyces marxianus NRRL Y-7571, immobilized in sodium alginate and activated coal,
ACKNOWLEDGEMENTS
The authors thank CNPq, CAPES and FAPERGS for the financial support of this work and scholarships.
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