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Universidade Metodista de São Paulo, CP 5002, São Bernardo do Campo, SP 09735-460, Brazil
Marcia R. Braga
Rita de Cássia L. Figueiredo-Ribeiro 1
Instituto de Botânica, Seção de Fisiologia e Bioquímica de Plantas, CP 4005, São Paulo, SP 01061-970, Brazil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Penicillium janczewskii, isolated from the rhizosphere of Vernonia herbacea, grows rapidly on media containing either sucrose or inulin, although inulin more than sucrose induced the production of inulinases. Three different extracellular ß-fructofuranosidases (two inulinases and one invertase) were purified from fungal cultures grown on sucrose or inulin, through precipitation with ammonium sulfate, and anion-exchange, hydrophobic interaction and gel filtration chromatographies. The optimum temperature of the three enzymes was approximately 60 C, optimum pH 4–5.5 and apparent molecular mass of 80 kDa. Km and Vmax values determined for invertase on sucrose were respectively 3.7 10–4 M and 7.9 10–2 µmol/min/mL, and on inulin 6.3 10–2 M and 2.09 10–2 µmol/min/mL. The values of km for the two inulinases were 8.11 10–4 and 2.62 10–3 M, being lower for inulin when compared to those obtained for sucrose. The inulinases did not produce oligofructans from inulin, indicating they are primarily exoinulinases. The differences found in inulinase induction patterns when inulin or sucrose was used seem to be related to modifications on the enzyme properties, mainly concerning substrate affinity.
Key words: ß-fructofuranosidases, enzyme purification, filamentous fungus, inulin, sucrose
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
The interest in inulin or inulo-oligosaccharides has increased in the past decade since the discovery of their benefits in human nutrition. The functional properties of inulin in food and nonfood products depend partly on the chain length of the polymer (Öngen-Baysal and Sukan 1996). Fructose is a sweetener largely used in food and beverage industries with a sweetening power 70% higher than sucrose. It is well tolerated by diabetics, improves iron absorption by children and aids the removal of ethanol from the blood of alcoholics (Pessoa-Jr and Vitolo 1999). Fructo-oligosaccharides (FOS) are regarded as functional foods, positively influencing the composition of the gut microflora, increasing the population of bifidobacteria and improving mineral absorption. FOS also have been used as soluble fiber because they apparently help prevent constipation and colon cancer (Nakamura et al 1997, Kaur and Gupta 2002).
Enzymes that hydrolyze ß-fructosyl linkages are of two types: (i) invertase (ß-D-fructofuranoside fructohydrolase, EC 3.2.1.26
[EC]
), which hydrolyses sucrose but is unable to cleave fructose from inulin; and (ii) inulinases (2, 1-ß-D-fructan:fructan hydrolase EC 3.2.1.7
[EC]
), which are active on sucrose but in addition release fructose from the nonreducing end of the fructose chain (Kaur et al 1992, Ettalibi and Baratti 2001). Many of these enzymes also hydrolyze levan, raffinose and stachyose, although differences may be noticed at the substrate specificity and affinity (Muller and Seyfarth 1997).
The production of ß-fructofuranosidases (inulinases) is widely distributed in some groups of microorganisms and its synthesis is dependent on growth conditions, mainly the carbon source (Pandey et al 1999, Pessoni et al 1999). These enzymes are classified according to their mode of action on inulin. Endo-inulinases (ß-D-fructan:fructan hydrolase, EC 3.2.1.7) produce inulo-oligosaccharides from inulin, and exo-inulinases (ß-D-fructopyranoside fructohydrolase, EC 3.2.1.8
[EC]
0) release fructose from the fructosyl terminal of inulin (Nakumura et al 1997). Fungal producers are of particular importance because they might contain several types of fructan-hydrolases exhibiting high activity and stability (Balayan et al 1996). Invertases and inulinases of fungi may be considered digestive enzymes, generally secreted to the environment where nutrient sources are available.
Protein secretion is a process of major importance to filamentous fungi and has attracted interest as a valuable source of industrial enzymes (Wallis et al 1997). Microbial inulinases play an important role in the hydrolysis of inulin for production of high-fructose syrups, FOS and ethanol (Zhang et al 2004, Skowronek and Fiedurek 2006). Therefore the search for novel inulinase fungal producers and the characterization of these enzymes have received increasing attention.
Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea, a species of the Asteraceae from the cerrado (Cordeiro-Neto et al 1997) is an efficient microorganism for the production of extracellular inulinases (Pessoni et al 1999). Fructose syrup, produced from inulin by hydrolysis with these enzymes, reduced by ca. 46% plasma glucose level in diabetic rats (Pessoni et al 2004).
Maintenance of P. janczewskii on inulin medium induces secretion of proteins with higher inulinase activity compared to a sucrose-containing medium. Differences in cell wall structure arising from growing P. janczewskii on sucrose or inulin as carbon sources was reported by Pessoni et al (2005). In the present work we describe properties of the extracellular inulinases and invertase influenced by the carbon source.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
Culturing.— –
Disks (9 mm diam) were taken from 8 d old cultures of P. janczewskii grown on PDA (potato-dextrose agar) (Pessoni et al 1999). Inocula were transferred to 500 mL Erlenmeyer flask containing 100 mL of the medium composed of (g/L): NaNO3 (3), KH2PO4 (1), KCl (0.5), MgSO4·7H2O (0.5), FeSO4·7H2O (0.01) and inulin (1%, w/v) from Vernonia herbacea or sucrose (Sigma) as carbon source. The cultures were incubated up to 15 d on an orbital shaker (140 rpm) at 28 C in the dark (Pessoni et al 1999). At the end of each incubation period mycelia from each flask was filtered under vacuum with glass fiber (Whatman) and exhaustively washed with distilled water to remove traces of media. Mycelia were lyophilized and weighed for growth evaluation (dry mass).
Enzyme purification.— –
Liquid culture medium was collected after 12 d of fungal growth (at maximal inulinase and invertase activities) and filtered under vacuum through a 0.45 µm membrane (Millipore). The filtrate was concentrated by ammonium sulfate ([NH4]2SO4) precipitation (30–80% saturation). After centrifugation (12 000 g for 20 min at 5 C), the precipitate was resuspended in Tris-HCl 0.02 M buffer, followed by overnight dialysis (12 000 cut-off membrane) at 4 C against the same buffer. After dialysis protein in the enzyme preparation was separated by anion exchange column Mono Q (HR 5/5, Pharmacia) on FPLC system (Akta-Purifier 10/100, Pharmacia-Biotech), previously equilibrated with Tris-HCl 0.02 M buffer (pH 7.8). The column was washed with the same buffer. The adsorbed enzyme was eluted at a flow rate of 1.0 mL/min in a linear gradient of 0–0.5 M NaCl. Fractions, which exhibited high inulinase or invertase activity, were pooled and lyophilized. The lyophilized samples were desalted by centrifugation through Biogel P6DG (Bio-Rad), saturated with 40% (NH4)2SO4 and loaded on an Butyl 650S hydrophobic interaction column (Toyopearl) in FPLC system, equilibrated with 0.05 M phosphate buffer (pH 7.2) containing 40% (NH4)2SO4. The enzymes were eluted at a flow rate of 2.0 mL/min with a linear 40–0% (NH4)2SO4 gradient in the same buffer. Fractions that showed high inulinase or invertase activities were pooled and lyophilized. Lyophilized samples were desalted by centrifugation through Biogel P6DG (Bio-Rad) and were loaded onto a Superose 12 HR (Pharmacia) gel filtration column in FPLC system to estimate the molecular weight. The column was calibrated with ribonuclease (15.7 kDa), ovoalbumin (45.0 kDa), bovine serum albumin (66.0 kDa) and aldolase (170.0 kDa). The void volume (Vo) was determined with blue dextran (2000 kDa). Optical densities of eluted fractions were measured at 280 nm and protein concentrations were determined by Bradford (1976) with a Bio-Rad protein assay kit and BSA as standard.
Enzymatic assays.— –
Inulinase and invertase activities were measured with a 1% (w/v) substrate as described by Pessoni et al (1999). Inulinase and invertase activities were determined by the quantification of reducing sugars released after incubation with 1% inulin (Sigma, average Mr 5 kDa) or sucrose (Sigma) respectively in a 0.1 M sodium acetate buffer (pH 5) at 55 C for 10 min. The reaction was stopped with the alkaline copper reagent of Somogyi-Nelson (Somogyi 1945). One unit (U) of inulinase activity was defined as the amount of enzyme that produces 1 µmol of fructose/min under the above conditions, using calibration curve obtained with a standard solution of fructose. One unit of invertase activity was considered the amount of enzyme that hydrolyzes 1 µmol of sucrose/min. An equimolar mixture of glucose and fructose was used as standard for the quantification of reducing sugars. Because inulinases also have invertase activity, the presence of these two enzymes was discriminated by the addition of 10 mM pyridoxal hydrochloride to the incubation mixtures containing sucrose as substrate. Pyridoxal hydrochloride is a well known invertase inhibitor in microbes and plants (Vega et al 1991, Cairns 1992, Obenland et al 1993).
Properties of inulinases and invertase.— –
Optimum enzyme pH was determined with two buffers: 0.15 M sodium acetate (pH 3–5) and 0.15 M citrate-phosphate (pH 6–7) at 55 C for 10 min. The optimum temperature was determined by measuring the enzyme activity at different temperatures (30–70 C) at pH 5 for 10 min. To determine kinetic parameters the enzyme preparation was incubated with different concentrations of inulin (0.25–4 mM, average Mr 5 kDa) or sucrose (0.7–58 mM) in 0.1 M sodium acetate at pH 5 for 10 min. The Km and Vmax values were determined by the method of Lineweaver-Burk. Substrate specificity of the enzymes was determined by incubation with sucrose, raffinose (Sigma), 1-kestose and nystose (a gift of N. Shiomi), and inulin from Helianthus tuberosus, Vernonia herbacea and Polymnia sonchifolia, prepared as described by Carvalho and Dietrich (1993), inulin from Viguiera discolor according to Isejima and Figueiredo-Ribeiro (1993), levan from Gomphrena macrocephala according to Shiomi et al (1996) and oligofructan Neosugar (Meiji Seika Kaisha). The concentrations of substrates were expressed as percentages (Xiao et al 1989) to compare the results with those obtained for fructan polysaccharides, which are polydisperse molecules. The concentrations of enzymatic extracts in incubation mixtures were 6–10 µg protein. To examine the patterns of hydrolysis incubation products were analyzed on a CarboPac PA-1 anion exchange column with a Dionex DX 300 gradient chromatography system (HPAEC/PAD). The gradient and chromatographic conditions were established according to Shiomi et al (1996).
Polyacrylamide gel electrophoresis and detection of activity.— –
Electrophoretic analyses were performed according to the method of Laemmli (1970). A 10% sodium dodecyl sulfate (SDS)-acrylamide gel was used for denaturing electrophoresis. Proteins were stained with Coomassie R-250 blue. The purity was judged by SDS-PAGE and the relative molecular weights of purified enzymes were determined by comparing the mobility with those of standard proteins (Bio-Rad).
Native electrophoresis was performed on 7% gel according to a method proposed by Chen et al (1996). After PAGE the gel was incubated with 0.5 M sucrose dissolved in 0.1 M sodium acetate buffer (pH 5) at 30 C for 30 min or in 1% inulin in 0.1 M sodium acetate buffer (pH 5) at 55 C for 30 min. The inulinase and invertase bands were stained with 0.2% triphenyl tetrazolium chloride (TTC) in 1M NaOH at 100 C for 5 min. The gel was fixed with 7.5% acetic acid.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). An increase in reducing sugars coincided with the exponential growth phase of the cultures indicating extracellular hydrolysis of the substrates (FIG. 1a, b). In the inulin-containing medium a marked decrease in reducing sugars of the culture media was observed after 9 d, indicating that the stationary growth phase started when exogenous free sugars no longer were available (FIG. 1b). Invertase activity was detected in the filtrate cultures mainly from 6 d (FIG. 2a), whereas inulinase activity increased from day 9 (FIG. 2b). Both activities reached maximum values about 12 d when high amounts of soluble proteins were detected in culture media. The total amount of protein in the culture medium was higher when the fungus grew on sucrose (FIG. 2a). The specific activities of inulinase and invertase were approximately 14 times higher when P. janczewskii was cultivated on inulin than on sucrose (date not shown).
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). The culture filtrate exhibited specific activities of 15 U/mg and of 14.4 U/mg for inulinase and invertase, respectively (I/S ratio 1.02). Chromatographic analysis of the crude enzyme on a Mono Q column yielded two peaks of protein (PI and PII), which coincided with the detection of inulinase and invertase activities (FIG. 3). The pooled fractions of active peaks PI and PII were applied on Butyl-Toyopearl column, which increased their specific activities (TABLE I). Butyl-Toyopearl column PII resulted in one active peak of inulinase (FIG. 4a), with Mr of ca. 80 kDa as determined by gel permeation chromatography on Superose 12HR. This was confirmed by SDS-PAGE, showing a single Coomassie-stained protein band (FIG. 4b). Native-PAGE after staining with 0.2% TTC showed that the 80 kDa band corresponded to the active protein (data not shown).
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). Incubation mixtures of both PI and PII peaks with sucrose in the presence of pyridoxal showed no enzymatic inhibition, indicating that only inulinases were present in the inulin grown fungal medium.
Purification of invertase.— –
An extracellular invertase was purified from P. janczewskii grown on sucrose (TABLE II). The enzyme preparation was not retained on the Mono Q column but yielded a single protein peak with invertase activity on Butyl-Toyopearl column (FIG. 4c). The Mr of this protein peak was ca. 80 kDa, as estimated by gel permeation chromatography on Superose 12 HR (TABLE II) and confirmed by SDS-PAGE (FIG. 4d). These steps resulted in a 26-fold purified invertase (TABLE II). Incubation mixtures of this protein peak with sucrose in the presence of pyridoxal showed more than 50% enzymatic inhibition, indicating that invertase predominated in the sucrose grown fungal medium.
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). PI showed maximal activity at pH 6 and pH 5.5 for inulin and sucrose hydrolysis, respectively (FIG. 5a) whereas in the case of PII the highest activity was detected at pH 5 for both substrates (FIG. 5b). The optimum pH for invertase was ca. 5 (FIG. 5c). The optimum temperature for PI was 40 C on sucrose and 40–60 C for inulin hydrolysis (FIG. 5d). Maximal activity of inulinase PII occurred at 55 C, this enzyme being less stable at lower or higher temperatures (FIG. 5e). The invertase activity was maintained at 55–65 C (FIG. 5f). With the exception of PI, no apparent significant differences among the enzymes were present in the other fractions.
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). Kinetic analyses were based on a Mr of 5 kDa for inulin. The invertase Km values were 3.7 10–4 M for sucrose and 6.3 10–2 M for inulin, indicating a higher affinity for sucrose. In contrast PI and PII purified from P. janczewskii grown on inulin showed higher affinity for inulin when compared to sucrose.
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). Inulinases (PI and PII) were capable of hydrolyzing sucrose, raffinose, and fructo-oligosaccharides and inulo-polysaccharides (FIG. 6a–b). In contrast invertase showed low ability to hydrolyze fructans from different sources but was capable of degrading ß-(2,1) fructo-oligosaccharides (FIG. 6c). HPLC analysis of products formed in the hydrolysis of substrates by PI and PII suggested a terminal (exo-) mode of action because only fructose monomers and a small amount of glucose were detected (FIG. 7). These results indicate that the purified enzymes are ß-(2,1) fructofuranosidases.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Rueda et al 2001, Pessoni et al 2005), which in turn can influence enzyme secretion. No differences in growth rates and sporulation were observed in Penicillium janczewskii when the fungus was grown on medium containing either sucrose or inulin, as previously reported. However hyphae from this fungus grown on inulin-containing medium were more delicate and exhibited thinner cell walls than those cultivated on sucrose (Pessoni et al 2002, 2005). An investigation indicated that inulin induced inulinase and invertase activities in culture filtrates of P. janczewskii (Pessoni et al 1999). In the present work we demonstrated that the production of ß-fructofuranosidases (invertases and inulinases) from P. janczewskii and their properties also were affected by the carbon source.
Many reports describe the use of sucrose as carbon source for inulinase production (Vandamme and Derycke 1983, Pandey et al 1999). As reported by (Pessoa-Jr and Vitolo (1999) concerning Kluyveromyces marxianus, inulin in the medium also resulted in higher invertase and inulinase activities in P. janczewskii when compared to sucrose. The activity of these enzymes increased exponentially 12 d then declined, a pattern that could be attributed to an active process of extracellular protein secretion instead of hyphal autolysis, as suggested in the case of Pycnoporus sanguineus (Quiroga et al 1995).
The procedures for purification of invertase and inulinases from P. janczewskii based upon precipitation of enzymes with (NH4)2SO4, followed by ion exchange column and hydrophobic interaction and gel permeation, resulted in a 26-fold purification of invertase while inulinases (PI and PII) were purified 15–24-fold (TABLES I, II). In addition to inulinase activity, the purified fractions PI and PII of P. janczewskii also contained some hydrolytic activity on sucrose. It is now admitted that inulinases purified from various sources still retain some invertase activity after a number of chromatographic processes, suggesting that the active site might have two parts, one involved in sucrose hydrolysis and the other in inulin breakdown (Nakamura et al 1997, Kochhar et al 1999).
The relative Mr of invertase and inulinases PI and PII from P. janczewskii, as determined by gel filtration and SDS-PAGE (single band), were ca. 80 kDa, similar to those found for an invertase from Aspergillus nidulans (Chen et al 1996) and an inulinase from Penicillium cyclopium (Balayan et al 1996) but smaller than that of the invertase isolated from Monographella nivalis (195 kDa) (Cairns et al 1995). The optimum acidic pH and temperature up to 50 C observed for fructosyl-hydrolases from P. janczewskii are consistent with the range of values observed for invertase and inulinases from other microorganisms (Pandey et al 1999). Various forms of inulinase and invertase showing differences in their pH optima, Km values and thermal stability are not uncommon in fungi and might help in adaptation to a variety of environments (Kaur et al 1992).
The purified invertase from P. janczewskii proved to be specific for ß-fructofuranosides with highest affinity for sucrose and raffinose, followed by oligofructans (neosugar and P. sonchifolia), but not hydrolyzed polyfructans as the inulin of Vernonia herbacea and Viguiera discolor and the phlein of Gomphrena macrocephala (FIG. 5c). These results are consistent with the mode of action of invertases (ß-D-frutofuranosidase fructohidrolase, EC 3.2.1.26
[EC]
), according to Baer et al (1998) and Naumov and Doroshenko (1998). On the other hand inulinases from P. janczewskii hydrolyzed predominantly ß-2,1-fructosyl-linkages, being capable of hydrolyzing not only inulin but also sucrose, raffinose and various inulo- and fructo-oligosaccharides. These enzymes showed a little activity against levan (FIG. 6a–b). Considering that these inulinases split off terminal fructose units from the inulin molecule and no fructosaccharides were produced by their action (FIG. 7), we concluded that these enzymes are exoinulinases (2,1-ß-D-fructan-fructano-hydrolases, EC 3.2.1.7
[EC]
). These exo-inulinase activities have been reported for enzymes isolated from Chrysosporium pannorum (Xiao et al 1989), Penicillium trzebinskii (Onodera and Shiomi 1992), Kluyveromyces marxianus and Bacillus licheniformis (Abelian and Manukian 1996).
In the present work we demonstrated that ß-D-fructofuranosidases produced by P. janczewskii present differences in some properties depending whether inulin or sucrose were used as carbon source. These differences concern mainly their affinity to the substrates, possibly related to small structural modifications in the proteins secreted. Several attempts to determine the N-terminal amino acid sequence of these purified enzymes by the Edman procedure were unsuccessful, presumably because the N-terminus was blocked (data not shown), as was found for a number of expressed eucaryotic proteins (Chen et al 1996). Therefore based on our data the possibility that the secreted enzymes are distinct proteins cannot be discarded. However slight differences in the substrate specificity of glycosidases involving three dimensional folds could converge to different catalytic strategies of a single protein (Davis et al 2005). This suggestion could explain the behavior of P. janczewskii concerning the use of different carbon sources for the production of enzymes, which share several physico-chemical properties but differ in their affinity to the substrates. Cloning and sequencing of invertase and inulinases from P. janczewskii would help to elucidate these questions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: ritarib{at}usp.br
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LITERATURE CITED |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). Data are the mean of triplicates ± standard deviation.
), and protein (
). Data are the mean of triplicates ± standard deviation.
); Activity on sucrose (
); NaCl 0.5 M (···); DO 280 nm (—); mAU = milliabsorbance units.
