Food Chemistry 120 (2010) 799–804
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Purification and characterisation of Saccharomyces cerevisiae external invertase isoforms Uroš Andjelkovic´ a,*, Srdjan Pic´uric´ b, Zoran Vujcˇic´ c a
Institute for Chemistry, Technology and Metallurgy, Department of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia Institute of Biochemistry II, University Clinic Johann Wolfgang Goethe-University Frankfurt, 60590 Frankfurt, Germany c Faculty of Chemistry, Department of Biochemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia b
a r t i c l e
i n f o
Article history: Received 23 March 2009 Received in revised form 3 November 2009 Accepted 9 November 2009
Keywords: Invertase Isoforms Saccharomyces cerevisiae Enzyme stability Deglycosylation Imobillization
a b s t r a c t 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. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction External invertase (b-fructofuranosid fructohydrolase, EC 3.2.1.26) from Saccharomyces cerevisiae is an important enzyme in the food industry; it catalyses the hydrolysis of sucrose into an equimolar mixture of glucose and fructose, known as invert sugar. Invert sugar obtained by this enzyme reaction is colourless and has a higher yield of conversion than the product that is obtained by acid hydrolysis (Danisman, Tan, Kaçar, & Ergene, 2004). Invert sugar has a lower crystallinity then sucrose, which is important in the food industry to ensuring that the products remain fresh and soft for a long time. In the production process of high-test molasses, the addition of external invertase is essential to prevent crystallization (Piggot, 2003). External invertase is also important in the production of high fructose syrup (Tomotani & Vitolo, 2007). Addition of external invertase increases the yield production of ethanol from molasses (Doelle & Doelle, 1989) and high-test molasses (Piggot, 2003). Invertase has been isolated from the different microorganisms (Bhatti, Asgher, Abbas, Nawaz, & Sheikh, 2006; Haq, Baig, & Ali, 2005). However, the external invertase from S. cerevisiae is commonly used in food industry because this yeast is non-pathogenic and non-toxicogenic (Food and Drug Adminis* Corresponding author. Tel.: +381 11 3282393; fax: +381 11 2636061. E-mail address:
[email protected] (U. Andjelkovic´). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.11.013
tration, 2001). External invertase is also the most commonly used model enzyme to study different immobilization matrices for the food industry due to its stability, high degree of glycosylation, no need for any cofactors and its commercial significance (Danisman et al., 2004; Milovanovic´, Bozˇic´, & Vujcˇic´, 2007). On the other hand, the application of the suitably immobilized external invertase in enzyme reactors for sucrose hydrolysis, on the industrial scale, is still in the development phase with search for more stable external invertase preparations ongoing. The main functional state of the external invertase is a homodymer with a molecular mass of 270 kDa (Neumann & Lampen, 1967). It is a very stable glycoprotein that dissociates only under denaturation conditions. External invertase dimers can also associate to form tetramers, hexamers and octamers (Esmon, Esmon, Schauer, Taylor, & Schekman, 1987). Approximately 50% of the external invertase mass is polymannan and 3% is glucosamine (Neumann & Lampen, 1967). The carbohydrate component is organised in the 9–10 asparagin linked oligosaccharide subunits of different lengths (Ziegler, Maley, & Trimble, 1988). External invertase also contains phosphate groups covalently bound to mannose (Trimble, Maley, & Chu, 1983). The external invertase carbohydrate moiety increases its thermal stability (Wang, Eufemi, Turano, & Giartosio, 1996), resistance against protease (Chu & Maley, 1980), solubility (Gascon, Neumann, & Lampen, 1968) and makes the enzyme extremely stable at the room temperature
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(Chu, Trimble, & Maley, 1978). External invertase exhibits a high degree of microheterogeneity, with respect to the carbohydrate component, and variable state of oligosaccharide chain phosphorylation (Colonna, Cano, & Lampen, 1975; Frevert & Ballou, 1982; Neumann & Lampen, 1967; Smith & Ballou, 1974). There are no references as to how this microheterogeneity influences the catalytic properties and stability of the external invertase. Here we show that the microheterogeneous external invertase preparations that give single bands on SDS–PAGE and that were obtained by frequently used published methods (Andersen & Jorgensen, 1969; Neumann & Lampen, 1967), are in fact mixtures of at least four isoforms which exhibit different stabilities and chemical reactivities. In this paper, we describe the purification and properties of these four external invertase isoforms. It is shown that there is no difference between the isoforms in the structure of the protein part of the molecule. Moreover, we have demonstrated that these four isoforms are present in the external invertase preparations of several S. cerevisiae industrial strains. Detailed study of isoforms is important in order to improve the efficiency of the food industry processes as well as for increasing the yield of the invertase immobilization procedures. Beside commercial benefit, explorations of external invertase isoforms have scientific importance for understanding the enzyme stability. 2. Materials and methods 2.1. Materials All chemicals were of analytical grade and obtained from Sigma Chemical Company (St. Louis, MO, USA). Four different industrial strains of baker’s yeast S. cerevisiae have been examined: Alltech Fermin (Senta, Serbia), Vrenje (Beograd, Serbia), Budafok (Budapest, Hungary), Ziko (Skopje, FYR Macedonia). Commercial external invertase (b-fructofuranosid fructohydrolase EC 3.2.1.26, Grade VII from baker’s yeast) from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Purification of yeast external invertase isoforms 2.2.1. Preparation of yeast external invertase extract Saccharomyces cerevisiae cells were suspended in deionized water (1:1 w/w). Toluene (3%) and sodium carbonate (1%) were added. The suspension was incubated for 4 days at room temperature allowing cell-autolysis to occur. The crude cell extract was centrifuged at 4000g for 20 min. The pH in supernatant was adjusted to 4.0 with 1 M sulphuric acid and allowed to sediment overnight at 4 °C. The sediment was removed by centrifugation as above. The external invertase was precipitated at 4 °C by addition of cold 96% ethanol to 50% (v/v) saturation. The pellet which contained the total invertase activity was completely dissolved in deionized water and dialysed overnight at 4 °C against deionized water. Material precipitated during dialysis was removed by centrifugation as before. Obtained supernatant, representing purified extract, has been used for further purification of the external invertase isoforms. 2.2.2. Ion-exchange chromatography Isoforms of external invertase were isolated by ion-exchange chromatography – QAE-Sephadex A-50 (Pharmacia, Uppsala, Sweden) column (3 15 cm) was equilibrated in 50 mM acetate buffer pH 4.5 at the room temperature. The purified extract was loaded on the equilibrated column. Non-bound proteins were washed out with the equilibration buffer and the external invertase was eluted with a step elution using equilibration buffer with conductivity adjusted with 1 M NaCl. The conductivity of buffers for step elution (determined in preliminary experiments) was: first step
4.90 mS/cm, second step 8.50 mS/cm, third step 10.00 mS/cm and fourth step 15.00 mS/cm. Fractions (3 mL) were collected and the A280 and invertase activity were monitored. 2.2.3. Gel chromatography Final purification of each of the isoforms was performed on the Sephacryl S-200 HR (Amersham Biosciences, Uppsala, Sweden) column XK (2.6 105 cm) equilibrated in 50 mM acetate buffer pH 4.5. The absorbance at 280 nm and the invertase activity were monitored for each fraction and those with the invertase activity were analysed with the SDS–PAGE. Afterwards, selected fractions containing only one pure external invertase protein band were pooled. Samples obtained by gel filtration were further dialysed against the deionized water overnight at 4 °C and afterwards lyophilised. All following experiments were carried out with those samples that represent isolated external invertase isoforms. The retention time was determined with Blue Dextran as void volume marker. 2.3. Polyacrylamide gel electrophoresis (PAGE) SDS–PAGE was performed on Hoefer SE 600 Ruby vertical system, 10% separation gel (pH 8.8) and 3% stacking gel (pH 6.8). The Tris/glycine buffer with 0.1% SDS was used according to the discontinuous system; gels were stained with 0.1% Coomassie brilliant blue (CBB) R-250 (Laemmli, 1970). Molecular weight was estimated by comparison with marker proteins (a-2-Macroglobulin (170 kDa), b-galactosidase (116 kDa), Phosphorylase b (97 kDa) Amersham-Pharmacia and PageRuler™ Fermentas). Native PAGE was performed in identical conditions as SDS–PAGE omitting SDS in each buffer and b-mercaptoethanol in sample buffer, and samples were not boiled. 2.4. Isoelectric focusing IEF was performed using 4% polyacrylamide gels containing ampholines of pH range from 3 to 10 (Amersham-Pharmacia, Uppsala, Sweden), according to the manufacturers instructions. The isoelectric point was evaluated by comparison with protein pI markers (Sigma, USA). Gels were stained by CBB R-250. 2.5. Periodate cleavage of carbohydrate from external invertase Carbohydrate was cleaved by incubating 0.05 mg of highly purified external invertase in 5 mL of 25 mM sodium phosphate buffer, pH 7.0, containing 50 mM sodium meta-periodate. Reaction vessel was wrapped in foil to protect the reaction mixture from light. After 6 h of incubation at room temperature (21 °C), 10 lL of glycerol were added and the reaction mixture was desalted on Sephadex G-25 column. Immediately after salt was removed, 10 lL of b-mercaptoethanol were added and samples were analysed by SDS–PAGE and Native PAGE. 2.6. Invertase activity assay and concentration determination Invertase (25 lL) was added to 0.3 M sucrose solution in 50 mM acetate buffer (475 lL), pH 4.5. After 5 min at 25 °C reaction was terminated by addition of 2,4-dinitrosalicylic acid reagent (500 lL) and mixture was boiled in wather bath for 5 min. Before mesuring absorbance at 540 nm, 4 mL of deionized water was added (Bernfeld, 1955). The standard curve was obtained with different concentrations (between 0.5 and 10 mM) of an equimolar mixture of D-glucose and D-fructose in 50 mM acetate buffer, pH 4.5. One unit of the invertase activity (U) corresponds to the amount of enzyme that catalyses the hydrolysis of 1 lmol of sucrose per 1 min under described assay conditions. Invertase
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concentration was obtained measuring absorbance at 280 nm (A280nm = 2.25 for 1 mg/mL invertase solution) (Trimble & Maley, 1977). 2.7. Determination of the kinetic constant The Michaelis–Menten constant (Km) for sucrose was determined using the described conditions of the invertase assay and a sucrose concentration in the range from 2.5 to 300 mM. The Km was calculated using the Lineweaver–Burk transformation of Michaelis–Menten equation. 2.8. pH and temperature optima To determine the pH optimum of each of the isoforms the described invertase assay was performed at 25 °C but in McIlvaine’s buffer (0.2 M sodium phosphate and 0.1 M citrate) of the different pH values in the range from 3.0 to 8.0. The temperature optima were determined using the described conditions of invertase assay at various temperatures (ranging from 10 to 70 °C). 2.9. Thermal stability Samples of the external invertase isoforms (15 U/mL) in deionized water were incubated at 60 °C. Aliquots (25 lL) were taken every 5 min and the residual activity was directly measured using the described conditions of invertase assay.
Fig. 1. QAE-Sephadex chromatography of S. cerevisiae external invertase purified extract. Conductivity of the elution buffer was: first step 4.90 mS/cm, second step 8.50 mS/cm, third step 10.00 mS/cm and fourth step 15.00 mS/cm. Arrows indicate position of individual invertase isoforms.
Table 1 Purification of the S. cerevisiae external invertase isoforms. Purification step
Volume (mL)
Total activity (U)
Yield (%)
Crude lysate Clarified and desalted lysate Acide precipitation Ethanol precipitation
800 1000 900 190
785 773 641 622
100 98.4 81.7 79.2
140 150 90 15
203 254 62 30
25.8 32.3 7.9 3.8
15 14 17 21
165 211 46 23
21.0 26.9 5.9 2.9
2.10. Immobilization of the external invertase The external invertase isoforms were immobilized on Eupergit C, industry proven carrier porous material containing oxirane groups reactive toward amino and sulfhydryl groups of enzymes (kindly provided by Röhm GmbH, Darmstadt, Germany). Thirty milligrams of Eupergit C was added to 2 mL samples of external invertase isoforms (diluted with deionized water to 32 U/mL) and shaken for 1 h at room temperature and afterward placed at 4 °C. After 7 days at 4 °C with occasional shaking, immobilizate was extensively washed with deionized water to remove the non-bound enzyme. Control samples of invertase were exposed to the same conditions omitting Eupergit C.
QAE-Sephadex EINV1 EINV2 EINV3 EINV4 Sephacryl S-200 HR EINV1 EINV2 EINV3 EINV4
Specific activity (U/mg)
pI
3160 2410 2470 1480
4.43 4.22 3.95 3.76
3. Results and discussion External invertase was isolated from four different industrial strains of baker’s yeast S. cerevisiae. These preparations were analysed and here presented results showed the presence of four external invertase isoforms. Therefore, this appeared to be an inherent feature of each strain, which is consistent with previously described external invertase polymorphism in S. cerevisiae strain X218O (Frevert & Ballou, 1982) and FH4C, LK2G12 and baker’s yeast (Colonna et al., 1975). 3.1. Purification of yeast external invertase isoforms Extract of the external invertase was prepared by autolysis of yeast cells. After 4 days of autolysis more than 80% of the external invertase was released. An extract with low concentrations of contaminants was prepared by acid precipitation of contaminants and the subsequent ethanol precipitation of the external invertase. Isolation of the external invertase isoforms from the purified extract was achieved on the QAE-Sephadex ion-exchange column. Resulting chromatogram is given in Fig. 1. Four different peaks with invertase activity were obtained applying the step elution (cf. Section 2 for detail). Each single peak obtained was separately re-chro-
matographed, on the same QAE-Sephadex column. However, additional peaks were not obtained and each separated peak exclusively gave a single peak with invertase activity (results not shown). This simple control experiment confirmed that four different samples of external invertase were obtained. The purity of the four peaks was checked by the SDS–PAGE and each peak with external invertase isoform contained one or two low molecular weight contaminants. Those contaminants, described earlier (Trimble & Maley, 1977) were removed by gel filtration on Sephacryl S-200 HR column. A summary of the purification and specific activity of the individual external invertase isoforms are listed in Table 1. Isoform EINV1 exhibits the highest specific activity (3160 U/mg) which is in a good agreement with the highest published values for external invertase preparation (Andersen & Jorgensen, 1969; Neumann & Lampen, 1967; Trimble & Maley, 1977). The achieved final yield for EINV1 and EINV2 is 21.0% and 26.9%, respectively, which is a very high purification yield. Previously published procedures (Andersen & Jorgensen, 1969; Neumann & Lampen, 1967) for external invertase purification did not segregate individual fractions from ion-exchange chromatography. According to these procedures all the fractions with invertase activity have been pooled together in one sample. This
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Fig. 2. Electrophoretic profiles of the isolated S. cerevisiae external invertase isoforms. (A) Native PAGE. (B) Isoelectric focusing; arrows indicate position of pI markers.
preparation of external invertase will be identified as total external invertase (EINV) in further text. 3.2. Electrophoretic analysis of external invertase isoforms SDS–PAGE analysis of external invertase isoforms showed that all four isoforms have one identical diffuse band. This SDS–PAGE profile is identical with the previously published profile for the preparation of total external invertase (Trimble & Maley, 1977; Rodriguez, Gomez, Gonzalez, Barzana, & Lopez-Munguia, 1997). The diffuse band in the SDS–PAGE is characteristic for external invertase and glycoproteins in general. Native PAGE of four isoforms (Fig. 2A) reveals differences between them. Electrophoretic mobility increases from EINV1 to EINV4, following the order in which they are eluted from ion-exchange column. Samples of EINV and the commercially obtained external invertase (EINV Sigma) have identical broad diffuse bands covering regions of all four isoforms (Fig. 2A). This, very broad diffuse band, is characteristic of external invertase samples prepared following published procedures (Andersen & Jorgensen, 1969; Neumann & Lampen, 1967). Separation of four external invertase isoforms on an ion-exchange column was possible due to the difference in the negative charge density at the surface of isoforms. This difference in negative charge between isoforms is confirmed by Native PAGE (Fig. 2A). On the other hand, identical SDS–PAGE profiles and the same retention volumes in gel filtration of all four isoforms demonstrate that there is no significant difference in molecular weight between them. Therefore, comparable molecular weight of the isoforms and differences in quantity of negative charge at the molecule surface is the main cause of different mass/charge ratio, i.e. mobility in Native PAGE. IEF of external invertase isoforms (Fig. 2B) also showed differences in pI values between four isoforms. The pI values are given in Table 1. EINV yielded a very broad band in pI range from 3.80 to 4.40. These results are in agreement with the published results (Colonna et al., 1975) for the preparation of external invertase prepared following the published methods (Andersen & Jorgensen, 1969; Neumann & Lampen, 1967). The acidity of the isoforms increases in the same fashion in which they elute from ion-exchange column. The differences in pI values between isoforms are higher than could be predicted on the basis of the amino acid sequence
of the external invertase (Taussig & Carlson, 1983). This difference between individual isoforms is presumably due to differences in the covalently bound phosphate to mannose (Frevert & Ballou, 1982; Trimble et al., 1983). The carbohydrate moiety was removed from all individual external invertase isoforms by periodate oxidation cleavage. The resulting proteins were analysed by electrophoresis in order to test potential differences in the protein part of the molecule. SDS–PAGE analysis of periodate oxidised external invertase isoforms showed that all four isoforms have one identical band at 60 kDa, which corresponds to the mass of a monomer without carbohydrate units (Fig. 3). The periodate cleavage of the carbohydrate from the external invertase gives results that can be compared with the published results of the carbohydrate removal by the endo-b-Nacetylglucosaminidase H (Chu & Maley, 1980; Trimble & Maley, 1977). Native PAGE analysis showed that differences in the mobility between the isoforms disappear after periodate treatment (results not shown). This result suggests that all four isoforms have the same protein moiety. It seems that the differences between the isoforms are due to the posttranslational modifications, which is in agreement with the previous reported observations (Colonna et al., 1975; Frevert & Ballou, 1982). 3.3. Determination of the kinetic constant, pH and temperature optima The Km was determined to be 25.6 mM, for all four isoforms, using a substrate range from 2.5 to 300 mM sucrose in 50 mM acetate buffer (pH 4.5). The same value was published for preparation of EINV (Gascon et al., 1968). All four isoforms exhibit an identical pH optimum from 3.5 to 5.0 and temperature optimum with the maximum activity at 60 °C, and the results obtained are also in agreement with those published for the preparation of EINV (Gascon et al., 1968). The lack of significant differences in the Km, pH and temperature optima and the molecular weight between external invertase isoforms shows that there is no difference in catalytic activity between isoforms. The absence of a difference in the enzymatic activity between external invertase isoforms is one aspect of enzymatic catalysis. Another, important aspect, particularly when transferred to the industrial application, is how the observed differences between isoforms affect the enzyme’s stability and its chemical reactivity.
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Fig. 3. SDS–PAGE profiles of periodate treated external invertase isoforms. Gel is silver stained.
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Fig. 5. Efficiency of immobilization of the yeast S. cerevisiae external invertase isoforms on Eupergit C.
3.5. Immobilization of the external invertase isoforms
Fig. 4. Time courses of inactivation of the yeast S. cerevisiae external invertase isoforms at 60 °C.
3.4. Thermal stability External invertase isoforms exhibit significant differences in thermal stability at 60 °C (Fig. 4). Isoform EINV1 is the most stable and had the highest activity (about 30% of the activity) amongst isoforms even after 35 min of incubation at 60 °C. On the other hand, EINV3 quickly lost its activity and after 20 min almost no activity could be detected. Thermal stability of EINV is same as EINV Sigma and was shown for both to be lower than the thermal stability of the isoform EINV1. It has been previously published that deactivation of the external invertase follows biphasic kinetic (Cavaille & Combes, 1995; Rodriguez et al., 1997; Vrabel, Polakovicˇ, Štefuca, & Baleš, 1997). Our obtained results implied that the cause of biphasic kinetics of denaturation could be caused by the existence of the four isoforms in the preparations of external invertase. Four isolated isoforms are different in stability with first order inactivation kinetics, showing that inactivation of each isoform is a one-reaction process.
Finally, to test whether the observed differences amongst the individual invertase isoforms might also lead to differences in the chemical reactivity, we have used Eupergit C which is commonly used matrix for immobilization of enzymes in food industry. Immobilization of the isoforms on Eupergit C occurs via formation of covalent bonds between epoxide functional groups of Eupergit C and amino groups of enzyme. Results (Fig. 5) demonstrate large differences between external invertase isoforms in the degree of binding to Eupergit C, revealing their significantly different chemical reactivities. This result indicated that 72% of the applied activity of isoform EINV1, 24% of isoform EINV2 and 6% of isoform EINV4 were bound, whilst the applied activity of EINV3 was not bound at all. Hence, for the optimisation of the covalent immobilization procedures for the external invertase it is necessary to test whether the external invertase preparation contains isoforms. The reactivity of the each isoform with matrix has to be tested. Loading only the most reactive isoform to the matrix will improve the yield of immobilization. Therefore it would be possible to reduce the amount of matrix, what is especially important when expensive matrix is applied. Moreover, the existence of external invertase isoforms with different chemical reactivity could be an explanation for the low yield of some invertase immobilization procedures. Differences in surface architecture between isoforms, i.e. different exposure of amino groups of the protein toward reactive oxirane groups of Eupergit C most probably causes their different chemical reactivity resulting in significant differences in the yield of immobilization. 4. Conclusions In the present study the four isoforms of the yeast S. cerevisiae external invertase were isolated and characterised. Highly purified preparations were obtained. The procedure presented here is reproducible, simple, has a low cost and can easily be adapted for the large scale purification. The four isolated external invertase isoforms showed substantially different thermal stability and chemical reactivity. The isoforms have the same catalytic properties. The deglycosylation studies showed that the protein part of the four isoforms was identical.
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