Induction and properties of (1 3)-β-D-glucanase from Aureobasidium pullulans

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1 Indian Journal of Biotechnology Vol 3, January 2004, pp Induction and properties of (1 3)-β-D-glucanase from Aureobasidium pullulans M S Dake*, J P Jadhav and N B Patil Department of Biochemistry, Shivaji University, Kolhapur , India Received 1 October 2002; accepted 24 March 2003 The structure determination of polysaccharides using specific enzyme(s) as a tool has been of great importance. Such an enzyme, (1 3)-β-D-glucanase was induced in Aureobasidium pullulans by yeast β-glucan as a potent inducer. The formation of enzyme was, however, repressed by glucose. The purified enzyme has molecular weight 230 kda, optimum ph 5.5 and optimum temperature 60 C. The enzyme hydrolyzes laminarin and yeast β-glucan. The k m value obtained for laminarin was 0.17%. The analysis of the hydrolyzed product demonstrates that the enzyme acts on β (1,3) linkages from the non-reducing end(s). Keywords: Aureobasidium pullulans, induction, glucanase, purification, extracellular enzymes, β-glucan Introduction β-glucanases are hydrolytic enzymes capable of causing lysis of cell walls. They are active on insoluble substrates, such as β-glucan components of fungal cell wall, laminarin and pachyman. They act both as exo or endo hydrolases. β-glucanases are further classified as β-1,3-glucanase, β-1,6-glucanase and β-1,4-glucanase on the basis of the type of glucosidic linkage cleaved by them. They have significant role in structural analysis of polysaccharides 1,2. β-glucanases have been found in higher plants 3 as well as in fungi 4 and bacteria 5. Six different β-1,3- glucanases have been purified from Saccharomyces cerevisiae 6. Strains of Bacillus and Streptomyces species are reported to be highly active in producing extracellular β-1,3-glucanases 7,8. Nonspecific β- glucanases, i.e. β-glucosidase and β-1,3-glucanase enzymes, have been purified from fungus Penicillium ochro-chloron 9. In yeast, (1 3)-β-glucanases are involved in morphogenetic events, such as cell budding, conjugation and sporulation 10,11. The cell wall of yeast undergoes continuous rearrangement of β-glucans during growth period. The process involves making and breaking of bonds between wall polymers, manipulated by β-glucanases through controlled hydrolysis. As a consequence, different glucanases are required at *Author for correspondence: Tel: , , ; Fax: , manjushadake@rediffmail.com different stages during cell life cycle. For their higher specificity and selective hydrolysis of cell wall polymers, β-glucanases are employed in the preparation of protoplasts 12. β-glucanases are also beneficial for production of alcoholic beverages like beer and wine. The β-glucanase enzymes present in such beverages hydrolyze β-glucans, thereby reduce the wort viscosity and improve filtration process as well as extraction yields. In view of the above, it is essential to elaborate on specific methods for purification of the glucanase enzymes. The present paper includes isolation, purification and characterization of exo-β-1,3- glucanase enzyme produced extracellularly from a saprophytic mold, Aureobasidium pullulans. The data reported here are concerned with the purification and kinetic properties of the enzyme which hydrolyzes laminarin; a linear β(1 3)-glucan. Materials and Methods Source (1 3)-β-D-Glucanase enzyme was purified from a saprophytic mold, Aureobasidium pullulans. Culture of Aureobasidium pullulans (NCIM-1041) was obtained from National Chemical Laboratory, Pune, India. Chemicals Biogel P-2, P-100, P-200 were purchased from Biorad. Laminarin, DEAE sepharose and Blue Dextran were purchased from Sigma Chemical Company, USA. Glucose oxidase kit was obtained

2 DAKE et al:β-glucanase IN AUROBASIDIUM PULLULANS 59 from Bio Lab. The chemicals used for electrophoresis were purchased from standard manufacturers. Culture Conditions The culture of Aureobasidium pullulans was routinely grown in a liquid medium containing 1% sucrose, 0.5% yeast extract, 0.2% K 2 HPO 4 ; 0.05% MgSO 4.H 2 O; 0.15% NaNO 3 and 1% yeast β-glucan as an inducer, at 27 C for 15 days of incubation with intermittent shaking of the culture flasks. The fungus culture was maintained on slants having the same medium with 2% agar at 4 C. The cultural filtrate containing the enzyme activity was dialyzed against 10 mm phosphate buffer at ph 7.0 under cold conditions to remove glucose and then used for further purification of enzyme. Enzyme Assays Glucanase Activity (1 3)-β-D-Glucanase activity was quantified by measuring the amount of glucose liberated from laminarin substrates using glucose oxidase peroxidase method specific for glucose 13. One unit of (1 3)-β- D-glucanase activity was defined as the amount of enzyme required to produce 1 μg of glucose per 30 min at ph 5.5 and at temperature 45 C. Amylase Activity The amylase activity was measured using 1% starch as a substrate and 50 mm sodium acetate buffer, ph 4.8 using dinitrosalicylic acid method 14. Analytical Procedures The glucose was measured by using glucoseoxidase peroxidase method specific for glucose. Total carbohydrate was estimated using the phenolsulphuric acid method 15. Proteins were estimated by Lowry s method 16. Reducing sugars were determined by using 3,5-dinitrosalicylic acid method. The dialyzed enzyme preparation was chromatographed using DEAE sepharose CL-6B ion exchange chromatography at 4 C. The column ( cm) was equilibrated with 10 mm sodium phosphate buffer, ph 7.0. The effluent fractions with β-1,3-glucanase activity, collected after ion exchange purification step, were concentrated by ultrafiltration and lyophilization. The enzyme was purified further using Biogel P-100 ( cm) and Biogel P-200 ( cm) columns successively as a molecular sieving technique. One ml of the enzyme solution was loaded onto Biogel P-100 and Biogel P-200 columns, equilibrated previously with 10 mm sodium phosphate buffer, ph 7.0. Elution was done using the same buffer with a flow rate of 10 ml/hr and 8 ml/hr for Biogel P-100 and Biogel P-200 columns, respectively. One ml fraction was collected and used for further analysis. All the operations of column chromatography were carried out at 4 C. The products of hydrolysis, resulting from the action of β- 1,3-glucanase on laminarin, were characterized further by Biogel P-2 gel filtration and descending paper chromatography. The column containing Biogel P-2 ( cm) was equilibrated with 10 mm phosphate buffer, ph 7.0 at room temperature. The reaction mixture contained 5 mg laminarin per ml, 10 U of β-glucanase enzyme solution per ml and 50 mm of citrate buffer, ph 5.5. The reaction mixture was incubated at 45 C for 24 hrs and then loaded on the Biogel P-2 column. After loading the reaction mixture, the elution of the column was carried out using the same buffer and 1 ml fraction was collected for further analysis of carbohydrates. Paper Chromatography The reaction mixture was subjected to descending paper chromatography using butanol:ethanol:water (4:1:5; v/v) and the separated sugars were detected using alcoholic sodium hydroxide and acetonic silver nitrate as spraying reagents. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) PAGE was performed using 5% (w/v) polyacrylamide gel ( cm) by the method of Laemmli 17. Sodium phosphate buffer (100 mm, ph 8.0) containing 0.1% (w/v) SDS was used as the running buffer. The molecular mask markers (Boehringer Manheim) consisting of aldolase (158 kda), β-amylase (200 kda), catalase (240 kda) and ferritin (450 kda) were used as standard proteins. Gels were stained with Amido Black 10B and destained with 7.5% (v/v) acetic acid. Results Production of Enzyme To achieve the maximum enzyme production, the culture conditions were standardized. For that different media, as given in Table 1, were used with variable percentage of carbon source and yeast β- glucan as an inducer, along with metal salts of Na, K and Mg. Medium No. 7, i.e 1% sucrose+1% yeast β- glucan+other media components, yielded higher

3 60 INDIAN J BIOTECHNOL, JANUARY 2004 activity of (1 3)-β-D-glucanase enzyme (Table 1). Therefore, this medium was selected and used for culturing A. pullulans. The culture was incubated for 15 days. At the end of incubation, the culture was filtered. The cultural filtrate contained both β-1,3- glucanase activity and amylase activity. The cultural filtrate was further centrifuged under cold conditions to remove the fungal mycelia and other residual mass. The clear cultural filtrate was dialysed at 4 C for 24 hrs. The dialysed enzyme was loaded onto the DEAE sepharose CL-6B column. β-(1,3)-glucanase activity did not bind and was recovered in the effluent. The absorbed proteins were eluted using a linear NaCl gradient (0 to 0.5 M) in equilibration buffer which contained amylase activity. Thus, two major peaks were obtained as shown in Fig. 1. The first peak showed β-1,3-glucanase activity, while the second peak indicated an amylase activity. Thus, β-glucanase activity was separated from amylase activity, so that about 85% anionic proteins were removed from β- glucanase enzyme, ultimately showing increase in the purification fold of the enzyme. After ion exchange chromatography, partially purified β-1,3-glucanase enzyme was concentrated by ultrafiltration and lyophilization. The concentrated enzyme solution was applied onto a Biogel P-100 column. β-1,3-glucanase activity was eluted in the void volume of the column with v e /v o ratio as 3. The protein contents were found to be uniformly distributed throughout the activity peak. These observations indicated that the enzyme was freed from any other protein contaminants (Fig. 2). The Table 1 Media with variable compositions producing (1 3)-β-D-glucanase activity. S. No. Medium composition Activity μg/1ml enzyme/30 minutes 1. Sucrose 1%+α-β-glucan substrate 2% as alkali insoluble mass isolated 318 from yeast Saccharomyces cerevisiae+common media components 2. Sucrose 0.2%+yeast β-glucan 0.2%+ common media components Nil [absence of growth of mold A. pullulans] 3. Sucrose 2.0%+β-glucans 0.2%+common media components Sucrose 10%+other media components Nil 5. Sucrose 2%+yeast β-glucan 0.5%+other media components Sucrose 5%+β-glucan 1%+other media components * Sucrose 1%+yeast β-glucan 1%+other media components 480 Fig. 1 DEAE sepharose ion exchange chromatography. Elution profile of (1:3)-β-D-glucanase.

4 DAKE et al:β-glucanase IN AUROBASIDIUM PULLULANS 61 fractions showing β-1,3-glucanase activity were concentrated and applied further onto Biogel P-200 column. Here, again β-1,3-glucanase activity was eluted in the void volume of the column with v e /v o ratio as 3. A single peak was obtained as shown in Fig. 3. Since, the fractionation range of Biogel P-200 is in the range of 30,000 to 200,000 Da, it is obvious that the enzyme has a molecular weight higher than 200,000 Da. Kinetic Properties Effect of ph and Temperature on β-1,3-glucanase Activity β-1,3-glucanase enzyme was optimally active between ph 3.5 to 6.5. The activity was not stable in acidic ph less than 2.0 as well as in alkaline ph above 8.0 (Fig. 4). Similarly, β-1,3-glucanase enzyme showed higher activity within temperature range of 40 to 60 C (Fig. 5). The enzyme showed negligible activity at 10 to 20 C and became totally inactive at 80 C. Thus, after 30 min of incubation, the enzyme showed maximum degradation of laminarin at ph 5.5 and at a temperature of 45 C. Effect of Substrate Concentration on the Enzyme Activity Enzyme activity was measured at different substrate concentrations from 1 to 10 mg/ml. Increase Fig. 2 Gel filtration chromatography on Biogel P-100 Fig. 3 Gel filtration chromatography on Biogel P-200. Elution profile of (1:3)-β -D-glucanase on Biogel P-200.

5 62 INDIAN J BIOTECHNOL, JANUARY 2004 in β-1,3-glucanase activity was observed with increase in laminarin substrate concentration up to 7 mg/ml, which thereafter remained constant. The results are expressed in the form of Lineweaver Burk plot as shown in Fig. 6. Michaelis menten constant (K m ) calculated for laminarin as a substrate was 1.7 mg/ml. Effect of Metal Ions The purified enzyme was incubated with 10 mm concentration of various metal ions in 50 mm citrate buffer, ph 5.5 at 45 C for 30 min. Table 2 indicates that Co 2+, Mn 2+ and Ca 2+ are the activators of the enzyme, and Hg 2+, Fe 2+ and Ag + are the strong inhibitors; while Mg 2+ and Na + slightly reduce the enzyme activity. Action Pattern of the Enzyme β-1,3-glucanase enzyme is able to act on laminarin and yeast β-glucan substrates. The hydrolysis products resulting from the action of β-1,3-glucanase on laminarin were analyzed by using Biogel P-2 gel filtration and descending paper chromatography techniques. Biogel P-2 gel filtration chromatography is very efficient in separation of oligosaccharides on the basis of their molecular size and number of sugar residues. The elution profile is depicted in Fig. 7. The obtained major peak lay between fraction numbers 35 th to 51 st just after the void volume. This peak indicates the presence of oligosaccharides resulting from the partially degraded laminarin substrate. The major product of (1 3)-β-D-glucanase hydrolytic action on laminarin was glucose, which was eluted between fraction numbers 80 th to 90 th. There was an absence of other laminarin oligosaccharides in the reaction mixture, which otherwise get eluted after the void volume. Thus, glucose is the major product of hydrolysis of β-1,3-glucanase action on laminarin. Table 2 Effect of metal ions on activity of β-1,3-glucanase enzyme Metal salt Effective concentration of metal ion (mm) Enzyme activity μg/ml/30 min Percentage enzyme activity Fig. 4 Effect of ph on activity of (1:3)-β-D-glucanase STD MgSO * COCl CuSO HgCl AgNO * CaCl FeSO * MnCl NaCl Fig. 5 Effect of temperature on activity of (1:3)-β-D-glucanase Fig. 6 Lineweaver Burk plot for (1:3)-β-D-glucanase

6 DAKE et al:β-glucanase IN AUROBASIDIUM PULLULANS 63 Fig. 7 Gel filtration chromatography on Biogel P-2. Elution profile of the products of hydrolysis released due to the action of (1:3)- β -D-glucanase on laminarin. Fig. 9 Comparative mobilities of standard protein markers and the glucanase enzyme: (A) Aldolase (158 kda), (B) β-amylase (200 kda), (C) β-glucanase (230 kda), (D) Catalase (240 kda), (E) Ferritin (450 kda) were used at concentration of 20 μg/10 μl homogeneous as confirmed by the results from SDS- PAGE. Nine μg of enzyme protein, estimated by Lowry method, was applied to a single tube gel along with other standard protein markers. The molecular weight of enzyme protein was calculated on the basis of the mobilities of the protein bands developed in tube gels. The estimated molecular weight of the enzyme was 230,000 Da (Fig. 8). Again, the presence of a single band under reducing and non-reducing conditions exhibits the homogeneity of the enzyme (Fig. 9). Fig. 8 Determintation of molecular weight of enzyme protein by polyacrylamide gel electrophoresis (PAGE). The pure protein standards are: (A) Aldolase (158 kda), (B) β-amylase (200 kda), (C) β-glucanase (230 kda), (D) Catalase (240 kda), (E) Ferritin (450 kda) were used at concentration of 20 μg/10 μl. The action pattern showed that β-1,3-glucanase is an exo enzyme. The reaction mixture, along with pure sugar samples, was spotted on chromatography paper Whatman No. 1. The solvent system was allowed to run for 72 hrs. The paper chromatographic analysis also revealed that the major hydrolysis product of enzyme action on laminarin was predominantly glucose. So the mode of action of (1 3)-β-Dglucanase is of exo type, which must be cleaving the oligosaccharide chain from nonreducing end releasing glucose units successively. Electrophoretic Properties: Molecular Weight The enzyme preparation, obtained following the above-mentioned purification steps, was Discussion β-1,3-glucanase enzyme has been purified from the culture filtrates of A. pullulans, a saprophytic mold. The enzyme is extracellular and inducible in nature. Further, the enzyme is capable of acting on yeast β- glucan and laminarin substrates. The enzyme action on β-glucans, an alkaline insoluble mass isolated from yeast, resulted in its solubilization by liberating glucose residues those can be estimated later. The enzyme is exo type in nature as per the paper chromatographic analysis, exhibiting glucose as the major hydrolysis product. The exo-1,3-β-glucanase, thus, cleaves the substrate from nonreducing end, liberating glucose residues. β-1,3-glucanase enzyme was purified by removal of α-amylase activity by ion exchange purification step using DEAE sepharose CL-6B column. For further purification, the concentrated enzyme was applied to Biogel P-100 column. The enzyme showed elution in the void volume of the column, showing that it has a molecular weight higher than 100,000 Da. Here, the enzyme also got separated from other

7 64 INDIAN J BIOTECHNOL, JANUARY 2004 protein contaminants, resulting in its purification. The enzyme further showed similar pattern of elution in Biogel P-200 column, exhibiting that its molecular weight is higher than 200,000 Da. The homogeneity and molecular weight of the enzyme was further determined by using SDS-PAGE. From the graph of mobility vs log of molecular weight as shown in Fig. 8, the moleuclar weight of β-1,3-glucanase was calculated to be 230,000 Da. Most of the β-glucanases, characterized from the other fungi, have the optimum ph between Similarly, the β-glucanase enzyme from A. pullulans has optimum ph 5.5. The enzyme activity increased progressively from 2.5 to ph 6.5, thereafter it decreased. Accordingly, β-glucanases showed increase in its activity with increase in temperature from 20 to 45 C. The enzyme was thermostable up to 70 C. The measured k m value of exo-1,3-β-dglucanase for laminarin was 0.17%. The enzyme activity remained constant above 7 mg/ml laminarin substrate concentration, indicating the saturation of substrate binding sites of enzyme due to higher substrate concentration. Study regarding the effect of metal ions on the enzyme activity indicated that Co 2+, Mn 2+ and Ca 2+ are the activators of enzyme, whereas Hg 2+, Fe 2+ and Ag + are the strong inhibitors. The action pattern of β-1,3-glucanase enzyme is revealed also by using Biogel P-2 gel filtration analysis. The reaction mixture containing β-1,3- glucanase enzyme and laminarin, when loaded on Biogel P-2 column after prolonged incubation period, showed the presence of glucose as a major hydrolysis product as well as the higher oligosaccharides those get eluted in the void of the column. Since, a major peak of only glucose was observed between fraction number 80 th to 90 th, it appears that the enzyme is of exo type in nature. However, a small peak adjacent to the left of the glucose peak, between fraction numbers 75 th to 80 th, was also noted, which may be elution of disaccharides that remained unhydrolyzed. This is because the substrate laminarin may consist of linkages other than (1 3)-β-D-glucosidic linkage. Alternatively, the terminal oligosaccharide residue may be degraded very slowly by the enzyme. References 1 Cabib E, Roberts R & Bowers B, Synthesis of the yeast cell wall and its regulation, Annu Rev Biochem, 51 (1982) Fleet G H, Cell walls in the yeast, vol IV, edited by A H Rose & J S Harrisson (Academic Press, New York) 1991, Wong Y S & Maclachan G A, 1,3-β-D-Glucanases from Pisum sativum seedlings. I. Isolation and purification, Biochim Biophys Acta, 571 (1979) Huotari F I, Nelson T E, Smith F & Kirkwood S, Purification of an exo-β-d-1,3-glucanase from Basidiomycetes species QM 806, J Biol Chem, 243 (1968) Mori H, Yamamoto S & Nagasaki S, Multiple form of lytic glucanase of Flavobacterium dormitator var. glucanolyticae and the properties of the main component enzyme, Agric Biol Chem, 41 (1977) Hien N H & Fleet G H, Separation and characterization of six (1 3)-β-glucanases from Saccharomyces cerevisiae, J Bacteriol, 156 (1983) Zemek J, Polysaccharide hydrolyzing enzymes in the Bacillus, Folia Microbiol, 26 (1981) Lilley G & Bull A T, The production of β-1,3-glucanase by a thermophilic species of streptomyces, J Gen Microbiol, 83 (1974) Jadhav J P, β-glucanases from Penicillium ochro-chloron: Their use in study of α-β glucan complex in Saccharomyces carlsbergenesis. PhD Thesis, Shivaji University, Kolhapur, India, Phaff H J, Enzymatic yeast cell wall degradation, Adv Chem Ser, 160 (1977) Phaff H J, A retrospective and current view on endogenous β-glucanases in yeast, in Proc 5 th Int Symp on Advances in Protoplast Research (Hungarian Academy of Sciences, Budapest, Hungary) 1979, Yamamoto S & Nagasaki S, Studies on microbial enzymes active in hydrolyzing yeast cell walls, J Ferment Technol, 50 (1972) Lloyd J B & Whelan W J, An improved method for the enzymatic determination of glucose in the presence of maltose, Anal Biochem, 30 (1969) Bernfeld P, Methods in enzymalogy, vol I (Academic Press, New York) Dubois M, Giles K A, Hamilton J K, Robers P A & Smith F, Colorimetric method for determination of sugars and related substances, Anal Chem, 28 (1956) Lowry O H, Rosebrough N J, Farr A L & Randall R, Protein measurement with the Folin phenol reagent, J Biol Chem, 193 (1951) Laemmli U K, Cleavage of structural proteins during the assembly of the head of bacteriophage T 4, Nature (Lond), 227 (1970) Pitson S M, Seviour R J & Dougall B M, Non cellulolytic fungal β-glucanases: Their physiology and regulation, Enzyme Microb Technol, 15 (1993)