MIRCEN Journal, 1988, 4, 425-430 Properties of 8-glucosidase purified from Aspergillus niger H.H. Yeoh*, T.K. Tan, S.L. Chua & G. Lim Department of Botany, National University of Singapore, Kent Ridge, Singapore OS11 Received 22 February 1988; accepted as revised 30 April 1988 Introduction B-Glucosidase (EC 3.2.1.21), the enzyme responsible for the hydrolysis of cellobiose to glucose, has received much attention primarily because the efficiency of this step is critical to the overall breakdown of cellulose (Woodward and Wiseman 1982; Parr 1983). Analyses of kinetic data of this enzyme from various fungi and yeasts, such as Aspergillus, Botryodiploidia, Candida, Coriolus, Dekkera and Saccharomyces showed variations in the kinetic properties of this enzyme (Woodward and Wiseman 1982; Blondin et al. 1983; Evans 1985; Kohchi etal. 1985; Dekker 1986). Studies have also shown that Aspergillus species are good B-glucosidase producers (Woodward and Wiseman 1982). We have screened some local strains of Aspergillus for B-glucosidase activity and in this paper we describe the purification and kinetic properties of a B~ slucosidase from Aspergillus niger USDB 0355, a strain that shows potential as a 00d source of B-glucosidase. Materials and methods Organism Aspergillus niger USDB 0355 was maintained on 4.5% (wv) Czapek Dox Agar slants. Six ml of spore suspension (1 x 10° spores/ml) were inoculated into 150 ml of 2% (wiv) cellulose medium (Zhu et al. 1982) and incubated in an orbital shaker (130 rev./ min) for 14 d at 28-29°C. About 1 lof culture was prepared for enzyme purification, Purification of B-glucosidase All stages were carried out at 4°C unless otherwise stated. The fungal culture (ca. 350 m)) was filtered to remove the mycelia. The extracellular B-glucosidase in the culture broth was then precipitated with (NHy)2SO, at 90% saturation. The precipitate was recovered by centrifugation, resuspended in a minimal volume (ca. 2 ml) of $0 mw *Corresponding author © Oxford University Press 1988426 H. H. Yeoh, T. K. Tan, §. L. Chua & G. Lim sodium citrate, pH 4.6. It was then eluted through Sephadex G-200 column (45 x 2 ‘em) with 10 ma sodium citrate, pH 4.6. Fractions of 4 ml each were collected, and those with B-glucosidase activity were pooled and concentrated to 2 ml by Millipore ultrafiltration unit Immersible CX-10. This was then separated on a DEAE, Sepharose CL-6B column (19 x 2 cm) by an initial washing with 100 ml 10 mat sodium citrate, pH 4.6, followed by a 400 ml linear gradient of 0 to 0.3 m NaCl in 10 mM sodium citrate, pH 4.6. Fractions (4 ml each) containing B-glucosidase were then collected and concentrated to about 5 mi, This was further fractionated by CM- Sepharose CL-IB chromatography (19 x 2 cm) employing the same elution system as, for DEAE Sepharose CL-6B chromatography. Fractions (4 ml each) active in enzyme activity were collected and used for purity determination and kinetic studies Enzyme assay B-Glucosidase was measured with p-nitrophenyl-B-glucoside (PNPG). The reaction mixture containing 5 ma PNPG and 50 ma sodium citrate, pH 4.6, was equilibrated at ‘60°C for 10 min before the enzyme preparation (10 to 50 il) was added. The total reaction volume was kept at 600 4. After 15 min, the reaction was stopped by the addition of 3 ml 100 mat NazCO3. The absorbance of the resulting mixture was read at 420 nm. A calibration curve was also prepared using p-nitrophenol as standard. Protein assay Protein was determined using a modified Lowry's method using bovine serum albumin as standard (Miller 1959). Electrophoresis Electrophoresis in different polyacrylamide gel concentrations (3.75%, 5%, 6% and 7.5% wiv) was carried out according to Hedrick and Smith (1968). This method was used to assess the purity of the enzyme preparation and estimate its molecular size. Albumin (66 kDa), alcohol dehydrogenase (150 kDa), apoferritin (443 kDa), urease (480 kDa) and thyroglobulin (669 kDa) were used as molecular weight markers, Kinetic studies ‘The kinetic properties of the purified enzyme were investigated using the assay procedure described above but with different substrate concentrations (0.1 to 10 mm) or in the presence of different inhibitor concentrations (0.1 to 10 my). The Ky, value was calculated using the method of Wilkinson (1961). Inhibition types were determined by plotting the Lineweaver-Burk plots and the K, values were calculated from plots of slopes/intercepts against inhibitor concentrations (Engel 1981), Results and discussion The purification of B-glucosidase from Aspergillus niger is summarized in Table 1. The enzyme was eluted from the DEAE-Sepharose CL-6B column with approximately 0.2 M NaCI and with a 112-fold purity. CM-Sepharose CL-6B chromatography was subsequently employed to recover the enzyme in an unbound form but this step did not enhance the purity of the preparation. ‘The final preparation of enzyme showed aProperties of p-glucosidase purified from Aspergillus niger 427 ‘Table 1 Purification of B-glucosidase from Aspergillus niger Fractions Total Total Specific. Purification Recovery protein* activity” activity® (fold) % (mg) Crude extract 38 616 16 1 100 90% satd (NH,):S0, 69 02s 9.0 6 7 Sephadex G200 chromatography 0.7 42.5 90 037 cy DEAE Sepharose CL-6B chromatography ig 239) ima 37 CM Sepharose CL-6B chromatography 0105 19.9) 930) 120 30 Starting material, ca. 350 mi culture trate © Total activity expressed as umol p-nitrophenol min © Specific ativty expressed ss uml p-nitrophenolmin/mg protein 120-fold purification and had a sp. act. of 0.2 mmol p-nitrophenol liberated/min/mg Protein at 60°C. Compared to other reported purifications of this enzyme, this method gave a reasonably good recovery (30%) of enzyme activity. The enzyme purified from other sources showed recovery ranging from 7.5% to 39% (Inamdar & Kaplan 1966; Umezurike 1971; Blondin et al. 1983; Kohchi er al. 1985). ‘The enzyme preparation gave a single band upon PAGE using different polyacrylamide gel concentrations and had a molecular size of 325 kDa in éomparison with other proteins of known molecular size. This value is comparable to those reported for B-glucosidase from Saccharomyces cerevisiae, Aspergillus fumigatus and Botryodiplodia theobromae (Woodward and Wiseman 1982). Fungal f-glucosidase of smaller molecular sizes have also been reported (Woodward and Wiseman 1982; Freer 1985), ‘The pH optimum for B-glucosidase activity was 4.6. However, at pH 4 and 5.5, the activities obtained were about 80% of its maximum activity at pH 4.6. This pH ‘optimum is similar to those reported for other Aspergillus spp., Candida wickerhamai and two Aspergillus niger strains but is lower than those of Saccharomyces and Candida spp. which have been reported to range from pH 5.7 to 6.8 (Woodward & ‘Wiseman 1982; Freer 1985; Kohchi et al. 1985; Okada 1985; Dekker 1986). The purified enzyme was optimal at 60°C when the assay was 30 min. With a 15 min assay, the temperature optimum of the enzyme was 65°C (Fig. 1). This suggested that the enzyme was not very stable at 65°C. Thus the temperature optimum of this enzyme was taken as 60°C and was used for all kinetic studies. This value is higher than those reported for the enzyme from Saccharomyces, Candida and an Aspergillus niger strain (Woodward and Wiseman 1982; Kohchi et af. 1985; Okada 1985) and is similar to one Aspergillus niger strain (Dekker 1986). However, itis lower than that of Thermoascus aurantiacus which has an optimum temperature of 70°C (Woodward and Wiseman 1982). Kinetic studies of B-glucosidase, carried out at pH 4.6 and 60°C, showed the enzyme followed the Michaelis-Menten rate laws with both PNPG and cellobiose as substrates (Figs. 2A and B). The Kp values for PNPG and cellobiose were 0.82 + 0.10 mst and 1.33 + 0.20 mw, respectively. At high PNPG concentrations, an428 HH. Yeoh, T. K. Tan, S. L. Chua & G. Lim | 4] ‘mmol p-nitrophenovmg protein Omer 6 ees 10 90 re 20 ee 20 00 Time (minutes) Fig. 1 Reaction profile of Aspergillus niger Blucosidase at two diferent temperatures. 0, °C, (mmol glucose/minimng proteny* (mmol p-nitrophenolminimg protelny* = 0 oan cremr ana 10 2 4 6 (p-nitrophenyl B-glucoside, mM" (cellobiose, mi" Flg.2_ Effect of PNPG (A) and cellobise (B) concentration on B-sucosidase activity of Aspergillus niger. inhibition of enzyme activity was observed (Fig. 2A). With cellobiose, substrate inhibition of the enzyme activity was less (Fig. 2B). Substrate inhibition of B- lucosidase activity has been reported for the enzyme from other fungal sources (Part 1983; Evans 1985). A range of Ky (PNPG) and K,, (cellobiose) values has been reported for -glucosidase from different fungal sources but these may be attributed to different conditions employed in its determination (Workman & Day 1982; Woodward and Wiseman 1982; Parr 1983; Evans 1985; Kohchi et al. 1985). The B-glucosidase was competitively inhibited by glucose with a K, value of 2.89 mat glucose. K; values ranging from 1.8 my to 6.5 mw glucose have been reported with ‘other preparations (Evans 1985; Kohchi et af. 1985). The value obtained from this study is similar to those of f-glucosidase from an Aspergillus niger strain and Dekkera intermedia (Blondin et al. 1983; Dekker 1986). Although glucose is a competitive inhibitor of many fungal B-glucosidases (Blondin et al. 1983; Evans 1985; Kohehi et al 1985; Dekker 1986) it has also inhibited Trichoderma viride B-glucosidase non-Properties of B-glucosidase purified from Aspergillus niger 429 competitively (Gong er al. 1977). On the other hand, B-glucosidase from Candida wickerhamii is unaffected by glucose (Freer 1985). Glucono-S-lactone, a known inhibitor of fungal G-glucosidase, inhibited the B- slucosidase non-competitively (mixed inhibition). The K, values characteristic of this inhibition were Kj = 0.7 ma glucono-8-lactone and Kix. = 14.2 mM glucono-3- lactone, when Kiy and Kiys values reflect the affinity of the inhibitor for the free ‘enzyme and the enzyme-substrate complex, respectively. With Chaetomium trilaterale B-glucosidase, glucono-8-lactone has been shown to be a competitive inhibitor (Uziie et al. 1985). Thus, like glucose, glucono-8-lactone can inhibit B-glucosidases by different mechanisms. However, from the respective K, values, glucono-B-lactone appeared to be a stronger inhibitor of the enzyme than glucose. References Buonpis, B., Raromanenina, R., ARNAUD, A. & Gatzy, P. 1983. Purification and properties of the B-glucosidase of a' yeast capable of fermenting cellobiose to ethanol: Dekkera intermedia van der Walt. European Journal of Applied Microbiology and Biotechnology 17, 1-6 Dekker, R. F. H. 1986. Kinetic, inhibition, and stability properties of a commercial B- plucosidase (cellobiase) preparation from Aspergillus niger and its suitability in the hydrolysis of lignocellulose. Biotechnology and Bioengineering 28, 1438-1442. Enott, P. C. 1981, Enzyme kinetics. The steady-state approach. 2nd edn. pp. 26-36. London: ‘Chapman and Hall Evans, C. S. 1985. Properties of the B-D-glucosidase (cellobiase) from the wood:-rotting fungus, Coriolus versicolor. Applied Microbiology and Biotechnology 22, 128-131, Faeex, S. N. 1985. Purification and characterization of the extracellular B-ghicosidase produced by Candida wickerhamii. Archives of Biochemistry and Biophysics 243, 515-522 Gono, C. S., Lavisci, M, R. & Tsao, G. T. 1977. Collobiase from Trichoderma viride Purification, Properties, Kinetics, and Mechanisms. Biotechnology and Bioengineering 19, 959-981. Hepmicx, J. L. « Swim, A. J. 1968. Size and charge isomer separation and estimation of molecular weights of protein by disc gel electrophoresis. Archives of Biochemisiry and Biophysics 126, 155-164, Ivampar, B. N, & KaPLan, J. G. 1966. Purification and properties of an inducible P-glucosidase ‘of bakers’ yeast. Canadian Journal of Biochemistry 44, 1099-1108, Koucut, C., Havastt, M. & Nacat, S. 1985. Purification and properties of B-glucosidase from Candida pelliculosa var, acearethertus. Agricultural and Biological Chemistry 49, 779-784. Matter, G.L, 1959, Protein determination for large aumbers of samples. Analytical Chemistry 31, 964, OxaDA, G. 1985. Purification and properties ofa cellulase from Aspergillus niger. Agricultural and Biological Chemistry 49, 1257-1265. Pars, S. R. 1983. Some kinetic properties ofthe P-D-glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose. Enzyme and Microbial Technology §, 457462. Umezunixe, G. M. 1971. The purification and properties of extracellular B-glucosidase from Botryodiplodia theobromae Pat, Biochimica et Biophysica Acta 227, 419428, Uzi, M., Matsuo, M. & Yasut, T. 1985. Possible identity of p-xylosidase and Bglucosidase of Chactomium rilaterale. Agricultural and Biological Chemisery 49, 1167-1173. Witxivsow, G. N, 1961, Statistical estimations in enzyme kinetics. Biochemical Journal 80, 324-332, ‘Woovwanp, J. WiseMaN, A. 1982. Fungal and other B-D-glucosidases - their properties and, applications. Enzyme and Microbial Technology 4, 73-79. Workwan, W. E. a Day, D. F, 1982. Purification and properties of Buglucosidase from Aspergilus terreus. Applied and Environmental Microbiology 44, 1289-1295, Zav, Y. S., Wo, Y. Q., Cit, W., TAN, C., Gao, J. He, Fel, J. X. & Si 1, CN. 1982,430. H. Yeoh, T. K. Tan, 8. L. Chua & G. Lim and regulation of cellulose synthesis in Trichoderma pseudokoningii mutants EA3-867 and N2-T8. Enzyme and Microbial Technology 4, 3-12. Summary Extracellular Brglucosidase from Aspergillus niger USDB (1355 was purified 120-fold. It gave a single band on PAGE and had an M, of 325 000. It was optimally active at 60°C and pH 4.6. It hhad Ky, values for p-nitrophenyl-B-glucoside and cellobiose of 0.82 + 0.10 ma and 1.33 + 0.20 ma, respectively. It was competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-B-lactone. Résumé Proprittés de la B-glucosidase purifiée d’Aspergillus niger La Bralucosidase extracellulaire d'Aspergilus niger USDB 0355 a été purfiée 120 fois. enzyme a révélé une bande unique sur PAGE et avait un M, de 325 kialtons, Elle était optiquement active & 60°C et & pH 4.6. Elle présentait des valeur des Km pour le p-nitrophenyl- Brglucoside et la celobiose, respectivement de 0.82 + 0.10 ma et de 1.33 + 20'mu. Elle ét complétement inhibée par le glucose et présentait une inhibition non-compétitive mixte parla slucono-S-lactone, Resumen Propiedades de ta glucosidasa purificada a partir de Aspergillus niger Se purficé la glucosidasa extracelular de Aspergillus niger USDB 0385 120 veces. Su electroforesis en gel de poliacrilamida (PAGE) resulté en una Gnica banda con peso molecular de 325000D. La actividad fue Optima a 60°C y pH 4.6. Los valores de la Km para el p- nitrofenol-lucosido y para la celobiosa fueron de 0.82 + 0.10 mu y 1.33 + 0.20 mu respectivamente. La glicosa inhibié competitivamente el enzima mientras que la glucono-B- lactona lo inhibié no competitivamente (inhibicién mixta).