J. Microbiol. Biotechnol. (2018), 28(5), 757–764 https://doi.org/10.4014/jmb.1802.02051 Research Article Review jmb Characterization of Three Extracellular β-Glucosidases Produced by a Fungal Isolate Aspergillus sp. YDJ14 and Their Hydrolyzing Activity for a Flavone Glycoside Jong Min Oh1, Jae Pil Lee1, Seung Cheol Baek1, Yang Do Jo2, and Hoon Kim1,2* 1Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea 2Department of Agricultural Chemistry, Sunchon National University, Suncheon 57922, Republic of Korea Received: February 28, 2018 Revised: March 9, 2018 A cellulolytic fungus, YDJ14, was isolated from compost and identified as an Aspergillus sp. Accepted: March 14, 2018 strain. Three extracellular β-glucosidases, BGL-A1, BGL-A2, and BGL-A3, were separated First published online using ultrafiltration, ammonium sulfate fractionation, and High-Q chromatography. The April 12, 2018 molecular masses of the three enzymes were estimated to be 100, 45, and 40 kDa, respectively, o *Corresponding author by SDS-PAGE. The optimum pH and temperature of BGL-A3 were 5.0 and 50 C, respectively, Phone: +82-61-750-3751; whereas the optimum pH and temperature of BGL-A1 and BGL-A2 were identical (4.0 and Fax: +82-61-750-3708; o o E-mail: [email protected] 60 C, respectively). The half-life of BGL-A3 at 70 C (2.8 min) was shorter than that of BGL-A1 and BGL-A2 (12.1 and 8.8 min, respectively). All three enzymes preferred p-nitrophenyl-β-D- glucopyranoside (pNPG) and hardly hydrolyzed cellobiose, suggesting that these enzymes were aryl β-glucosidases. The Km of BGL-A3 (1.26 mM) for pNPG was much higher than that of BGL-A1 and BGL-A2 (0.25 and 0.27 mM, respectively). These results suggested that BGL-A1 and BGL-A2 were similar in their enzymatic properties, whereas BGL-A3 differed from the two enzymes. When tilianin (a flavone glycoside of acacetin) was reacted with the three enzymes, the inhibitory activity for monoamine oxidase, a target in the treatment of neurological disorders, was similar to that shown by acacetin. We conclude that these enzymes may be useful in the hydrolysis of flavone glycosides to improve their inhibitory activities. pISSN 1017-7825, eISSN 1738-8872 Copyright© 2018 by Keywords: Aspergillus sp. YDJ14, extracellular β-glucosidases, aryl β-glucosidases, flavone The Korean Society for Microbiology glycoside hydrolysis and Biotechnology Introduction β-glucosidases [2]. Fungal β-glucosidases are used in bioflavorings and in β-Glucosidases (E.C. 3.2.1.21) catalyze the hydrolysis of the development production of novel carbohydrate foods, β-1,4-glycosidic linkages to release nonreducing terminal animal feeds, bioethanol, and pharmaceuticals [4-6]. They glucosyl residues from disaccharides, oligosaccharides, are also employed in the hydrolysis of isoflavone glycosides and alkyl or aryl β-glucosides [1, 2]. According to their [7]. Recently, fungal β-glucosidase from several Aspergillus substrate specificity, enzymes are divided into three species, including A. fumigatus [8], A. saccharolyticus [9], groups: (i) aryl β-glucosidases, which hydrolyze only aryl A. niger [10], A. ochraceus [11], A. oryzae [12], A. terreus [13], β-glucosidic linkages; (ii) cellobiases, which hydrolyze and Aspergillus sp. [15], have been characterized. The cellobiose; and (iii) broad-substrate specificity β-glucosidases, production of active β-glucosidases from microbial compost which hydrolyze a wide range of substrates with different communities has also been studied [16]. bonds [3, 4]. Most of the β-glucosidases are broad specificity Many researchers have studied the hydrolysis of flavonoid May 2018 ⎪ Vol. 28⎪ No. 5 758 Oh et al. glycosides by β-glucosidases, with the majority of studies described previously [15]. The concentrations of protein were focusing on isoflavone glycosides from soybean [17-19], analyzed by the Bradford method [26]. Sodium dodecyl sulfate– and flavanone glycosides from citrus extracts [20]. However, polyacrylamide gel electrophoresis (SDS-PAGE) was carried out little information is available on flavone glycosides. using 11.5 % polyacrylamide gels [27]. Previous research demonstrated that acacetin, a flavonoid, Activity Staining was a potent inhibitor of monoamine oxidases (MAOs), Activity staining for β-glucosidase activity in active High-Q catalyzing the oxidative deamination of monoamine chromatography fractions was performed in a gel after SDS-PAGE neurotransmitters and serving as a target for the treatment and renaturation, as previously described, except for the use of for neurological disorders [21]. The same study showed MUG instead of MUC [15, 28]. that the inhibitory activity of tilianin, a glycoside of acacetin, was lower than that of acacetin. Enzyme Assay In this report, three β-glucosidases were separated The activity of β-glucosidase was assayed in a reaction mixture from Aspergillus sp. YDJ14, a cellulolytic fungal strain of 2 mM pNPG in 50 mM sodium citrate buffer (pH 5.0) as isolated from compost, and their biochemical properties substrate, with the reaction at 50oC stopped after 30 min [15]. One were characterized. The potential applications of these unit of β-glucosidase activity was defined as the amount of β-glucosidases in the hydrolysis of flavone glycosides and enzyme that generated 1 μmol of p-nitrophenol in 1 min. Substrate in improving the inhibitory activity of a pharmaceutical specificity was analyzed using 0.2 mM pNPG, pNPC, pNPX, cellobiose, and xylobiose as small substrates, and 0.5% CMC, enzyme were also investigated. β-glucan, and xylan as polysaccharides. The substrates pNPG, pNPC and pNPX were analyzed by measuring the amounts of Materials and Methods p-nitrophenol released. The others were analyzed by measuring the amounts of reducing sugar released using the DNS method Chemicals and Enzymes [29]. One unit of enzyme activity was defined as the amount of Glucose, cellobiose, xylobiose, p-nitrophenyl-β-D-glucopyranoside enzyme that liberated 1 μmol of reducing sugar per minute under (pNPG), p-nitrophenyl-β-D-cellobioside (pNPC), p-nitrophenyl-β- the conditions as above. D-xylopyranoside (pNPX), 4-methylumbelliferyl-β-D-glucopyranoside (MUG), carboxymethyl-cellulose (CMC), barley β-glucan, birchwood Biochemical Characterization of the Purified Enzyme xylan, dinitrosalicylic acid (DNS), trypan blue, and other chemicals The optimum pH for enzyme activity was analyzed using were purchased from Sigma-Aldrich (USA). Acacetin and tilianin 50 mM universal buffer (from pH 3.0 to 8.0), and the optimum (acacetin 7-glucoside) were isolated from Agastache rugosa, and temperature was determined from 30oC to 80oC in 50 mM of MAO inhibition was assayed using recombinant human MAO-A, sodium citrate buffer (pH 5.0). Thermostability was analyzed by as described previously [21]. preincubation of the enzyme without substrate for up to 1 h at 60oC or 70oC. The effects of various cations (Na+, K+, Ca2+, Mg2+, Isolation and Identification of the Fungal Strain Mn2+, Zn2+, Fe2+, Cu2+, Co2+, and Ba2+) at 2.0 mM, and 10% A compost sample was obtained from the Compost Factory isopropanol, 1% Triton X-100, and 1% SDS on enzyme activity [22], and cellulolytic fungi were isolated on PDA (potato dextrose were determined. The Km and Vmax values for the enzymes were agar) plates containing 0.4% CMC (PDA/CMC) or xylan (PDA/ determined using Lineweaver-Burk plots at five concentrations of xylan), as described previously [15]. Based on the halo size around pNPG (0.1–4.5 mM) at 50oC. the colony, a strain was selected and named YDJ14. The isolated strain was identified by 18S rRNA sequencing by Solgent (Korea). Binding Analysis with Insoluble Avicel Sequence similarity was searched, and a phylogenetic tree was The binding properties of the enzymes to carbohydrate were constructed using the BLASTN program [23, 24] on the NCBI web analyzed as described previously, with slight modifications [30]. site. The nucleotide sequence of the YDJ14 isolate was deposited Briefly, the crude extract was mixed with 1.5% Avicel in 0.3 ml of in GenBank under the accession number MG976613. 50 mM sodium citrate buffer, pH 5.0, by shaking for 40 min on ice. The mixtures were then centrifuged. The amount of unbound Enzyme Production and Isolation enzyme was determined by measuring the cellulase activity in the o The isolate was cultured at 30 C, with shaking at 150 rpm in supernatant. 200 ml of potato dextrose broth, as described previously [15], with some modifications (i.e., 21 days of culture to analyze the Hydrolysis of a Flavone Glycoside and Analysis of MAO-A extracellular β-glucosidase activity in the culture supernatant). Inhibitory Activity The proteins were separated by ultrafiltration, ammonium sulfate Tilianin (100 μM), a flavone glycoside, was reacted for 6 h at fractionation (40–60%), and High-Q column chromatography, as 50oC with approximately 0.1 U of each β-glucosidase in 50 μl of J. Microbiol. Biotechnol. Three β-Glucosidases from Aspergillus sp. YDJ14 759 illustrated in Fig. 1. The isolate was named Aspergillus sp. YDJ14. Production and Separation of β-Glucosidases The Aspergillus sp. YDJ14 isolate produced the maximum amount of β-glucosidase at 21 days after inoculation. When the culture supernatant was concentrated to approximately 20-fold, the yield was 85.3% (Table 1). High-Q chromatography of the ammonium sulfate fractionate showed major (fractions from 36 to 40) and minor (fractions from 46 to 48) activity peaks (Fig. 2A). In SDS-PAGE analysis, the fractions in the major peak exhibited two kinds of protein bands at Fig. 1. Phylogenetic tree of the strain YDJ14. each side of the maximum peak (fraction 38); a protein band from fraction 37 (100 kDa) and a protein band from 0.1 M sodium phosphate buffer (pH 6.0) [15]. After boiling for fraction 40 (45 kDa). The protein band from fraction 47 in 5 min and centrifugation, the supernatant was added to 450 μl of the minor peak was 40kDa (left of Fig.