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A Thesis for the Degree of Master of Science
Purification and Characterization of
β-Glucosidase from Seeds of Pumpkin
(Cucurbita moschata)
호박씨 유래 β-glucosidase의 분리·정제 및
특성 규명
February, 2013
Kim, Eui Young
Department of Agricultural Biotechnology
Seoul National University
Purification and Characterization of
β-Glucosidase from Seeds of Pumpkin
(Cucurbita moschata)
지도교수 장 판 식 이 논문을 석사학위 논문으로 제출함 2013년 2월 서울대학교 대학원 농생명공학부 김 의 영 김의영의 석사 학위 논문을 인준함 위 원 장 이 형 주 (인) 부위원장 장 판 식 (인) 위 원 문 태 화 (인)I
Abstract
The objective of this study was purification and characterization of β-glucosidase (EC 3.2.1.21), catalyzing the hydrolysis of β-glucosidic bonds, from Pumpkin seed (Cucurbita moschata). The β-glucosidase was purified by fast protein liquid chromatography using Hitrap DEAE-sepharose FF, Hitrap Q-sepharose XL, and HiPrep 16/60 Sephacryl S-100 Hiresolution column. The 8.24-fold purified enzyme had a specific activity of 16.62x10-2
unit/mg protein against p-nitrophenyl-β-D-glucopyranoside (pNPG). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis showed β-glucosidase from pumpkin seed was dimeric structure with molecular mass of 48.1 kDa, the sum of 28.8 and 19.3 kDa. The molecular mass estimated by SDS-PAGE was analogous with that of 42.8 kDa estimated by gel permeation chromatography using Sephacryl S-100 Hiresolution column. The optimum temperature and pH of the enzyme were 70°C and pH 4.0, respectively. The enzyme was stable in the range of pH 2.0 to 10.0 and under 60°C, respectively. The Km, Vmax, and kcat of β-glucosidase
employing p-nitrophenyl-β-D-glucopyranoside (pNPG) as substrate were 2.22 mM, 0.078 unit/mg protein, and 13.29 min-1, respectively. From the result of zymogram analysis, the enzyme had the activity in the condition not
II
that subunits were departed but that subunits were associated. It was composed of 364 amino acids, and the secondary structure of β-glucosidase from seeds of pumpkin (Cucurbita moschata) consists of α-helix (26.10%), antiparallel β-sheet (20.17%), parallel β-sheet (8.22%), β-turn (18.16%), and random coil (27.34%) in the stable condition and it was affected by temperature and pH. There is a possibility that the enzyme have the characteristics of pH reversible inactivation according to the study of the effect on the enzyme activity and stability.
The purified β-glucosidase could cleave β-glucosidic bonds in phytochemicals and enhance the bioavailability of phytochemicals. Moreover, it was found that pumpkin seed-derived β-glucosidase has exceptional stability at wide range of pH in pre-experiment. Therefore the purified β-glucosidase could be applied to the food industry and pharmaceutical industry.
Keywords: β-glucosidase; pumpkin seeds; Cucurbita moschata; enzyme
kinetics; circular dichroism
III
Contents
Abstract ··· І Contents ··· ІІІ List of tables··· V List of figures ··· VІ 1. Introduction ··· 12. Materials and Methods ··· 5
2-1. Materials ··· 5
2-2. Preparation of crude enzyme ··· 6
2-3. Purification using fast protein liquid chromatography ··· 7
2-4. Analysis of β-glucosidase activity and protein concentration ··· 9
2-5. Polyacrylamide gel electrophoresis ··· 11
2-6. Determine of molecular mass ··· 12
2-7. Effects of temperature and pH on the enzyme ··· 12
2-8. Enzyme kinetics ···13
2-9. Zymography···14
2-10. Amino acid analysis ···14
IV
3. Results and Discussion ···17
3-1. Purification of β-glucosidase seeds of pumpkin (Cucurbita moschata) ···17
3-2. Characterization of β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 22
3-2-1. Determination of molecular mass ··· 22
3-2-2. Effects of pH and temperature on the enzyme ··· 24
3-2-3. Enzyme kinetics ··· 30
3-3. Effects of interaction between subunits on the enzyme activity ··· 32
3-4. Structural characteristics of β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 35
4. Conclusion ··· 42
5. References ··· 44
V
List of tables
Table 1. Summary of purification of β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 20
Table 2. Amino acid composition of β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 38
Table 3. Content (%) of structure elements in the β-glucosidase at different temperatures ··· 39
Table 4. Content (%) of structure elements in β-glucosidase at different pH ··· 41
VI
List of figures
Fig. 1. Comparison of β-glucosidase activity and protein concentration in crude enzyme extracts from various agricultural products (■, β-glucosidase activity; □, protein concentration) ··· 4
Fig. 2. Schematic procedure for the purification of β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 10
Fig. 3. Purification of β-glucosidase from seeds of pumpkin by (a) anion exchange chromatography with Hitrap DEAE FF, (b) anion exchange chromatography with Hitrap Q XL, (c), and (d) gel permeation chromatography with HiPrep 16/60 Sephacryl S-100 Hiresolution ··· 19 Fig. 4. (a) SDS-PAGE and (b) native-PAGE analysis of purified β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 21
Fig. 5. Determination of molecular mass of β-glucosidase from seeds of pumpkin (Cucurbita moschata) using GPC equipped with HiPrep 16/60 Sephacryl S-100 Hiresolution (●: standard proteins, ○: β-glucosidase). Ve and Vo mean the elution volume of each protein and the void volume, respectively ··· 23
VII
Fig. 6. Effects of pH on the activity of the purified β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 26
Fig. 7. Effects of temperature on the activity of the purified β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 27
Fig. 8. pH-stability of the purified β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 28
Fig. 9. Thermo-stability of the purified β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 29
Fig. 10. Hanes-Woolf plot for the determination of the kinetic constants for the hydrolysis catalyzed by β-glucosidase from seeds of pumpkin (Cucurbita moschata) ··· 31
Fig. 11. Subunit organization of glucosidase. M, marker protein; lane 1, β-glucosidase was reduced with 2-mercaptoethanol, denatured with SDS, and subjected to SDS-PAGE; lane 2, β-glucosidase was denatured with SDS in the absence of 2-mercaptoethanol and subjected to SDS-PAGE; lane 3, β-glucosidase was reduced with 2-mercaptoethanol in the absence of SDS and subjected to native-PAGE ··· 33
Fig. 12. Zymogram profiles of β-glucosidase migrated on SDS-PAGE (lane 2) and native-PAGE (lane 4) using MUG as substrate and SDS- and
VIII
native-PAGE profile stained with Coomassie Brilliant Blue R-250 (lane 1, 3) ··· 34
Fig. 13. CD spectra of β-glucosidase from seeds of pumpkin at pH 2.0 ( ), 4.0 ( ), and 8.0( ) ··· 40
1
1. Introduction
β-Glucosidase (β-D-glucoside glucohydrolase, E.C. 3.2.1.21) catalyzes the hydrolysis of β-glucosidic linkage at the non-reducing end of the molecules, which results in liberation of free glucose unit and the corresponding aglycone (Riou, Salmon, Vallier, Günata, & Barre, 1998). From the extensive studies, it has been reported that β-glucosidase plays important roles in metabolic pathways such as defense against pests (Bell, 1981) and activation of phytohormones in plants (Matsuzaki & Koiwai, 1986; Schliemann, 1984).
On the one hand, the enzyme can be applied to wine or juice processing (Ogawa, Yoshida, Kariya, Ohnishi, & Ikeda, 2002; Wang & Ma, 2005) and producing functional food with enhanced bioavailability (Ribeiro, Mandarino, Carrao-Panizzi, de Oliveira, Campo, Nepomuceno, et al., 2007) in food and pharmaceutical industries. For example, it has been well known that the enzyme has an ability to convert glycosylated forms of isoflavones into aglycone moieties (Day, DuPont, Ridley, Rhodes, Rhodes, Morgan, et al., 1998; Ioku, Pongpiriyadacha, Konishi, Takei, Nakatani, & Terao, 1998; Setchell, Borriello, Hulme, Kirk, & Axelson, 1984), which are absorbed more efficiently than glycosylated forms in gastrointestinal tract (Izumi,
2
Piskula, Osawa, Obata, Tobe, Saito, et al., 2000)
From these reasons, there have been efforts to discover novel sources for β-glucosidase with not only high activity as a catalyst but also desired characteristics (e.g. tolerant to heat in food processing or pH-stable for oral administration) from various plants and microorganisms. It is obvious that β-glucosidases from edible plants are more favorable than microbial-derived ones in terms of safety and approval for food additives.
In the preliminary study, crude extracts from eighteen agricultural products were tested to screen suitable sources for the β-glucosidase. The result indicated that three crops including pyogo mushroom, almond, and pumpkin seeds possess relatively higher β-glucosidase activities than the others (Fig. 1). Among the crops selected by β-glucosidase assay, there has been no report on β-glucosidase from pumpkin (Cucurbita moschata) seeds. Moreover, it was found that pumpkin seed-derived β-glucosidase has exceptional stability at wide range of pH in pre-experiment.
Based on these backgrounds, pumpkin seeds were selected and assessed as a novel source for β-glucosidase. Hence, the primary objective of this study is to purify β-glucosidase form pumpkin seeds and investigate enzymatic characteristics involving optimum conditions, thermo- and
pH-3
stability, and kinetics. As a further aim, it has been attempted to elucidate the relations between pH-tolerant characteristic and the secondary structure of β-glucosidase from pumpkin seeds.
4 Cash ewnu t Kidn ey be an Small red b ean Pistac hio Pine n ut Pean ut Perill a see d Sunfl ower seed Big bl ack b ean Yello w be an Small blac k bea n Form ented soyb ean Blac k bea n Pump kin se ed Black seed Almo nd Sesa me se ed Pyog o mus hroo m D-A bs or ba nc e at 4 0 0 nm fo r en zy m e ac tiv ity ( ■ ) 0.0 0.2 0.4 0.6 0.8 D-A bs or ba nc e at 5 95 n m fo r pr ot e in c on ce nt ra tio n (□ ) 0.0 0.2 0.4 0.6 0.8 Enzyme activity Protein concentration
Fig. 1. Comparison of β-glucosidase activity and protein concentration in crude enzyme extracts from various agricultural products (■, β-glucosidase activity; □, protein concentration).
5
2. Materials and Methods
2-1. MaterialsSeeds of pumpkin (Cucurbita moschata) were purchased from a farm located in Daesan-eup, Seosan-si, Chungcheongnam-do, Korea and harvested in 2010. The seeds were stored at 4°C before study. p-Nitrophenyl-β-D-glucopyranoside (pNPG, ≥98%), Trizma®base (≥99.9%), N,N,N′,N′-tetramethylethylenediamine, 2-mercaptoethanol (≥99%), sodium dodecyl sulfate (SDS), glycerol (≥99%), and glycine (≥99%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Hydrochloric acid (35~37%) and sodium phosphate dibasic (anhydrous) were obtained from Daejung Chemicals & Metals Co. (Shihueng, Gyeonggi-do, Korea). Ammonium sulfate (99.5%), citric acid (99.5%), sodium carbonate (anhydrous), sodium chloride (99.5%), and triton X-100 were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). Bio-Rad protein assay kit, N,N’-methylenebis-acrylamide (30% N,N’-methylenebis-acrylamide/bis 37.5:1), ammonium persulfate, bovine serum albumin, and bromophenol blue were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Acetic acid (100%) was purchased from J.T. Backer Co. (Phillipsburg, NJ, USA).
4-6
Methylumbelliferyl-β-D-glucoside (MUG) was purchased from Anaspec, Inc. (Fremont, CA, USA).
2-2. Preparation of crude enzyme
Two hundred grams of fresh seeds of pumpkin were ground with a blender and homogenized with 1.2 L of 20 mM Tris-HCl (pH 7.0) overnight at 4°C. After the mixture was centrifuged at 14,000×g for 1 h, the supernatant was filtered through a 3 μm membrane filter (Advantec MFS, Inc., Tokyo, Japan). Ammonium sulfate was added to the filtrated supernatant to obtain 10% saturated solution, and then it was stirred overnight at 4°C and centrifuged at 14,000×g for 1 h at 4°C. The precipitate was discarded and the supernatant was brought to 80% saturation. After the centrifugation at 14,000×g for 1 h at 4°C, the precipitate was dissolved in a small volume of 20 mM Tris-HCl (pH 7.0) and dialyzed against the same buffer over 24 h with at least five changes of dialyzing media to remove ammonium sulfate using dialysis membrane with a molecular weight cutoff (MWCO) of 12-14 kDa (Fisherbrand®, Fisher Scientific, Pittsburgh, PA, USA). The dialyzed solution was concentrated, filtered through a 0.45 μm membrane filter, and used as a crude enzyme source in the following
7
experiments.
2-3. Purification using fast protein liquid chromatography (FPLC)
The purification procedure was performed according to the flow scheme shown in Fig. 2 and all steps were carried out at 4oC using a fast protein liquid chromatography system (GE Healthcare, Uppsala, Sweden) with UV detector at 280 nm. The crude enzyme was applied to the Hitrap DEAE-sepharose FF column (1.6 × 2.5 cm) equilibrated with 50 mM Tris-HCl buffer (pH 9.0) at a flow rate of 5 mL/min and then eluted stepwise with 0.05, 0.10, 0.15, 0.20, and 1.0 M NaCl. Fractions with the highest β-glucosidase activity were collected and concentrated with desalting by ultrafiltration with a 10 kDa MWCO membrane (Millipore, Billerica, MA, USA). The pooled β-glucosidase peak was loaded onto a column of Hitrap Q-sepharose XL (0.7 × 2.5 cm) equilibrated with 50 mM Tris-HCl buffer (pH 8.0) at a flow rate of 1.0 mL/min after and filtration with 0.45 μm membrane filter. The column was eluted stepwise with 0.05, 0.10, 0.15, 0.20, and 1.0 M NaCl and fractions with the highest β-glucosidase activity were collected, concentrated by ultrafiltration, and purified with a HiPrep 16/60 Sephacryl S-100 (1.6 × 60 cm) column after filtration with 0.20 μm membrane filter. The column was washed in 50 mM Tris-HCl buffer (pH 7.0) containing 0.15 M NaCl at a
8
flow rate of 0.4 mL/min. Fractions containing β-glucosidase activity was pooled, concentrated, and used for all the biochemical and structural characterizations.
9
2-4. Analysis of β-glucosidase activity and protein concentration
β-Glucosidase activity was determined using pNPG as substrate by measuring the initial rate of p-nitrophenol (pNP) formation, as indicated by an increase in absorbance at 400 nm. Reaction mixture (4 mL) containing 5 mM pNPG in the McIlvaine buffer (50 mM citrate-100 mM sodium phosphate buffer, pH 5.0) was incubated with the enzyme solution at 70oC. Each aliquot (0.5 mL) of reactant was collected at 10-minute intervals for 30 min and mixed with 0.5 mL of 400 mM Na2CO3 to stop the reaction. One
unit of the enzyme activity is defined as the amount of enzyme required to release 1 μmol of pNP per minute. Protein was determined according to the Bradford method (Bradford, 1976). A standard curve was settled using bovine serum albumin.
10
Fig. 2. Schematic procedure for the purification of β-glucosidase from seeds of pumpkin (Cucurbita moschata).
11 2-5. Polyacrylamide gel electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE were performed on a 12% resolving gel and a 5% stacking gel according to the method of Laemmli (Laemmli, 1970) where 2-mercaptoethanol was used as reducing agent (Grebeshova, Salsedo-Torres, & Hidalgo, 1999). Samples were prepared by mixing purified enzyme with sample buffer containing 4% SDS, 10% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol, and 0.05% bromophenol blue and boiled for 2 min at 100oC before loaded. All PAGE analyses were performed using Hoefer SE 250 mini-gel system (GE Healthcare) at a room temperature at a constant current of 20 mA. After electrophoresis, protein bands were stained with Coomassie Brilliant Blue R-250 (Cleveland, Fischer, Kirschner, & Laemmli, 1977). Native-PAGE was done excluding SDS in stacking buffer, resolving buffer, and sample buffer.
To elucidate the interaction between two subunits, PAGE was conducted in three conditions; first, β-glucosidase was reduced with 2-mercaptoethanol, denatured with 4% SDS at 25 °C, and subjected to SDS-PAGE. Secondly, it was denatured with 4% SDS in the absence of 2-mercaptoethanol followed by SDS-PAGE. Finally, 2-mercaptoethanol was used to reduce the
β-12
glucosidase in the absence of SDS, and the protein was subjected to native-PAGE.
2-6. Determination of molecular mass
The molecular mass of the enzyme was determined by SDS-PAGE and gel permeation chromatography on a HiPrep 16/60 Sephacryl S-100 (1.6 × 60 cm) column. SDS-PAGE was performed in the method of Laemmli (Laemmli, 1970) on a 12% resolving gel and a 5% stacking gel. The HiPrep 16/60 Sephacryl S-100 (1.6 × 60 cm) column was previously equilibrated with 50 mM Tris-HCl (pH 7.0) containing 0.15 mM NaCl and calibrated with molecular mass standards as follows: bovine serum albumin (66 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa).
2-7. Effects of temperature and pH on the enzyme
The effects of pH and temperature on the enzyme activity were determined using pNPG as substrate by measurement of the relative activity.
13
The optimum pH was determined by measuring the activity at the range of pH 2.0 to 11.0 using Britton-Robinson buffer at 70oC, while the optimum temperature for the enzyme activity in McIlvaine buffer (pH 4.0) was measured at temperatures from 40 to 80oC.
pH-stability of the enzyme was estimated by measurement of the residual activity of each enzyme solution after incubation at various pH in the range of pH 2.0 to 12.0 using Britton-Robinson buffer for 24 h at 4oC. To observe the thermo-stability of the enzyme, the residual activity was measured after incubation at various temperatures (30-90oC) in McIlvaine buffer (pH 4.0) for 30 min.
2-8. Enzyme kinetics
Michaelis-Menten constant (Km) and maximum velocity (Vmax) were
determined with β-glucosidase at pH 4.0 and 70oC using pNPG as a substrate ranging of 0.5 to 12 mM. The enzyme concentration was 0.5 mg/mL of reaction mixture in all cases. The kinetic parameters were evaluated from Hanes-Woolf plot.
14 2-9. Zymography
β-Glucosidase activity was also revealed by zymography. For this, SDS-and native-PAGE were performed with the method of Laemmli (Laemmli, 1970), but samples were both not heated before. After SDS-PAGE, the gel was submerged in 50 mM Tris–HCl (pH 8.0) containing 1.0% triton X-100 for 1 h, with constant agitation to get rid of SDS allowing the renaturation of proteins. The gel was then incubated in McIlvaine buffer (pH 4.0) for 15 min for equilibration and was superposed on 1.0% agar containing 10 mM of MUG. After incubation at 37oC for 24 h, the active protein bands were visualized by UV light.
2-10. Amino acid analysis
The amino acid composition of β-glucosidase from seeds of pumpkin (Cucurbita moschata) was analyzed using a high-performance liquid chromatograph (HPLC) instrument (Agilent 1200LC, Agilent Technologies, Inc., CA, USA) equipped with a C18 column (5 μm, 4.6 ×150 mm), fluorescence detector (excitation wavelength of 340 nm, emission wavelength of 450 nm) and a UV detector (338 nm). The β-glucosidase from
15
seeds of pumpkin was hydrolyzed in 6 N HCl at 130°C for 24 h. A calibration chromatogram was established for 21 known amino acids (aspartate, glutamate, serine, histidine, glycine, threonine, arginine, alanine, tyrosine, valine, phenylalanine, isoleucine, γ-aminobutyric acid, leucine, lysine, proline, asparagine, glutamine, norvaline, sarcosine, and hydroxyproline). The mobile phase A and B were 20 mM sodium phosphate monobasic (pH 7.8) buffer and water/acetonitrile/methanol (10:45:45 v/v/v) at 1.5 mL/min flow rate for 30 min.
2-11. Circular dichroism
Circular dichroism (CD) is being increasingly recognized as a valuable technique for examining the structure of proteins in solution(Kelly, Jess, & Price, 2005). Measurements were performed on homogeneous β-glucosidase at a concentration of 0.1 mg/mL. CD spectrometer (Chirascan™-plus, Applied Photophysics, Ltd., Leatherhead, Surrey, UK) was performed in 10 mM sodium acetate (pH 4.0) in the Far-UV regions (190-260 nm), path length (0.5 mm), and bandwidth (1.0 nm). To study the effects of temperature on the second structure of β-glucosidase, CD was measured at
16
different temperatures in the range of 30 to 85oC. The estimated percentages of secondary structure were calculated from the CD spectra with the by CDNN secondary structure analysis software.
17
3. Results and Discussion
3-1. Purification of β-glucosidase from seeds of pumpkin (Cucurbita
moschata)
β-Glucosidase was purified from seeds of pumpkin by anion exchange chromatography and gel permeation chromatography according to the procedure summarized in Table 1. The overall yield of the purification was around 5.54% with a purification fold of 8.24. The specific activity of 16.62x10-2 unit/mg was obtained from the homogeneous β-glucosidase activity from seeds of pumpkin. Fig. 3 shows the elution pattern of the enzyme on the three steps of chromatography. To confirm degree of purification, gel permeation chromatography was performed again and one peak meaning purified protein was detected (Fig. 3-(d)) and the homogeneity of the enzyme was indicated in native-PAGE profile (Fig. 4-(b)). The electrophoretic pattern of β-glucosidase from seeds of pumpkin in SDS-PAGE showed two dominant bands with molecular masses of 28.8 and 19.3 kDa (Fig. 4-(a)).
18 (a) Elution volume (mL) 0 100 200 300 400 A b so rp ti o n a t 28 0 n m ( m A u ) 0 100 200 300 400 500 600 b-G lu co s id a s e a c ti vi ty ( u n it /m L ) 0.00 0.01 0.02 0.03 N a C l (m o l/ L ) 0 20 40 60 80 100 120 Absorption at 280 nm (mAu)
b-Glucosidase activity (unit/mL) NaCl (mol/L) (b) Elution volume (mL) 0 50 100 150 200 250 300 A b s o rp ti o n a t 2 8 0 n m ( m A u ) 0 100 200 300 400 b-G lu c o si d a se a ct iv it y (u n it /m L ) 0.00 0.01 0.02 0.03 0.04 0.05 N aC l ( m o l/L ) 0 20 40 60 80 100 120 Absorption at 280 nm (mAu)
b-Glucosidase activity (unit/mL) NaCl (mol/L)
19 (c) Elution volume (mL) 0 20 40 60 80 100 120 A b so rp ti o n a t 28 0 n m ( m A u ) 0 10 20 30 40 50 60 b-G lu c o si d a se a ct iv it y (u n it /m L ) 0.00 0.01 0.02 0.03 0.04 0.05 0.06 Absorption at 280 nm (mAu)
b-Glucosidase activity (unit/mL)
(d) Elution volume (mL) 0 20 40 60 80 100 120 A b so rp ti o n a t 28 0 n m ( m A u ) 0 10 20 30 40 Absorption at 280 nm (mAu)
Fig. 3. Purification of β-glucosidase from seeds of pumpkin by (a) anion exchange chromatography with Hitrap DEAE FF, (b) anion exchange chromatography with Hitrap Q XL, (c) and (d) gel permeation chromatography with HiPrep 16/60 Sephacryl S-100 Hiresolution.
20
Table 1. Summary of purification steps of the β-glucosidase from seeds of pumpkin (Cucurbita moschata)
Step Total protein (mg) activity Total (unit)
Specific activity
(unit/mg) Yield (%) Purification fold
Crude extract 2237.5 45.151 0.0202 100.00 1.00 10-80% (NH4)2SO4 precipitation 852.8 32.211 0.0378 71.34 1.87 DEAE-sepharose FF 129.6 11.473 0.0885 25.41 4.39 Q-sepharose XL 68.0 6.231 0.0916 13.80 4.54 Sephacryl S-100 Hiresolution 15.1 2.503 0.1662 5.54 8.24
21
Fig. 4. (a) SDS-PAGE and (b) native-PAGE analysis of purified β-glucosidase from seeds of pumpkin (Cucurbita moschata).
22
3-2. Characterization of β-glucosidase from seeds of pumpkin (Cucurbita
moschata)
3-2-1. Determination of molecular mass
SDS-PAGE and gel permeation chromatography on HiPrep 16/60 Sephacryl S-100 Hiresolution were carried out to estimate the molecular mass of β-glucosidase from seeds of pumpkin. The purified enzyme has a molecular mass of approximately 42.8 kDa (Fig. 5), corresponding to that determined by SDS-PAGE (48.1 kDa). It was similar to those of Carica
papaya (54 kDa) (Dickman, Patil, & Kolattukudy, 1982) and Plumeria
obtusa (54 kDa) (Souza, Elias, Simeoni, de Paula, Gomes, Guerra, et al.,
2012) but, it was smaller than those of Prunus avium (68 kDa) (Gerardi, Blando, Santino, & Zacheo, 2001), Prunus dulcis (almond, 135 kDa) (Dale, Ensley, Kern, Sastry, & Byers, 1985), and Vanilla planifolia (201 kDa) (Odoux, Chauwin, & Brillouet, 2003). The purified enzyme gave two bands with molecular mass of 28.8 and 19.3 kDa (Fig. 4-(a)) and eluted as a single symmetrical peak associated with the β-glucosidase activity from gel permeation chromatography (Fig. 3-(d)). From these results, the native β-glucosidase appeared to be a heterodimeric protein.
23
Ve/Vo
1.2 1.4 1.6 1.8 2.0L
o
g
(M
W
)
4.0 4.2 4.4 4.6 4.8 5.0 Carbonic anhydrase Ribonuclease A Bovine serum albuminOvalbumin
Fig. 5. Determination of molecular mass of β-glucosidase from seeds of pumpkin (Cucurbita moschata) using GPC equipped with HiPrep 16/60 Sephacryl S-100 Hiresolution (●: standard proteins, ○: β-glucosidase). Ve and Vo mean the elution volume of each protein and the void volume, respectively.
24
3-2-2. Effects of pH and temperature on the enzyme
Optimum pH and temperature of purified β-glucosidase were estimated by using pNPG as a substrate. Fig. 6 shows the effect of pH on the activity of the enzyme. The optimum pH for β-glucosidase activity was found to occur at pH 4.0. β-Glucosidase was inactivated at pH 2.0 and over pH 7.0 and at pH above 5.0, the activity decreased very rapidly. The effect of temperature on the β-glucosidase activity was determined by assaying enzyme activity at different temperatures at pH 4.0. The profile of effect of temperature on the enzyme activity is shown in the Fig. 7. Under optimum pH condition, the purified enzyme showed 90% maximum enzyme activity at temperatures over the range of 60-70oC and had a temperature optimum of 70oC.
For investigation of the pH-stability, the purified enzyme was incubated in at various pH values. The pH-stability profile (Fig. 8) shows that the enzyme is highly stable over a broad pH range. In that profile, the residual activities over 80% were retained between pH 2.0 and 10.0 after pre-incubation for 24 h at 4oC. Especially, the enzyme retained about 93% of its original activity at pH 2.0.
25
9. It was investigated by measuring the residual activity after incubation in the McIlvaine buffer (pH 4.0) for 30 min at temperatures ranging of 30 to 90°C. The enzyme was highly stable at temperatures below 50oC but was
inactivated at higher temperatures over 70oC. The residual activity at the 70oC, the optimum temperature, was about 70%. That means the enzyme is not stable at the optimum temperature.
26 pH 1 2 3 4 5 6 7 8 9 10 11 12 R el at iv e ac ti vi ty ( % ) 0 20 40 60 80 100 120
Fig. 6. Effects of pH on the activity of the purified β-glucosidase from seeds of pumpkin (Cucurbita moschata).
27 Temperature (oC) 30 40 50 60 70 80 90 R el a ti ve a ct iv it y (% ) 30 40 50 60 70 80 90 100 110
Fig. 7. Effects of temperature on the activity of purified β-glucosidase from seeds of pumpkin (Cucurbita moschata).
28 pH 1 2 3 4 5 6 7 8 9 10 11 12 13 R e si d u al a ct iv it y (% ) 0 20 40 60 80 100 120
Fig. 8. pH-stability of the purified β-glucosidase from seeds of pumpkin (Cucurbita moschata).
29 Temperature (oC) 20 30 40 50 60 70 80 90 100 R es id u al a ct iv it y (% ) 0 20 40 60 80 100 120
Fig. 9. Thermo-stability of the purified β-glucosidase from seeds of pumpkin (Cucurbita moschata).
30 3-2-3. Enzyme kinetics
The kinetic parameters of purified enzyme (Vmax, Km, kcat, and kcat/Km)
were determined from Hanes-Woolf plot (Fig. 10) for pNPG under optimum conditions (pH 4.0, 70oC). The Vmax, Km, kcat and kcat/Km values were 0.078
31 Concentration of pNPG (mM) -4 -2 0 2 4 6 8 10 12 14 [p N P G ] / I n it ia l v el o ci ty 0 50 100 150 200
Fig. 10. Hanes-Woolf plot for determination of the kinetic constants for the hydrolysis catalyzed by β-glucosidase from seeds of pumpkin (Cucurbita
32
3-3. Effects of interaction between subunits on the enzyme activity
The electrophoretic mobility of the β-glucosidase under both reducing and denaturing conditions migrated as a dimer (Fig. 11-(a)). Under denaturing (in the presence of SDS) but nonreducing conditions (no 2-mercaptoethanol), the enzyme also migrated as a dimer (Fig. 11-(b)). When the enzyme was reduced with 2-mercaptoethanol and then subjected to native-PAGE (in the absent of SDS), the enzyme corresponded to the monomeric molecular mass (Fig. 11-(c)). These results indicated that the β-glucosidase was not composed of disulfide-linked dimers. Two subunits may associate in the linkage broken easily by SDS to form the native structure.
To check the enzyme has activity whether to exist as the dimer form meaning native form or a monomer form for each subunit, zymography was conducted both in the condition that the enzyme was in a monomer form after SDS-PAGE and in the native condition after native-PAGE without staining. The result of zymography was shown in Fig. 12. Only the native-form enzyme showed a distinct activity on solid (lane 4), for the fluorescence from the 4-Methylumbelliferone (MU) was shown under UV. The enzyme had the activity only in the native form subunits associated.
33
Fig. 11. Subunit organization of glucosidase. M, marker protein; lane 1, β-glucosidase was reduced with 2-mercaptoethanol, denatured with SDS, and subjected to SDS-PAGE; lane 2, β-glucosidase was denatured with SDS in the absence of 2-mercaptoethanol and subjected to SDS-PAGE; lane 3, β-glucosidase was reduced with 2-mercaptoethanol in the absence of SDS and subjected to native-PAGE.
34
Fig. 12. Zymogram profiles of β-glucosidase migrated on SDS-PAGE (lane 2) and PAGE (lane 4) using MUG as substrate and SDS- and native-PAGE profile stained with Coomassie Brilliant Blue R-250 (lane 1, 3).
35
3-4. Structural characteristics of β-glucosidase from seeds of pumpkin (Cucurbita moschata)
To analyze the secondary structure, the investigation about the total number of amino acid was performed as prerequisite. The amino acid composition of β-glucosidase from seeds of pumpkin is shown in Table 2. Among the 21 amino acids, the 15 amino acids were detected. Tryptophan, asparagine, glutamine, methionine and several amino acids can be destroyed in the pretreatment and not be detected for the small amount. The number of residues of each amino acid was calculated on the basis of a monomer molecular mass of 42.8 kDa. The total number of amino acids is 364. The most abundant essential amino acid in the seeds of pumpkin (Cucurbita
moschata) was glycine (Gly).
Far-UV CD spectra of β-glucosidase from seeds of pumpkin (Cucurbita
moschata) were recorded between 190 and 260 nm. The far-UV CD of a
protein generally reflects its secondary structure content. α-Helix proteins have negative bands at 222 and 208 nm and a positive band at 193 nm and proteins with β-sheet have a negative band at 218 nm and a positive band at 195 nm, whereas disordered proteins such as β-turn and random coil have negative bands at 189 nm and 198 nm and positive bands at 210 nm and 212
36
nm, respectively (Greenfield, 2007). The secondary structure of β-glucosidase from seeds of pumpkin consists of α-helix (26.10%), antiparallel β-sheet (20.17%), parallel β-sheet (8.22%), β-turn (18.16%) and random coil (27.34%). To know the effect of the temperature on the secondary structure, the experiments were carried out at different temperatures in the range of 30 to 85oC. Contents (%) of structure elements in β-glucosidase at different temperatures are shown in the Table 3. As the temperature increased, α-helix and parallel β-sheet contents became smaller and antiparallel β-sheet contents became bigger except 85oC. The contents of the structure of the enzyme were affected only slightly by change of the temperature between 35 and 80oC, but structural changes were larger at temperature as high as 85oC.
In the pH 2.0, the enzyme was inactivated, but the residual activity of the enzyme retained highly at pH 2.0 at the result of effect of pH on the stability. It means that enzyme inactivated at pH 2.0 can be partially reactivated by incubation at pH 4.0. It can be explained by assuming that a lowering of the pH causes a reversible conformational change of the β-glucosidase molecule to a form that is no longer enzymatically active. To reveal that, it is necessary to investigate that the enzyme has the active form of the secondary structure. So, to study the effect of pH on the secondary structure of β-glucosidase, CD
37
experiments were performed at the various pH of 2.0, 4.0, and 8.0. It was clarified that pH affects the secondary structure of the enzyme and there is certain structure in which the enzyme have the activity.
38
Table 2. Amino acid composition of β-glucosidase from seeds of pumpkin (Cucurbita moschata)
Amino acid Integer Amino acid Integer
Aspartate (Asp) 38 Valine(Val) 23 Glutamate(Glu) 30 Proline(Pro) 21 Serine (Ser) 27 Phenylalanine (Phe) 11 Histidine(His) 10 Isoleucine(Ile) 16 Glycine (Gly) 67 Leucine(Leu) 28 Threonine(Thr) 20 Lysine (Lys) 20 Arginine (Arg) 10 Methionine (Met) N.D.
Alanine (Ala) 39 Cysteine (Cys) N.D. Tyrosine (Tyr) 2 Tryptophan (Trp) N.D.
39
Table 3. Content (%) of structure elements in the β-glucosidase at different temperatures
Temperature (ºC) α-Helix (%) Antiparallel β-sheet (%) Parallel β-sheet (%) β-Turn (%) Random coil (%) 30 26.10 20.17 8.22 18.16 27.34 40 25.09 21.13 8.21 18.11 27.45 50 23.55 23.55 8.13 18.01 26.78 60 21.70 27.68 7.86 17.95 24.82 70 21.30 28.31 7.81 17.92 24.67 80 21.46 28.41 7.75 17.90 24.49 85 19.11 26.91 8.57 17.14 28.28
40 Wavelength (nm) 190 200 210 220 230 240 250 260 C ir cu la r d ic h ro is m ( m d e g ) -10 -8 -6 -4 -2 0 2 4 6 8 10 pH 2 pH 4 pH 8
Fig. 13. CD spectra of β-glucosidase from seeds of pumpkin at pH 2.0 ( ), 4.0 ( ), and 8.0( ).
41
Table 4. Content (%) of structure elements in β-glucosidase at different pH
pH α-Helix (%) Antiparallel β-sheet (%) Parallel β-sheet (%) β-Turn (%) Random coil (%) 2.0 23.06 41.39 4.99 18.85 11.70 4.0 25.69 20.46 8.18 18.17 27.50 8.0 33.06 11.73 8.16 17.96 29.08
42
3. Conclusion
β-Glucosidase has been purified from seeds of pumpkin (Cucurbita
moschata). The 8.24-fold purified enzyme had a specific activity of 0.1662
unit/mg protein against p-nitrophenyl-D-glucopyranoside (pNPG). β-Glucosidase from seeds of pumpkin was dimeric structure with molecular mass of 48.1 kDa estimated by the SDS-PAGE, analogous with the molecular mass of 42.8 kDa estimated by gel permeation chromatography. The optimum temperature and pH of the enzyme were 70°C and pH 4.0, respectively. The enzyme was stable in the range of pH 2.0 to 10.0 and under 60°C, respectively. The Km, Vmax and kcat value of β-glucosidase for the
substrate pNPG were 2.22 mM, 0.078 unit/mg protein and 13.29 min-1,
respectively. It was composed of 364 amino acids, and the secondary structure of β-glucosidase from seeds of pumpkin (Cucurbita moschata) consisted of α-helix (26.1%), antiparallel β-sheet (20.17%), parallel β-sheet (8.22%), β-turn (18.16%) and random coil (27.34%) in the stable condition. The secondary structure of the enzyme was affected by temperature and pH. As it is possible that the enzyme have the characteristics of pH reversible inactivation according the study of the effect on the enzyme activity and stability, it is necessary that a further study should be carried out about that.
43
The purified glucosidase from seeds of pumpkin differs from other β-glucosidase in that it possesses a respectively higher activity in the acidic condition than most β-glucosidases purified from other sources do. This now sets the stage for more detailed investigations of β-glucosidase from seeds of pumpkin (Cucurbita moschata), such as cloning of the full-length gene followed by protein engineering studies. The present results should contribute to better industrial production of β-glucosidase.
44 5. References
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47
국문초록
이 연구의 목적은 β-glucosidic 결합의 가수분해를 촉매 하는 호박씨(seeds of Cucurbita moschata) 유래 β-glucosidase(EC 3.2.1.21)를 추출 및 정제하고 특성을 규명하는 것이다. β-glucosidase는 DEAE-sepharose Fast Flow, Q-sepharose XL, Sephacryl S-100 Hiresolution column 등을 장착한 fast protein liquid chromatography로 정제하였다. 정제된 β-glucosidase는 native-PAGE를 통해 순수하게 정제되었음을 확인하였다. 순수한 β-glucosidase는 기질 p-nitrophenyl-β-D-glucopyranoside에 대 하여 16.62x10-2 unit/mg의 specific activity를 가졌으며, 8.24의 정제도를 나타냈다. Gel permeation chromatography에 의하여 측 정된 분자량은 42.6 kDa이었으며, SDS-PAGE에서 나타난 두 밴드 의 분자량의 합(48.2 kDa)과 비교하였을 때, native 상태의 β-glucosidase는 두 개의 subunit을 가진 dimer임을 확인할 수 있었 다. 두 개의 subunit은 SDS-PAGE에서 disulfide bond를 환원하는 2-mercaptoethanol 없이, SDS만 존재할 경우에도 두 개의 band 로 나타났다. 이로써 두 subunit 간의 결합은 disulfide bond가 아 님을 확인할 수 있었다. 4-Methylumbelliferyl-β-D-glucoside를 기질로 하여 zymography를 실시한 결과 호박씨 유래 β-glucosidase는 dimer 상태에서 효소의 활성을 가지지만, dimer가 각각의 monomer로 분리되었을 때에는 효소의 활성을 잃는 것을
48 확인할 수 있었다. 효소의 최적 온도와 pH는 상대적으로 70°C와 pH 4.0임을 확인하였고, Km과 효소의 효율성을 나타내는 kcat 값은 각각 2.22 mM과 13.29 min-1임을 확인하였다. β-Glucosidase는 pH 2-10의 구간에서 24시간 동안 80% 이상의 안정성을 보였으며, 온도 60oC 이하에서 30분동안 80% 이상의 안정성을 보였다. 구성 아미노산 분석 결과, β-glucosidase는 약 364개의 아미노산으로 구성되어 있었으며, circular dichroism으로 2차 구조를 분석한 결 과, α-helix의 구조는 25.69%, antiparallel β-sheet는 20.46%, parallel β-sheet는 8.18%, β-turn은 18.17%, random coil은 27.5% 를 포함하고 있었다. 온도와 pH에 따라 2차 구조가 변하였으며, 온도보다 pH 조건에 따라 더 많은 변화를 관찰할 수 있었다. 이러 한 특성은 pH에 따라 활성을 나타내는 특정한 2차 구조가 있음을 밝히는데 기초 연구자료가 될 것으로 사료된다. 본 연구의 결과를 바탕으로 추가적인 연구를 진행할 수 있으며, 낮은 pH를 갖는 식 품 등 여러 산업에 적용될 수 있을 것으로 사료된다.
