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Purification and Characterization of a <TEX>${\beta}$</TEX>-Glucosidase from Aspergillus niger and Its Application in the Hydrolysis of Geniposide to Genipin
Purification and Characterization of a ${\beta}$-Glucosidase from Aspergillus niger and Its Application in the Hydrolysis of Geniposide to Genipin
Journal of Microbiology and Biotechnology. 2014. Jun, 24(6): 788-794
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : January 27, 2014
  • Accepted : March 08, 2014
  • Published : June 28, 2014
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About the Authors
Guohong Gong
ghgong@ipp.ac.cn
Zhiming Zheng
Hui Liu
Li Wang
Jinshan Diao
Peng Wang
Genhai Zhao

Abstract
An extracellular β-glucosidase from Aspergillus niger Au0847 was purified to homogeneity by precipitation with ammonium sulfate, anion exchange, and gel filtration. The purified protein was composed of two subunits with molecular masses of 110 and 120 kDa. Au0847 β-glucosidase exhibited relatively high thermostability and pH stability, and its highest activity was obtained at 65℃ and pH 4.6, respectively. As a potential metalloprotein, its enzymatic activity was potently stimulated by manganese ion and DTT. The β-glucosidase displayed avid affinity and high catalytic efficiency for geniposide. Au0847 β-glucosidase has potential value as an industrial enzyme for the hydrolysis of geniposide to genipin.
Keywords
Introduction
Genipin is a naturally occurring iridoid compound, whose structure ( Fig. 1 ) was determined in the early 1960s [8 , 9] . Genipin is potentially valuable as a biomaterial with pharmaceutical applications, as a fingerprint reagent, and as a colorimetric reagent that is transformed to a blue pigment in the presence of suitable amino acids [1 , 6 , 13] . As a prospective natural cross-linker for protein, collagen, gelatin, and chitosan, it displays markedly lower toxicity and higher biocompatibility than glutaraldehyde and other commonly used synthetic compounds [5 , 20] . Pharmaceutically, genipin has anti-inflammatory effects, choleretic action for liver disease control, and can inhibit the protein UCP2 in the treatment of type 2 diabetes [12 , 21] .
Genipin occurs naturally in the fruits of plants, including Genipa americana L., Gardenia jasminoides Ellis, and Eucommia ulmoides Oliv. However, it is present at a low concentration (ca. 0.01% in gardenia fruit). On the other hand, its β-glycoside derivative, geniposide, is relatively abundant in the fruit of G. jasminoides (ca. 4%). It is conceivable that large-scale preparation of genipin could be achieved through β-glucosidase-catalyzed hydrolysis of geniposide extracted from gardenia fruit ( Fig. 1 ).
β- D -Glucosidase (E.C. 3.2.1.21) catalyzes the hydrolytic reaction mainly for β-(1-4)- and -(1-6)-glucosidic bonds of β- D -glucopyranosides, and belongs to glycoside hydrolase family GH1 and GH3 based on amino acid sequence similarities [10 , 11] . β-Glucosidases prepared from different plants or microorganisms possess different physicochemical properties of structure, molecular mass, substrate specificity, kinetic parameters, activation, and inhibition effects of metal ions. A higher affinity of substrate and higher specific activities are essential for industrial applications of an enzyme. To transform geniposide to genipin efficiently, the suitable β-glucosidase must possess high activity and higher affinity for the geniposide substrate.
In this study, we purified and characterized an extracellular β-glucosidase from an Aspergillus niger Au0847 mutant that was generated by low-energy ion beam implantation, which abundantly expresses the enzyme. The purified enzyme exhibited relatively high thermostability and higher affinity to geniposide. Its application in the hydrolysis of geniposide to genipin was also studied.
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β-Glucosidase-catalyzed transformation of geniposide extracted from gardenia fruit to genipin.
Materials and Methods
- Strain and Culture Conditions
Aspergillus niger Au0847, a mutant generated by low-energy ion beam implantation, which produces β-glucosidase in high-yield, was isolated and screened in our laboratory [7] . The cells were grown in seeding medium containing 1.2% glucose, 1% CaCO 3 , 3.0 g/l KH 2 PO 4 , 4.0 g/l NaNO 3 , 2.0 g/l (NH 4 ) 2 SO 4 , 0.5 g/l MgSO 4 ·7H 2 O, 0.5 g/l CaCl 2 , 0.0075 g/l FeSO 4 ·7H 2 O, 0.0025 g/l MnSO 4 ·H 2 O, 0.002 g/l ZnSO 4 , and 0.003 g/l CoCl 2 (pH 5.5–6.0). After culturing at 30℃ and 200 rpm for 18 h, 10% of the seeding culture was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of fermentation medium.
- Production and Purification of β-Glucosidase
The Au0847 strain was incubated at 33℃ with shaking at 200 rpm in the fermentation medium containing 5% corn cob powder, 4% wheat bran, 0.6% (NH 4 ) 2 SO 4 , 0.6% yeast extract powder, 0.6% glucose, and 1% CaCO 3 . After incubating for 4 days, the mycelia-free cultured broth (crude enzyme liquid) was collected by filtration and used for purification. After salting out using (NH 4 ) 2 SO 4 , the pellet was resuspended in buffer A (20 mM sodium phosphate, pH 7.0), desalted with a semipermeable membrane with a molecular weight cut-off of 3000, filtered with a 0.22 μm Millipore filter, and loaded into a DEAE-Sepharose Fast Flow anionic exchange column (1.6 cm × 20 cm) with an ÄKTA explorer 100 fast protein liquid chromatography protein purification system (GE, USA). Elution was performed with a linear 0–0.4 M gradient of NaCl in buffer A. The flow rate was 0.4 ml/min. The active fractions were applied to a Sephacryl S-200HR gel filtration column (HiPrep 16/60, 1.6 cm × 60 cm) with buffer B (50 mM sodium acetate, pH 4.8) at a flow rate of 1.0 ml/min. The protein was assessed by an enzyme activity assay, and its purity and molecular mass were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE.
- Enzyme Activity and Protein Assays
The β-glucosidase activity was measured using 1% salicin in 0.2 M sodium acetate buffer (pH 4.8) as the substrate. First, 0.5 ml of enzyme solution was incubated with 2 ml of salicin solution and allowed to react at 50℃ for 30 min. Then, 2.5 ml of 3,5-dinitrosalicylic acid (DNS) was added and boiled for 10 min. The solution volume was increased to 25 ml with water prior to determination of glucose content at 540 nm. One unit (U) of enzyme activity was defined as the amount of enzyme needed to hydrolyze salicin to 1 μmol of glucose in 1 min. The protein concentration was assayed by the method of Bradford [3] at 595 nm with bovine serum albumin as the standard.
- Mass Spectrometry and Protein Identification
The purified protein after trypsin digestion in gel was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Thermo LTQ apparatus. The Mascot search engine ( http://www.matrixscience.com ) was used to search for peptide fingerprints against predicted peptides in the NCBInr database. Protein scores greater than 75 were significant ( p < 0.05).
- Effects of Temperature and pH on Au0847 β-Glucosidase Activity and Stability
The optimal temperature of Au0847 β-glucosidase was tested between 40℃ and 75℃ by the standard enzyme assay described above. The thermal stability was assessed by incubating the enzyme at 60℃, 65℃, and 70℃ for 60 min prior to determination of the residual activity. The optimal pH was tested at pH values ranging from 2.2 to 8.0 (Na 2 HPO 4 -citric acid buffer). The pH stability was determined by incubating the enzyme in buffers of pH 2.2-8.0 for 24 h, and adjusting the pH to 4.8 prior to assay of residual enzyme activity.
- Kinetic Parameters of Au0847 β-Glucosidase
The Michaelis-Menten kinetics data (k m , V max ) of β-glucosidase were determined from Lineweaver-Burk plots [14] using 1–10 mM of salicin, geniposide, cellobiose, and 0.5–5 mM p -nitrophenyl-β- D -glucopyranoside ( p NPG) in 0.2 M sodium acetate buffer (pH 4.8) as substrates. The concentration of glucose hydrolyzed from the substrate was detected using a SBA-40C biological sensing analyzer (Shandong Academy of Science, China) [4] .
- Effects of Inhibitors and Activators on Au0847 β-Glucosidase Activity
The β-glucosidase was incubated with different additives, including final concentrations of 5 mM of metal ions and 10 mM of sodium dodecyl sulfonate (SDS), ethylene diamine tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), and dithiothreitol (DTT). The residual activity was measured using the DNS method.
- Transformation of Geniposide to Genipin with Au0847 β-Glucosidase
The solid crude enzyme (freeze-dried precipitate after (NH 4 ) 2 SO 4 salting out) was applied for the hydrolysis reaction. The solid enzyme (360 U) was incubated with 50 ml of 8.3 g/l geniposide (dissolved in buffer A) in 250 ml flasks at 30℃. All the experiments were carried out in triplicate.
- Analysis of Geniposide and Genipin
High-performance liquid chromatography (HPLC; Waters, USA) with a Kromasil C18 column (200 mm × 4.6 mm, 5 μm) was performed to analyze the concentrations of geniposide and genipin. A mobile phase of methanol/water (45:55 (v/v)) was used with a flow rate of 1.0 ml/min and column temperature of 25℃. The sample injection volume was 20 μl and the detection wavelength was 238 nm. Standards of geniposide and genipin (98% purity) were supplied by the Center of Delta Natural Organic Compounds (China).
Results
- Purification of Au0847 β-Glucosidase
At first, the collected crude enzyme liquid was purified by (NH 4 ) 2 SO 4 salting out. The recovery of β-glucosidase increased with saturation of (NH 4 ) 2 SO 4 , with the highest recovery obtained at a saturation of 80%. After separating with procedures of anionic exchange and gel filtration, the active fraction was analyzed by SDS-PAGE and native-PAGE ( Fig. 2 ). Two closely migrating bands of the purified β-glucosidase after gel filtration were present on the SDSPAGE gel ( Fig. 2 A, lane 2). The molecular mass of the two proteins was estimated as 110 and 120 kDa using gel filtration calibrated markers. The doublet was unlikely to be have been separated by the aforementioned purification procedure. It is more conceivable that they represented two subunits of β-glucosidase that had been denatured by SDS and other denaturants. To explore this possibility, the same fraction was analyzed by native-PAGE, which revealed only a single band ( Fig. 2 B, lane 2). The results showed that β-glucosidase was constructed of two subunits with molecular masses of 110 and 120 kDa. Furthermore, the polypeptides of the two purified subunits were identified by MS/MS. The two protein bands presented different peptide mass fingerprints. Therefore, they were not one subunit with different amounts of glycosylation. The obtained seven polypeptides (RIGADSTVLLK; DLANWNVEK; GIQDAGVVATAK; ITLQPSEETK; YYYVSEGPYEK; HYIAYEQEHFR; NGVFTATDNWAIDQIEALAK) were used to search against the NCBInr database for peptide matches with the Mascot program. The protein was identified as a β-glucosidase belonging to glycoside hydrolases family 3 (GH3), and the polypeptides matched well with the amino acid sequence of β-glucosidase from Aspergillus niger (GenBank: AFW 98805; 841 aa). The β-glucosidase purification is summarized in Table 1 . The purification factor reached about 16 and achieved 78% recovery.
Purification of β-glucosidase.
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Purification of β-glucosidase.
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PAGE analysis of purified Au0847 β-glucosidase. (A) SDS-PAGE with an 8% separating gel. (B) Native-PAGE with a 12% separating gel. Lane assignments: lane 1, protein marker; lane 2, purified β-glucosidase from gel filtration. Arrows: indicating target bands of SDS-PAGE and native-PAGE, respectively.
- Effects of Temperature and pH on Au0847 β-Glucosidase Activity and Stability
The optimal temperature of purified β-glucosidase was 65℃ ( Fig. 3 A). It was quite stable at temperatures below 60℃. The enzyme activity decreased with increased temperature, with 50% activity evident at 70℃ for 15 min ( Fig. 3 B).
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Effect of temperature on Au0847 β-glucosidase activity and stability. (A) Thermoactivity of the Au0847 β-glucosidase. The maximum activity obtained at 65℃ was considered as 100%. (B) Thermostability of the Au0847 β-glucosidase was carried out by incubating the enzyme at different temperatures 60, 65, and 70oC for 60 min prior to measurement of residual activity. The non-heated enzyme represented the control (100%).
β-Glucosidase displayed high activity at pH values ranging from 4 to 4.8, with an optimum of pH of 4.6 ( Fig. 4 A). Its enzyme activity was quite stable at pH values ranging from 2 to 6 at room temperature for 24 h ( Fig. 4 B).
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Effect of pH on Au0847 β-glucosidase activity and stability. (A) Effect of pH on Au0847 β-glucosidase activity. The maximum activity obtained at pH 4.6 represented 100%. (B) Effect of pH on the stability of Au0847 β-glucosidase incubated in different pH buffers for 24 h prior to measurement of residual activity. The enzyme activity before incubation represented 100%.
- Kinetic Parameters of Au0847 β-Glucosidase
The Michaelis-Menten kinetic parameters (K m , V max ) of β-glucosidase toward various substrates were determined by Lineweaver-Burk plots ( Table 2 ). The K m and V max of the enzyme toward geniposide was calculated to be 2.933 mmol/l and 0.372 mmol·l -1 ·min -1 . The catalytic efficiency (k cat /K m ) for geniposide was 2.83 × 10 4 mol -1 ·l·s -1 .
Kinetic parameters of Au0847 β-glucosidase toward various substrates.
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Kinetic parameters of Au0847 β-glucosidase toward various substrates.
- Inhibitors and Activators of Au0847 β-Glucosidase Activity
The effects of some metal ions and agents on the activity of β-glucosidase are summarized in Fig. 5 A. Of the divalent cations investigated, Mn 2+ stimulated the activity of β-glucosidase to 163%, followed by Fe 2+ at 151%. The enzyme activity increased to 227% when the Mn 2+ concentration was 10 mM ( Fig. 5 B). On the other hand, Cu 2+ inhibited the activity, whereas Ca 2+ , Zn 2+ , Mg 2+ , Co 2+ , and Ba 2+ had no appreciable effects. Likewise, the enzyme activity was not inhibited or stimulated by the univalent cations Na + , K + , and Li + . The presence of SDS and DMSO did not inhibit the enzyme activity, whereas EDTA inhibited the enzyme activity up to 27%. The β-glucosidase showed significant enhanced activity in the presence of DTT.
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Effects of inhibitors and activators on Au0847 β-glucosidase activity. (A) Effects of some metal ions and agents on the activity of β-glucosidase. (B) The activation effects with Mn2+ of different concentrations.
- Transformation of Geniposide to Genipin with Au0847 β-Glucosidase
After (NH 4 ) 2 SO 4 salting out, the solid crude enzyme (freeze-dried precipitate) was applied for the hydrolysis reaction. Geniposide was hydrolyzed entirely when incubated with Au0847 β-glucosidase for 6 h, accompanied with the rapid increase of genipin concentration. The conversion rate reached a maximum of 98% after incubation for 4 h, followed by a slight decrease as the transformation process was prolonged ( Fig. 6 ).
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Transformation of geniposide to genipin with Au0847 β-glucosidase. The 360 U solid β-glucosidase was incubated with 8.3 g/l geniposide at 30℃ for 24 h, and periodically acquired samples were quantified by HPLC.
Discussion
β-Glucosidases isolated from different microbial sources exhibit a high degree of variability with respect to physicochemical properties and substrate specificity. In a previous study, we had isolated and screened a high-yield mutant, Aspergillus niger Au0847, generated by low-energy ion beam implantation [7] . In this study, the physicochemical properties and substrate specificity of the β-glucosidase were further defined. After precipitation with ammonium sulfate, anion exchange, and gel filtration, Au0847 β-glucosidase was purified to homogeneity. Two polypeptide bands of very similar staining intensity showed up close together on a gel. A doublet often indicates the presence of two polypeptides that share the same amino acid sequence for the most part, although one may have extra residues and/or they may differ in a few key positions. In nature, such polypeptides typically form multiple-subunit proteins or are part of a “family” of isoenzymes that perform similar functions but are each specialized. In the upper separation procedures, native-PAGE was contributed to verify the presence of two subunits of the enzyme rather than isoenzymes.
Au0847 β-glucosidase exhibited relatively high thermostability. The optimal temperature and such stability were higher than the enzyme from Aspergillus niger purified by Wang et al . [17] . Its enzyme activity was quite stable at pH values ranging from 2 to 6, implying that it would be suitable for applications conducted in an acidic condition. The β-glucosidase displayed greater affinity toward p NPG and geniposide than salicin and cellobiose. The catalytic efficiency (k cat /K m ) for geniposide was similar to the enzyme purified by Wang et al . [17] . The results indicated the potential of Au0847 β-glucosidase as an industrial enzyme for the hydrolysis of geniposide to genipin.
It is interesting that Mn 2+ strongly stimulated the enzymatic activity, and the positive effect reached a maximum (up to 227%) while the concentration of Mn 2+ increased to 10 mM. The result indicated that Mn 2+ is specifically recognized by some residues of Au0847 β-glucosidase. The activity of the β-glucosidase stimulated with these ions (Mn 2+ and Fe 2+ ) was similar to other glucosidases from Stachybotrys microspora [16] and Thermoanaerobacterium thermosaccharolyticum [15] . The chelating agent EDTA inhibited the enzyme activity up to 27%, indicating that Au0847 β-glucosidase may be a metalloprotein. The reducing agent DTT was not an inhibitor but a strong activator, suggesting that disulfide bonds are not essential for the enzyme and the inhibition effect of sulfhydryl oxidant in enzyme molecules was eliminated by disoxidation of DTT. The effects of salts on β-glucosidase are complex and highly specific [2] , so further work should be carried out to investigate their effects on the structure, stability, and activity of the purified enzyme.
The β-glucosidase displayed greater affinity toward geniposide than salicin and cellobiose. When it was applied to transform geniposide to genipin, the conversion rate reached a maximum of 98% in a short time (4 h), followed by a slight decrease as the transformation process was prolonged. The decreased conversion rate likely reflected the reaction between partial genipin and free amino acids in the crude enzyme [8 , 13] . The microbial preparation of genipin could be manipulated by direct inoculation of the relevant β-glucosidase-producing microbial strain into a fermentation medium containing fruit powder of a plant species like G. jasminoides [18] , or use of residual liquid after extraction of the yellow pigment. It may be a convenient and cost-efficient process, since no or few extra nutrients are needed in the fermentation medium. However, the process is time-consuming, requiring over 70 h, is easily affected by genipin loss, and involves the costs of separation and purification. In order to reduce genipin hydrolysis and byproduct formation, Yang et al . [19] developed a method for transformation of geniposide into genipin by immobilizing β-glucosidase in a two-phase aqueous-organic system. Compared with the one-phase system, the yield of genipin (maximum 63.08%) increased 30% and the reaction time decreased 29% in the two-phase system. In our study, the preparation of high-purity genipin and a high conversion rate were benefitted by relatively pure geniposide and an effective enzyme with higher affinity to geniposide. The hydrolysis reaction was performed at a relatively low reaction temperature in a short time, which helps for reducing byproduct formation. Further studies are needed to enhance the transformation of elevated concentrations of the substrate by chemical modification and immobilization of the β-glucosidase.
Acknowledgements
We thank Mrs. Gao Wu, the Research Centre for Life Sciences, University of Science and Technology of China, for her assistance in MS analysis. This work was supported by the Academy President Fund of Hefei Institutes of Physical Science, Chinese Academy of Sciences (No. Y29YJ23132).
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