Unless specified otherwise, all chemicals were of analytical grade. Solubilized crystalline cellulose was obtained from Kyokuto Seiyaku Co., Ltd, Japan. Avicel [(R) RH-101], 4-methylumbelliferyl-β-D-glucoside (MUG) and carboxymethyl cellulose (CMC) were products of Sigma Chemical Co., (St. Louis, Mo, USA). Cellobiose, xylose, glucose, sucrose, galactose and maltose were purchased from Wako Pure Chemical Industries, Ltd, Japan. 4-Nitrophenyl-β-D-glucopyranoside monohydrate (p NPG) was purchased from Tokyo Chemical Industry Co., Ltd, Japan. Corn stover was collected from Yingkou city, Liaoning Province in China. Wheat bran and bagasse were obtained from private companies.
Wood chip of Jatropha carcass, branch and leaves of J. carcass, wood chip of Manihot esculenta, branch and leaves of M. esculenta, coconut shell, sugarcane, and rice straw were used as lignocellulosic sources for degradation in Vietnamese National Park (Ba Be and Cuc Phuong). One month later, lignocellulosic sources were dug up. All strains that would be screened were isolated from degraded biomass samples and washed soil collected. Isolated strains were inoculated on solubilized crystalline cellulose (CC) plates and CMC plates to cultivate for two weeks (Deguchi et al. 2007). The microbes that could grow on CC and CMC were picked up and inoculated onto malt extract agar (MEA).
Screening of β-glucosidases-producing strains
The first step of screening
For primary screening, strains from MEA were plated on potato dextrose agar (PDA) medium in a 9-cm diameter Petri dish and incubated at 30°C for 5 days. Then the colonies were inoculated on β-glucosidases (EC 188.8.131.52) screening agar containing 1% of CMC, 0.5% of MUG, 1.5% of agar, and Mandels salts (Daenen et al. 2008). The cultures were incubated at 30°C for 3 days. Then the plates were observed under UV light. Colonies which showed fluorescence were sorted out. It is because methylumbelliferyl (MU) which was released from MUG by β-glucosidases can emit fluorescence when induced by UV light.
The second step of screening
For secondary screening, the mycelium of the β-glucosidases-producing isolates obtained from the primary screening was transferred to a new PDA medium in a 9-cm diameter Petri dish and incubated at 30°C. Once the fungus covered most of the PDA plate, agar plates with mycelium were transferred to a sterile blender containing 25 ml of sterile water and homogenized for 30 s. Ten ml of the fungal homogenate was used to inoculate into β-glucosidases secondary screening medium containing 1% corn stover + 1% wheat bran in 100 ml, pH 5.0 Mandels salts medium with KH2PO4 2 g l-1, (NH4)2SO4 1.4 g l-1, urea 0.69 g l-1, CaCl2·2H2O 0.3 g l-1, MgSO4·7H2O 0.3 g l-1, and 1 ml trace elements solution composing of MnSO4 1.6 g l-1, ZnSO4 2 g l-1, CuSO4 0.5 g l-1, CoSO4 0.5 g l-1 (Saibi et al. 2011) then incubated at 30°C, 150 rpm for 5 days. Crude enzyme extract was obtained by centrifuging the liquid medium at 20 000 g, 4°C for 20 min and collecting the supernatant for confirming the β-glucosidases activity.
β-Glucosidases activity towards p-nitrophenyl-β-D-glucopyranoside (p NPG) was measured with use of amount of p-nitrophenol (p NP) liberated from p NPG by using a calibration curve at 410 nm (Cai et al. 1998). The reaction mixture contained 0.5 ml, 2 mM p NPG in 50 mM sodium acetate buffer (pH 5.0) and an appropriately diluted enzyme solution 0.125 ml. After incubation at 45°C for 10 min, the reaction was stopped after adding 1.25 mL, 1 M Na2CO3, and the color that formed as a result of p NP liberation was measured at 410 nm. One unit of β-glucosidases activity was defined as the amount of enzyme required to liberate 1 μmol of p NP per minute under the assay conditions. Specific activity is defined as the number of units per milligram of protein.
Cellobiase activity was assayed using cellobiose as substrate. The enzymatic reaction mixtures (1 ml) containing 0.25 ml of enzyme solution and 0.75 ml of 0.5% cellobiose in 50 mM sodium acetate buffer (pH 5.0) were incubated for 30 min at 50 C. And then the mixtures were heated at 100 C for 5 min to stop the reaction. The amount of glucose released was measured by Bio-sensor (Oji Scientific Instruments Co., Itd). One enzyme unit was defined as the amount of enzyme that produced 1 μmol of glucose per minute.
Protein concentration determination
Protein concentrations in the enzyme preparations were determined with application of the method of Bradford (Bradford 1976) with reference to a standard calibration curve for bovine serum albumin (BSA).
DNA extraction and PCR amplification from cultures
Mycelia cultured on malt extract agar were harvested with a spatula, and DNA was extracted with use of a PrepMan® Ultra Reagent (Life Technologies, Carlsbad, California, USA). ITS-5.8S rDNA (ITS) and the D1/D2 regions of LSU rDNA (LSU) were amplified with the KOD FX (Toyobo, Osaka, Japan), and with primers ITS5 (GGAAGTAAAAGTCGTAACAAGG) and NL4 (GGTCCGTGTTTCAAGACGG) (O'Donnell 1993; White et al. 1990). The mixture was processed by following the manufacturer’s instructions of kit. The DNA fragments were amplified in a T-gradient thermal-cycler (Biometra, Göttingen, Germany). Thermal-cycling program for LSU and ITS was: initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 56°C for 30 s, extension at 68°C for 1 min and a 4°C soak. Amplified DNA was purified with use of the Agencourt® AMPure® Kit (Agencourt Bioscience, Beverly, Massachusetts, USA).
Sequencing reactions were performed with the BigDye® Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA), and with primers NL1 (GCATATCAATAAGCGGAGGAAAAG) and NL4 (GGTCCGTGTTTCAAGACGG) for LSU on the T-gradient thermal-cycler (Biometra). This thermal-cycler program was employed: initial denaturation at 96°C for 1.5 min, 35 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s, extension at 60°C for 1.5 min and a 4°C soak. Sequencing reaction products were purified with the Agencourt® CleanSEQ® Kit (Agencourt Bioscience) and sequenced with the ABI PRISM® 3730 Genetic Analyzer (Applied Biosystems). Contiguous sequences were assembled with ATGC software (Genetyx, Tokyo, Japan).
DNA was analyzed with use of CLUSTAL W (Thompson et al. 1994). Based on the EF-1α sequence of Fusarium genus (O'Donnell et al. 2012), phylogenetic tree was generated with use of the neighbor-joining algorithm in the MEGA ver5.0. Concordance of the EF-1a gene datasets was evaluated with the partition-homogeneity test implemented with MEGA (Tamura et al. 2011), using 1 000 random repartitions. The fungus was determined to be most closely related to Fusarium proliferatum by comparing it with related strains in GenBank. And the NBRC deposition number is NBRC109045.
Effect of different carbon sources on β-glucosidases production by F. proliferatum
The mycelium stored on PDA medium was transferred to new PDA medium in 9-cm diameter Petri dish and incubated at 30°C for 5 days. Once the fungus covered most of the PDA plate, agar plates with mycelium were transferred to a sterile blender containing 25 ml of sterile water and then homogenized for 30 s. Ten ml of the fungal homogenate was used to inoculate 100 ml of liquid pre-cultures, pH 7.0. Liquid pre-cultures were made according to the modified Mandels medium with and without 0.69 g L-1 urea supplemented with 0.1% of yeast extract and 1% of glucose (Saibi et al. 2011). After 3 days, the mycelium homogenate made by a sterile blender was used to inoculate the modified Mandels medium which containing 2% carbon source with and without urea as following, wheat bran, corn stover, 1% wheat bran + 1% corn stover, bagasse, CMC, Avicel cellulose, sucrose, cellobiose, glucose, xylose, galactose and maltose. β-Glucosidases production by F. proliferatum in shaking flask batch cultures was carried out at 30°C and 150 rpm. Samples were withdrawn at different times during 12 days, and then centrifuged at 20 000 g for 20 min. Supernatants as crude enzyme were assayed for β-glucosidases activity, determined for pH, and analyzed by zymogram. Each culture was carried out in triplicate.
Electrophoresis and zymogram
Zymography is an electrophoretic technique for detection of purified or partly purified β-glucosidase. Zymography is based on SDS-PAGE that includes a substrate such as MUG or p NPG, which can be degraded by β-glucosidases. The degradation product emits fluorescence or produces change of color during the reaction period. However, this is not a practical method to assay β-glucosidases existing in the crude enzyme because various β-glucosidases existing in the crude enzyme caused overlapping fluorescence bands. A modified method that combines effective isolation with identification was developed to overcome the limitation of zymogram in the application on crude enzyme.
Step1: add the loading buffer for SDS-PAGE to the crude enzyme solution that was produced by incubating F. proliferatum in corn sotver + wheat bran based medium and glucose based medium, but the mix was not heated at a temperature of 100°C (Laemmli 1970). The mix of the crude enzyme and loading buffer was injected into the gel. Each sample was injected into four different wells and then the electrophoresis was applied.
Step2: After the electrophoresis, the first column of each sample was cut out of the gel and then treated with Coomassie Brilliant Blue (CBB) staining. The remaining gel was soaked in 20 mM, pH8.5 Tris–HCl buffer for two hours in order to remove SDS, so that the activity can be regained. The buffer was replaced every 30 min.
Step3: The first column that had been treated with CBB staining was used as a marker to cut the protein bands of the second column. The protein bands cut out of the second column were soaked in 20 mM p NPG for 10 min at a temperature of 45°C with the aim of active staining, and then 1.25 ml of 1 M Na2CO3 solution were added. If the color of the bands changes from colorlessness to yellow, it means that β-glucosidases exist in the bands.
Step4: Corresponding bands were cut out of the third and the fourth column based on positions of active bands of the second column. The cuts containing β-glucosidases were soaked in acetate buffer (0.05 M, pH5.0), and were crushed and separated by centrifugation. The supernatant was taken out and mixed with the same volume of loading buffer and then was analyzed with SDS-PAGE. Protein was stained with silver stainIIkit (Wako Pure Chemical Industries, Ltd, Japan).
Partial purification of β-glucosidase
Fine and dried powder of ammonium sulfate was added, over ice, into the crude extract enzyme to 50% saturation. And then the mix was still stirring at 4°C for 30 min. After centrifugation (42 500 g, 60 min), supernatant was decanted and the precipitate was discarded. Ammonium sulfate was added to bring the supernatant to 80% saturation. The latter was stirred overnight at 4°C and then centrifuged again. The precipitate was dissolved and dialyzed against 20 mM Tris–HCl buffer (pH 8.5). The dialyzed enzyme solution was centrifuged to remove the insoluble component and applied on the DEAE sepharose CL-6B column (1.5*20 cm) equilibrated with 20 mM Tris–HCl buffer (pH 8.5). The nonadsorbed protein fraction was eluted from the column with starting buffer (100 mL), and the adsorbed enzyme was collected through 5-stepwise elution chromatography (sodium chloride concentration: 0.1 M, 0.15 M, 0.2 M, 0.25 M and 0.3 M in the same buffer). There are two active peaks eluted from DEAE-Sepharose CL-6B at about 0.15 M and 0.25 M NaCl. The active fractions (0.15 M NaCl) were pooled and concentrated by a Centrifugal Filter Devices (Millipore Corporation Billerica, MA, USA), and then chromatographed separately on a superdex 75 column (1.5*60 cm) equilibrated with 20 mM Tris–HCl buffer (pH 8.5). The proteins were eluted with the same buffer at a flow rate of 1 mL min-1.