The Czapek Dox (CD) medium consisted of sucrose 30 g/L, NaNO3 3 g/L, K2HPO4 1 g/L, MgSO4·7H2O 0.5 g/L, KCl 0.5 g/L, FeSO4 0.01 g/L and CuSO4 0.01 g/L. The potato dextrose agar (PDA) medium was composed of peeled potato extract 200 g/L, glucose 20 g/L and agar 20 g/L. The fungal minimal medium (MM) was prepared as described by Penttilä et al. (1987). The fermentation medium (FM) contained sucrose 80 g/L, yeast extract 15 g/L, KCl 0.5 g/L, MgSO4·7H2O 0.5 g g/L, K2HPO4 5 g/L, NaNO3 2 g/L.
Isolation and preliminary screening of β-fructofuranosidase producing fungi
Different soil samples were collected from Shandong Xingguang Sugar Group Co. Ltd., P. R. China, a private enterprise engaging in extracting sucrose from sugar-cane with a history of nearly 20 years. To isolate fungal strains, the soil samples were serially diluted and spread on CD agar plates mentioned above and incubated at 30 °C for 3 days. The cultures were purified by repeated transfer to new fungal MM agar plates. Then the single colonies were grown in PDA plates at 30 °C for 6 days. The spores were washed with sterilized physiological sodium chloride solution. To rapidly determine the ability of these fungi to produce FFase, an indirect colorimetric plate assay for evaluation of FFase was used, which involved in the detection of glucose released from sucrose. This method entailed a glucose oxidase–peroxidase (Dingguo Corp., Beijing, China) coupled reaction using phenol and 4-aminoantipyrine for determination of glucose with the formation of pink halos around the fungal colony (Dominguez et al. 2006). In detail, 1 μL of spores (108/mL) was spotted on the CD agar plates and incubated at 30 °C for 8 h. Then 10 mL of the biochemical reaction system, composed of glucose oxidase (GOD, 10 U/mL), horseradish peroxidase (POD, 1 U/mL), 4-aminoantipyrine (0.16 mg/mL) and phenol (1 mg/mL) in the citric phosphate buffer (pH 5), was placed on the plates. After 10 min, the formation of pink halos around the colonies indicated the ability of the fungi to produce FFase.
Secondary screening of the FFase producers with transfructosylation activity
The spore suspensions (108 spores) of the candidate fungi were inoculated into 30 mL of FM media. After incubation at 30 °C for 48 h, the cultures were centrifuged and the cells were collected for rapid FOS synthesis reaction. The reaction mixture (5 mL) consisted of 25% (W/V) sucrose (2 mL) as the substrate, 0.1 M citrate buffer (pH 5.0, 3 mL) and adequate amount of the collected cells. The enzymatic reaction was carried out at 50 °C for 2 h with moderate shaking and terminated by heating the mixture in boiling water for 10 min. Then qualitative identification of FOS production and composition was conducted using the thin layer chromatography (TLC) according to the method described by Zhang et al. (2017).
Morphological classification and molecular identification
The taxonomic assignment of the target fungus was followed for observation and identification. Macroscopical characters were assessed at 6 days on PDA plates. To determine the microscopic characteristics of the fungus, 1 μL of spores (108/mL) were inoculated on CD plates and the glass cover slips were inserted 2 cm far from the spores. After incubation at 30 °C for 3 days, the slips were used for observation of the conidial heads, conidiophores and conidia shapes under a microscope. Each strain was identified in genus level according the standard methods provided by Pitt and Hocking (2009). Experiments were conducted thrice with 3 replicate plates.
The fungal mycelia were harvested from MM medium after cultivation of 3 days at 30 °C. Then the total genomic DNA was extracted using the DNeasy Plant Mini-Kit (Qiagen, Valencia, CA, USA). The internal transcribed spacer (ITS) sequence of fungal nuclear ribosomal DNA was amplified by PCR using ITS-F (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS-R (5′-TCCTCCGCTTATTGATATGC-3′) as primer pair. The PCR product was separated by electrophoresis and then isolated and sequenced. The fungal ITS sequence was deposited into GeneBank Data Library with the accession number KY705016. A homology search was performed with GenBank database and the homologous sequences were selected for phylogenetic analysis by using the neighbor-joining (NJ) method. Then an NJ tree was constructed using MEGA version 5.0. A bootstrap analysis was performed with 1000 replications as confirmation of each clade.
Fungal growth and FFase production on different carbon sources
108 spores were inoculated in 100 mL of CD liquid media with 3% sucrose replaced by 1% sucrose, maltose, glucose, fructose, xylose or glycerol as carbon sources. The cultures were taken out every 12 h to measure dry weight of mycelia and FFase activity. The FFase activity was determined according to the method of Hidaka et al. (1988). One unit of activity (U) was defined as the amount of enzyme required to produce 1 μmol of glucose per min. To further investigate the ability of the fungus to produce FFase on sucrose, 109 spores were inoculated in 300 mL of FM containing 8% sucrose as carbon source. The cultures were taken out every 12 h to measure dry weight of mycelia, supernatant pH and FFase activity.
Transfructosylation reaction and chromatography analysis
For FOS synthesis, a reaction mixture consisting of 20% sucrose (w/v) and fungal cells (6 units of enzyme per g sucrose) in 50 mM citrate phosphate buffer (pH 5.5) was stirred at 50 °C. The mixture was taken at appropriate times and treated in boiling water for 5 min to terminate the reaction. Then the sample was centrifuged at 1, 600 g for 10 min. The supernatant was collected and subjected to TLC to examine the homogeneity of the resulting products. To further analyze the effects of substrate concentration on the transfructosylation activity, different concentrations of sucrose (w/v) (0.2, 2, 5, 20 and 50%) were used for FOS synthesis. Quantitative analysis of the reaction products was carried out using high-performance liquid chromatography (HPLC, LC-6A, Shimadzu, Japan) with a Agilent Zorbax NH2 column (5 μm, 4.6 mm × 250 mm) (Agilent Technologies, Santa Clara, CA) coupled with a refractive index detector (2414, Waters, USA). The mobile phase was acetonitrile: water (70:30, v/v) at a flow rate of 1.0 mL/min. The identification and quantification of each FOS was carried out by a calibration curve built with authentic standards of kestose, nystose and fructosyl nystose (Sigma Corp., St Louis, MO).
Production of FFase using sugarcane molasses as carbon source
For inoculation, 109 spores were added into 300 mL of the culture medium and cultivated at 30 °C and 200 rpm. The culture medium consisted of the basal medium and various amounts of sugarcane molasses [2, 5,10, 15 and 20% (w/v)]. The basal medium contained yeast extract 15 g/L, KCl 0.5 g/L, MgSO4·7H2O 0.5 g/L, K2HPO4 5 g/L, NaNO3 2 g/L. After incubation for 48 h, the cells were collected and examined for the FFase activity. All experiments were conducted in triplicate.