Microorganism and chemicals
Geotrichum candidum S12 (CCTCC AF2012005), previously isolated from soil and stored in the China Center for Type Culture Collection (Wuhan, China), was used in the study.
Methanol, ethanol, 1-propanol, n-butanol, isobutanol, hexanol, and isoamyl alcohol were purchased from Sinopharm Chemical Reagent Co. Ltd (Ningbo, China). All the other chemicals were purchased from Sigma Chemical Co. (USA).
The commercial GDH from yeast, purchased from Evergrande Parkson Biological Technology Development co. Ltd (Beijing, China), was used in the study for comparison.
Preparation of G. candidum and enzyme extract
Geotrichum candidum S12 was cultivated aspreviously described (Zhang et al. 2013a) with some modifications. The concentration of hexanol in the medium was changed to 1.5 g/L. After cultivation, the cells were collected by centrifugation and stored at −20 °C before further study. To make an enzyme extract, 50 g of the frozen cells were ground in threefold of liquid nitrogen, and extracted with 1 L of citrate buffer (0.1 mmol/L, pH 5.8) for 30 min at 4 °C. Centrifugation at 8910×g for 30 min was performed to remove the cellular debris, and the supernatant was collected and used asanenzyme extract.
Purification of putative enzyme fraction
The enzyme fraction exhibiting the highest activity towards hexanol was isolated and purified from the above prepared enzyme extract by ammonium sulfate [(NH4)2SO4] precipitation, MonoQ anion-exchange chromatograph, and Sephacryl S-200 gel filtration chromatography (Zhu et al. 2012). In brief, 30 and 70% saturation of (NH4)2SO4 was used. The precipitated protein fraction by (NH4)2SO4 treatment was loaded onto a MonoQ10/100 column (1.6 cm × 40 cm; GE Healthcare, Germany) using AKTA purifier TM 100, and eluted using a linear gradient program with 0–1.4 mol/L NaCl in 0.1 mmol/L citrate buffer, pH 5.8. The fractions showing activity towards hexanol were further purified via gel filtration chromatography with a Sephadex S-200 column (1.8 cm × 100 cm; GE Healthcare, Germany). The column was equilibrated with 5 volumes of 0.1 mmol/L citrate buffer, pH 5.8. Proteins were eluted at a flow rate of 1 mL/min and 1 mL fractions were collected. The fractions showing activity towards hexanol were pooled, concentrated by dialysis and lyophilization (CS110-4 Labogene, Denmark), and then the protein concentration (Bradford 1976) and enzyme activity towards hexanol were measured.
The protein fractions showing the highest activity towards hexanol were freeze-dried to powder form and stored at −20 °C. Before using, the enzyme powder was prepared in 0.1 mmol/L citrate buffer (pH 5.8) at 0.11 mg/mL with an activity of 3802 U/mg for characteristics analysis.
HPLC and polyacrylamide gel electrophoresis analysis of the protein fraction
The HPLC measurement was carried out using a HPLC (SPD-20A, SHIMADZU Japan), on a 5 μm, 150 × 4.6 mm i.d. Wondasil-C18 column (SHIMADZU, Japan) using eluent of 0.1 mmol/L potassium phosphate buffer, pH 7.0. The purification was monitored by OD value at 280 nm based on previously reported methods (Kim et al. 1988).
Native polyacrylamide gel electrophoresis (Native-PAGE) was employed to determine the purity and relative molecular weight of the enzyme as described by Davis (1964). After electrophoresis, the protein bands on the gel were stained with coomassie brilliant blue R-250 and dehydrogenase-specific dyeing solution was implemented as described previously (Zhang et al. 2013a). In this way, the protein bands showing activity to hexanol and NADP+ were displayed.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was also performed on the obtained enzyme fraction with the hexanol-degrading activity to determine the purity and molecular mass of the enzyme, using the method reported by Laemmli (1970), (Zhu et al. 2012).
MALDI-TOF-MS analysisof the purified protein fraction
The protein fraction with the highest hexanol-degrading activity [Fig. 1f (51.4 kDa)] was manually excised from the SDS-PAGE gel and then identified by matching using reference gels/maps and peptide mass fingerprinting using a Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometer (MALDI-TOF MS, Applied Biosystems) (Klepsch et al. 2009). The identification of enzyme according to the peptide mass fingerprinting data was performed by MASCOT search program in the SwissProt database (Barash and Mor 1973), after excluding the trypsin autolysis products from the control spectrum.
Assays of the enzyme activities
Activity towards hexanol and other higher alcohols
The enzyme activity towards hexanol and other higher alcohols was measured according to the decrease of its concentration by gas chromatography (GC). The enzyme reaction and GC analysis methods were performed as described previously (Zhu et al. 2012) except that the reaction conditions were 30 °C for 60 min. One unit (U) of the purified enzyme is the amount of enzyme required to reduce 1 μmol of hexanol per hour under these assay conditions.
Activity towards glutamate and α-ketoglutarate
The enzyme activity measurement was performed at 30 °C. The enzyme activity was measured by following the change in absorbance at 340 nm as described by Choudhury and Punekar (2007). One unit (U) of activity is defined as the amount of enzyme required to reduce/oxidize 1 μmol NADP+/NADPH/min.
Enzymatic properties analysis
Substrate specificity
The substrate specificity was investigated by separately testing methanol, ethanol, 1-propanol, isobutanol, hexanol, isoamyl alcohol, glutamate, and α-ketoglutarate as substrates for the purified enzyme fraction. For each substrate, the enzyme activity was measured with different alcohol concentrations ranging from 10 to 50 mmol/L; glutamate and NH4
+ concentrations-from 5 to 50 mmol/L; NADP+ and NADPH concentrations -from 0.01 to 0.1 mmol/L; and α-ketoglutarate concentrations—in the interval from 0.5 to 5 mmol/L. The Michaelis-Menten constant (K
m) and the maximum rates of the reaction (V
max) for the different substrates were determined by plotting the activity data as a function of the substrate concentration according to the method of Lineweaver and Burk (1934). The activity of the GDH from yeast towards different substrates was also determined for comparison.
Conditions and factor influence measurement
The influence of pH and temperature on enzyme activity and stability was tested using hexanol as the substrate. For the enzymatic conditions tests, seven levels of pH from 2.2 to 8.0 were tested for pH influence using 0.1 mol/L phosphate buffer, and different levels of temperatures from 20 to 50 °C were used to evaluate the effect of temperature on enzyme activity. For enzyme stability conditions, similar levels of pH and temperature were tested according to the residual enzyme activity after incubation in phosphate buffer at pH 4.0.
To determine the effect of metal ions on the enzyme activity, Fe3+, Ba2+, Ca2+, Mn2+, Fe2+, Pb2+, K+, Mg2+, and Zn2+ were added into the reaction system (pH 4.0, 30 °C) at 5, 10, or 50 mmol/L by the addition of FeCl3, BaCl2, CaSO4, MnCl2, FeSO4, Pb(NO3)2, KCl, MgSO4, or ZnSO4, EDTA, DTT, ATP and ADP were also added to the reaction system to test the effects of inhibitors.