**3.5.2 Temperature**

256 Gel Electrophoresis – Advanced Techniques

(Table 5). It is interesting that only the denominated ADH 1 to 3 have activity with ethanol as a substrate, being the ADH3 the enzyme with the highest activity (Table 5). Surprisingly not a single one activity was revealed when *cis*-naphthalene-diol and NAD+ were used as a

DD activities were assayed only in crude cell-free extracts of aerobically-naphtahalene grown mycelia cells because at these moments, we were strongly interested in the NDD

In all cases, the DD activities were tested in cell-free extracts of *M. circinelloides* YR-1.grown in 0.5% of naphthalene as sole carbon source for 22 h at 28 ºC. Cytosolic fraction was separated in a native electrophoresis and the amount of protein loaded per track equalized and was equivalent to 300 g, NADP+ as electron acceptor and 100 mM *cis-*naphthalene-diol

Band intensity (relative units)a *cis*-naphthalene-diol Ethanol Enzyme NADP+ NAD+ NADP+ NAD+ *Rmb* DD 1 104.2 0.0 0.0 0.0 0.21±0.006 DD 2 1.2 0.0 0.0 0.0 0.40±0.010 DD 3 3.4 0.0 0.0 0.0 0.61±0.020 DD 4 3.2 0.0 0.0 0.0 0.69±0.030 DD 5 1.2 0.0 67.6 0.4 0.90±0.010 iDD1 56.8 0.0 0.0 0.0 0.56±0.010 iDD2 18.2 0.0 0.0 0.0 0.66±0.050 iDD3 16.3 0.0 3.6 0.4 0.73±0.010 iPDD1 82.9 0.0 0.0 0.0 0.23±0.006 iPDD2 103.5 0.0 0.0 0.0 0.78±0.003 iNDD 100.0 0.0 0.0 0.0 0.67±0.008 ADH1 0.0 0.0 1.2 3.1 0.42±0.008 ADH2 0.0 0.0 45.2 0.0 0.84±0.003 ADH3 0.0 0.0 74.8 0.0 0.94±0.010 Table 5. Activities of *cis-*naphthalene-diol and alcohol dehydrogenase of cytosolic fraction of *M. circinelloides* YR-1 grown in the best inducer for each one. Densitometric analysis was carried out as described in Materials and Methods. The enzymatic determination was on the

gel with *cis-*naphthalene-diol or ethanol as the substrate and NADP+ or NAD+ as the

a Relative units were obtained by densitometry, using the value from iNDD as 100% when

The Fig. 3 shows that the optima pH value for all five activities expressed with nnaphthalene as carbon source and NADP+ and naphthalene-diol in the enzymatic reaction was 8.5. It is noticeable that only the iNDD show activity at pH 3 and little DD activities

substrate (Table 5).

electron acceptor.

**3.5.1 pH dependence**

naphthalene was the carbon source.

activity.

**3.5 General properties of NDD activities** 

as enzyme substrate were used to reveal the zymograms.

The effect of the temperature on DD activities was tested on cytosolic fraction in a range of temperatures oscillating between 4 and 45 ºC. The optimum temperature was 37 ºC, notice that even at 45 ºC the activity band corresponding to the iNDD can be seen in the zymogram (Fig. 4). It is important to specify that the background in the lanes for activity revealed at 37 or 45 ºC were darker because of the incubation temperature.

Fig. 4. Temperature effect on dihydrodiol dehydrogenase activity present in cytosolic fraction of *M. circinelloides* YR-1. Each track was cut and activity developed at the indicated temperature.

#### **3.5.3 Requirement of different divalent ions**

Different divalent ions were used to prove if some of them were required for DD activities. The Fig. 5 shows that only Ca2+ had an enhancing effect on DD activities meanwhile the other divalent metals tested and also EDTA were inhibitory Fe2+ > Zn2+ > EDTA> Mg2+.

Polyacrylamide Gel Electrophoresis an Important Tool for the

Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms 259

enzyme it is the one that shows in a majority of substrates, 14 out of 18, being ethanol, propane-1-ol, benzyl alcohol and sorbitol the substrates where the activity did not show, but when show its intensity is really low (Fig. 7). In contrast, the band with the highest intensity is the DD2 enzyme when propane-1,2,3-triol was the substrate (Fig. 7, lane 10), even when this enzyme cannot be seeing when cis-naphtalene-diol is used as a substrate (Fig. 7, lane 18). As this DD2 enzyme there are others enzymes that did not show with cis-naphthalenediol and can be seen with others substrates, as it is DD3 and iPDD2, that shows with three and four different substrates respectively (as an example see Fig. 7, lane 10). It is surprising that the iPDD2 enzyme that only showed when phenanthrene was the carbon source to growth the fungus, it is present here depending on the substrate used, propane-1,2,3-triol, 2 methyl propane-1-ol, ethylene-glycol and benzyl alcohol (Fig. 7, lanes 10, 11, 13 and 15).

In the particular case of NDD1, it was present only when naphthalene was the carbon source (Fig. 7) but it was absent in all other aromatic hydrocarbons used as carbon source (Fig. 1) and this enzyme practically only uses *cis-*naphthalene-diol as substrate (Fig. 7, lane 18). In the case of iDD3 and DD5, both present broad substrate specificity, showing a special preference for short-chain alcohols, including 1-decanol (Fig. 7). There are five bands that show with different

Fig. 7. Substrate specificity of constitutive and inducible DD activities of cytosolic fraction of *M. circinelloides* YR-1 grown on naphthalene, revealed by activity-stained gels. A variety of substrates were used with NADP+ as electron acceptor. All substrates were tested at 100 mM of final concentration. Lane *1* methanol*; 2* ethanol*; 3* propane-1-ol*; 4* propane-2-ol*; 5* butane-1-ol*; 6* pentane-1-ol*; 7* 1*-*decanol*; 8* 1*-*hexadecanol*; 9* hexane-1,2,3,4,5,6- hexaol*; 10* propane-1,2,3-triol*; 11* 2-methyl propane-1-ol*; 12* 1-octadecanol*; 13* ethylene-glycol*; 14* poly-ethylene-

Many studies have been done on NAD+-dependent *cis*-dihydrodiol dehydrogenases (DD) in bacteria (Jouanneau & Meyer, 2006; Van Herwijnen et al., 2003). In the case of NADP+-

glycol 3350*; 15* benzyl alcohol*; 16* cholesterol*; 17* sorbitol*; 18 cis*-naphthalen-diol*.* 

**4. Discussion and conclusion** 

substrates, but its intensity is really low and they were not taken in account (Fig. 7).

Fig. 5. Divalent ions effect on dihydrodiol dehydrogenase activity present in cytosolic fraction of *M. circinelloides* YR-1. Each track was cut and stained adding to the reaction mixture the divalent ion indicated at the concentrations described in Materials and Methods.

#### **3.5.4 Pyrazole effect**

Pyrazole is a well known ADH competitive inhibitor (Pereira et al., 1992) and this is the principal reason we decide to prove its effect on the different DD activities present in crude cell-free extracts obtained from *M. circinelloides* YR-1 mycelia grown in naphthalene as the sole carbon source. As can be seen in Fig. 6, pyrazole has a little inhibitory effect on the different DD's. In addition, iNDD showed a mild decrease in the level of its activity when measured by staining for activity in gels in presence of pyrazole (Fig. 6)

Fig. 6. Pyrazole effect on dihydrodiol dehydrogenase activity present in the cytosolic fraction of *M. circinelloides* YR-1 grown in naphthalene. Each track was cut and stained adding or not to the reaction mixture 1.0 mM pyrazole.

#### **3.5.5 Substrate specificity of the different inducible and constitutive dihydrodiol dehydrogenase activities**

To test the substrate specificity we chose naphthalene as the carbon source in the culture media since we can observe two constitutive band of activity (DD1 and DD5) and three inducible bands (iDD1, iDD3 and iNDD) (Fig. 1, Table 2 and 3). A variety of substrates were tested in the gel making the assay with NADP+ as electron acceptor. The constitutive DD1

CaCl2 MgSO4 ZnSO4 FeSO4 EDTA No addition

iNDD

Ion added [1 mM]

Fig. 5. Divalent ions effect on dihydrodiol dehydrogenase activity present in cytosolic fraction of *M. circinelloides* YR-1. Each track was cut and stained adding to the reaction mixture the divalent ion indicated at the concentrations described in Materials and

Pyrazole is a well known ADH competitive inhibitor (Pereira et al., 1992) and this is the principal reason we decide to prove its effect on the different DD activities present in crude cell-free extracts obtained from *M. circinelloides* YR-1 mycelia grown in naphthalene as the sole carbon source. As can be seen in Fig. 6, pyrazole has a little inhibitory effect on the different DD's. In addition, iNDD showed a mild decrease in the level of its activity when

> Pyrazole1 mM (-) (+)

measured by staining for activity in gels in presence of pyrazole (Fig. 6)

Fig. 6. Pyrazole effect on dihydrodiol dehydrogenase activity present in the cytosolic fraction of *M. circinelloides* YR-1 grown in naphthalene. Each track was cut and stained

**3.5.5 Substrate specificity of the different inducible and constitutive dihydrodiol** 

To test the substrate specificity we chose naphthalene as the carbon source in the culture media since we can observe two constitutive band of activity (DD1 and DD5) and three inducible bands (iDD1, iDD3 and iNDD) (Fig. 1, Table 2 and 3). A variety of substrates were tested in the gel making the assay with NADP+ as electron acceptor. The constitutive DD1

iNDD

adding or not to the reaction mixture 1.0 mM pyrazole.

**dehydrogenase activities** 

Methods.

**3.5.4 Pyrazole effect** 

enzyme it is the one that shows in a majority of substrates, 14 out of 18, being ethanol, propane-1-ol, benzyl alcohol and sorbitol the substrates where the activity did not show, but when show its intensity is really low (Fig. 7). In contrast, the band with the highest intensity is the DD2 enzyme when propane-1,2,3-triol was the substrate (Fig. 7, lane 10), even when this enzyme cannot be seeing when cis-naphtalene-diol is used as a substrate (Fig. 7, lane 18). As this DD2 enzyme there are others enzymes that did not show with cis-naphthalenediol and can be seen with others substrates, as it is DD3 and iPDD2, that shows with three and four different substrates respectively (as an example see Fig. 7, lane 10). It is surprising that the iPDD2 enzyme that only showed when phenanthrene was the carbon source to growth the fungus, it is present here depending on the substrate used, propane-1,2,3-triol, 2 methyl propane-1-ol, ethylene-glycol and benzyl alcohol (Fig. 7, lanes 10, 11, 13 and 15).

In the particular case of NDD1, it was present only when naphthalene was the carbon source (Fig. 7) but it was absent in all other aromatic hydrocarbons used as carbon source (Fig. 1) and this enzyme practically only uses *cis-*naphthalene-diol as substrate (Fig. 7, lane 18). In the case of iDD3 and DD5, both present broad substrate specificity, showing a special preference for short-chain alcohols, including 1-decanol (Fig. 7). There are five bands that show with different substrates, but its intensity is really low and they were not taken in account (Fig. 7).

Fig. 7. Substrate specificity of constitutive and inducible DD activities of cytosolic fraction of *M. circinelloides* YR-1 grown on naphthalene, revealed by activity-stained gels. A variety of substrates were used with NADP+ as electron acceptor. All substrates were tested at 100 mM of final concentration. Lane *1* methanol*; 2* ethanol*; 3* propane-1-ol*; 4* propane-2-ol*; 5* butane-1-ol*; 6* pentane-1-ol*; 7* 1*-*decanol*; 8* 1*-*hexadecanol*; 9* hexane-1,2,3,4,5,6- hexaol*; 10* propane-1,2,3-triol*; 11* 2-methyl propane-1-ol*; 12* 1-octadecanol*; 13* ethylene-glycol*; 14* poly-ethyleneglycol 3350*; 15* benzyl alcohol*; 16* cholesterol*; 17* sorbitol*; 18 cis*-naphthalen-diol*.* 
