**3. Results**

#### **3.1 Sub-cellular distribution of dihydrodiol dehydrogenase activity**

The first approach was to know the sub-cellular distribution of the dihydrodiol dehydrogenase (DD) activity by means of a differential-centrifugation procedure and the spectrophotometer detection of the DD activity from *M. circinelloides* YR-1 grown in

340 nm in a Beckman DU-650 spectrophotometer. One unit of enzyme activity was defined as the amount required reducing 1 µmol of NAD+ or NADP+ per minute at 25ºC. Specific dihydrodiol dehydrogenase (DD) activity was expressed as units per milligram of protein. For DD activity in gels we developed an appropriate methodology because there is not any report in the literature about the detection of these enzymes by means of electrophoretic zymograms, so for we modified the method described for Nikolova & Ward (1991) for alcohol dehydrogenase. Briefly, after non-denaturing 6% (w/v) PAGE, described above, the activity was revealed as follows. The gel was submerged for 120 min in 4 ml of 0.5 M Tris-HCl buffer pH 8.5 containing 0.5 mg phenazine methosulphate (PMS), 7.5 mg *p*-nitro-blue tetrazolium (PNBT), 14.34 mg NADP+ or NAD+, 1 mM EDTA, 1 mM DTT and 100 mM of (1R, 2S)-*cis*-1, 2-dihydro-1, 2-naphthalene-diol as substrate. After incubating at 25 °C for 30 min (in dark) with gentle shaking at 80 rpm, the dihydrodiol dehydrogenases or ADH

When substrate specificity of DD was tested, different single alcohols were added to the mixture reaction at a final concentration of 100 mM. The following substrates were tested: *N-*decanol, *n-*hexadecanol, *n*-octadecanol, hexane-1,2,3,4,5,6-hexaol, benzyl alcohol, cholesterol, *cis-*naphthalene-diol, ethylene-glycol, poly-ethylene-glycol 3350, and sorbitol, were previously dissolved in dioxan and others were prepared in water: methanol, ethanol, propane-1-ol, propane-2-ol, butane-1-ol, pentane-1-ol, propane-1,2,3-triol and methyl

The pH, optimal temperature, substrate specificity, and effect of cations, EDTA and pyrazole were performed after a non-denaturing gel, 6% acrylamide, loading 300 µg of protein. The pH determination was performed from 3 to 9 with citrate buffer for 3 to 5, phosphate buffer for 5 to 7 and Tris/HCl buffer for 7 to 9. The temperature effect was tested in a range of 4 to 45 °C, using a freezer or metabolic bath at the desired temperature. The cation effect was tested using 1 mM of CaCl2, MgSO4, ZnSO4 and FeSO4, and for the EDTA, 1mM was also used. The assays were performed in the presence of *cis-*naphthalene-diol as substrate and NADP+ as electron acceptor; the enzymatic activity was measured over a range of pH values

Protein concentration was measured according Lowry et al. (1951), using bovine serum albumin as standard. Phenylmethanesulphonyl fluoride and *cis*-naphthalene-diol were purchased from Sigma (St. Louis, MO, USA), the alcohol used as substrates were from J.T.

Densitometric analysis was performed in a Gene Genius Bio-Imaging System V. 6.05.01,

The first approach was to know the sub-cellular distribution of the dihydrodiol dehydrogenase (DD) activity by means of a differential-centrifugation procedure and the spectrophotometer detection of the DD activity from *M. circinelloides* YR-1 grown in

SYNGENE, Synoptics Systems. Software used was Gene Tools V. 3.06.02, Syn. Ltd.

electro-morphs were observed as blue-dark bands.

in the forward reaction dihydrodiol diol.

Baker (Phillipsburg, NJ, USA). All reagents were analytical grade.

**3.1 Sub-cellular distribution of dihydrodiol dehydrogenase activity** 

propane-1-ol*.* 

**2.5 Miscellaneous** 

**3. Results**

different carbon sources, using a variation of the method described by Bergmeyer (1983). For this purpose we use the commercial substrate *cis*-naphthalene-diol. If the low speed supernatant is compared, the enzymatic activity was almost 8 times higher when naphthalene rather than glucose was the carbon source and NADP+ was used as electron acceptor (Table 1).


Table 1. NADP+ or NAD+-dependent dihydrodiol dehydrogenase activities present in subcellular fractions of *Mucor circinelloides* YR-1 grown on different carbon sources. Mycelial cells, grown in the indicated carbon sources, were broken (Braun) and fractions obtained by differential-centrifugation. DD activity of the different fractions was measured with *cis*naphthalene-diol as substrate and NADP+ or NAD+ as electron acceptor. The values are the means of three independent experiments with triplicate determinations. a ND, no detected.

This suggests that at least some of the detected activity could be inducible, and as can be seen, the major enzymatic activity is present in the soluble fractions. When NAD+ was used as electron acceptor, the activity found in the low speed supernatant when the fungus was grown in glucose as a carbon source is more than 3 times higher than the one present when naphthalene was used, and more than ten times higher if compared with the activity obtained with ethanol as a carbon source.

These results enhance the interest to investigate how many different activities will be revealed by electrophoretic zymograms in the cytosolic fraction of the fungus when it grown on different carbon sources.

#### **3.2 Use of zymograms to reveal the presence of several dihydrodiol dehydrogenase activities in cytosolic fraction of** *M. circinelloides* **YR-1 grown on different carbon sources**

Aerobically mycelium grown in different carbon sources (see Materials and Methods) was used to obtain the corresponding cytosolic fraction and each one was run on no-denaturing polyacrylamide gels and stained for NADP+-dependent dihydrodiol dehydrogenase activity with *cis*-naphtalen-diol as substrate.

Polyacrylamide Gel Electrophoresis an Important Tool for the

Carbon source *Rm*a of DD constitutives

fraction of *Mucor circinelloides* YR-1 grown in different carbon sources.

Carbon source *Rma* of DD inducibles

fraction of *Mucor circinelloides* YR-1 grown in different carbon sources.

others enzymes where referred to this value (Table 4).

*M. circinelloides* **YR-1** 

Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms 253

D-Glucose 0.22±0.04 0.4±0.03 0.62±0.01 0.69±0.02 0.90±0.01 Glycerol - - - - - Ethanol - - - - *n*-Decanol 0.21±0.01 - 0.61±0.02 0.69±0.03 0.89±0.03 *n*-Pentane - - - 0.7±0.03 *n*-Decane 0.21±0.01 - - - *n*-Hexadecane - - - - - Naphthalene 0.21±0.01 - - - 0.89±0.006 Anthracene 0.22±0.006 - - - 0.89±0.02 Phenanthrene - **-** - **-** 0.89±0.01 Pyrene - - - - 0.89±0.01 Table 2. Constitutive NADP+-dependent dihydrodiol dehydrogenase activities in cytosolic

<sup>a</sup>*Rm*; was calculated as described in Materials and Methods section as its standard deviation.

D-Glucose - - - - - - Glycerol - - - - - - Ethanol - - - - - *n*-Decanol 0.55±0.002 0.66±0.005 - - *n*-Pentane 0.55±0.001 0.66±0.003 0.74±0.002 - - *n*-Decane - - - - - *n*-Hexadecane - - 0.73±0.010 - - 0.66±0.005 Naphthalene 0.56±0.003 - 0.73±0.020 - - 0.67±0.008 Anthracene - - - - - 0.67±0.008 Phenanthrene - - 0.74±0.010 0.23±0.006 0.78±0.003 0.67±0.008 Pyrene - - 0.74±0.004 0.22±0.020 - 0.67±0.008 Table 3. Inducible NADP+-dependent dihydrodiol dehydrogenase activities in cytosolic

<sup>a</sup>*Rm*; was calculated as described in Materials and Methods section as its standard deviation.

With the comparing purpose the activity showed for the denominated iNDD enzyme that is induced when naphthalene was used as a carbon source was taking as a 100% and the

In all cases *cis*-naphthalene-diol and NADP+ were used in the enzymatic assay. When phenanthrene was used as a carbon source there are four different inducible enzymes, iDD3, iPDD1, iPDD2 and iNDD, being iPDD1 and iPDD2 best induced by this carbon source, and

**3.3 Bands intensity of dihydrodiol dehydrogenase activities in cytosolic fraction of** 

iDD1 iDD2 iDD3 iPDD1 iPDD2 iNDD

1 2 3 4 5

Fig. 1. Dihydrodiol dehydrogenase enzymatic activity present in cytosolic fraction of *M. circinelloides* YR-1 grown in different carbon sources*.* Mycelia in lane 1 was grown for 12 h and lanes 2-11 were grown for 22 h at 28ºC on sMMP medium added with the carbon source indicated. *Lanes*: *1,* D-glucose; *2*, glycerol; *3*, ethanol; *4*, *n-*decanol; *5*, *n*-pentane; *6, n*-decane; *7*, *n*-hexadecane; *8,* naphthalene; *9,* anthracene; *10,* phenanthrene; *11,* pyrene, at the concentrations described in Material and Methods. The extracts were electrophoresed and stained as described in Materials and Methods. In all cases 300 g of protein were loaded in each lane. In this gel 100 mM *cis-*naphthalene-diol was the substrate and NADP+ the electron acceptor. These results are representative gels and mycelia were grown up and run on the gels at least three times. The induction patterns were always reproducible.

Under the conditions tested, five bands were seen in glucose as carbon source (Fig. 1, lane 1) which were considered as constitutive dihydrodiol dehydrogenases and identified with a number (1-5) considering their decreasing *Rm* (DD1-5) (Table 2), and six inducible bands of activity were detected, depending of the carbon source in the culture media used for growth (Fig. 1; Table 3). One of the inducible bands (iDD1) was seen when *n*-decanol or *n-*pentane or *n-*hexadecane or naphthalene was the carbon source (Fig. 1, lanes 4, 5, 7 and 8 respectively, Table 3).

A second inducible NADP+-dependent dihydrodiol dehydrogenase activity was seen when *n*-decanol or *n-*pentane was the carbon source (iDD2) (Fig. 1, lanes 4 and 5; Table 3). The third inducible enzymes (iNDD) was seen only when aromatic hydrocarbons were used as sole carbon source in the growth media (Fig. 1, lanes 8 to 11, Table 3). A fourth inducible naphthalene-diol dehydrogenase (iDD3) was induced by some of the alcohols, alkanes and aromatic polycyclic compounds tested (Fig. 1, lanes 3, 4, 6, 8, 10 and 11; Table 3). When phenanthrene was used as the carbon source, two new bands with different relative motilities were revealed, iPDD1 and iPDD2, (Fig. 1, lane 10; Table 3). The iPDD1 was also observed when pyrene was the carbon source (Fig. 1, lane 10 and 11; Table 3).


Table 2. Constitutive NADP+-dependent dihydrodiol dehydrogenase activities in cytosolic fraction of *Mucor circinelloides* YR-1 grown in different carbon sources.

<sup>a</sup>*Rm*; was calculated as described in Materials and Methods section as its standard deviation.


Table 3. Inducible NADP+-dependent dihydrodiol dehydrogenase activities in cytosolic fraction of *Mucor circinelloides* YR-1 grown in different carbon sources.

<sup>a</sup>*Rm*; was calculated as described in Materials and Methods section as its standard deviation.

#### **3.3 Bands intensity of dihydrodiol dehydrogenase activities in cytosolic fraction of**  *M. circinelloides* **YR-1**

With the comparing purpose the activity showed for the denominated iNDD enzyme that is induced when naphthalene was used as a carbon source was taking as a 100% and the others enzymes where referred to this value (Table 4).

In all cases *cis*-naphthalene-diol and NADP+ were used in the enzymatic assay. When phenanthrene was used as a carbon source there are four different inducible enzymes, iDD3, iPDD1, iPDD2 and iNDD, being iPDD1 and iPDD2 best induced by this carbon source, and

252 Gel Electrophoresis – Advanced Techniques

Fig. 1. Dihydrodiol dehydrogenase enzymatic activity present in cytosolic fraction of *M. circinelloides* YR-1 grown in different carbon sources*.* Mycelia in lane 1 was grown for 12 h and lanes 2-11 were grown for 22 h at 28ºC on sMMP medium added with the carbon source indicated. *Lanes*: *1,* D-glucose; *2*, glycerol; *3*, ethanol; *4*, *n-*decanol; *5*, *n*-pentane; *6, n*-decane; *7*, *n*-hexadecane; *8,* naphthalene; *9,* anthracene; *10,* phenanthrene; *11,* pyrene, at the concentrations described in Material and Methods. The extracts were electrophoresed

and stained as described in Materials and Methods. In all cases 300 g of protein were loaded in each lane. In this gel 100 mM *cis-*naphthalene-diol was the substrate and NADP+ the electron acceptor. These results are representative gels and mycelia were grown

up and run on the gels at least three times. The induction patterns were always

observed when pyrene was the carbon source (Fig. 1, lane 10 and 11; Table 3).

Under the conditions tested, five bands were seen in glucose as carbon source (Fig. 1, lane 1) which were considered as constitutive dihydrodiol dehydrogenases and identified with a number (1-5) considering their decreasing *Rm* (DD1-5) (Table 2), and six inducible bands of activity were detected, depending of the carbon source in the culture media used for growth (Fig. 1; Table 3). One of the inducible bands (iDD1) was seen when *n*-decanol or *n-*pentane or *n-*hexadecane or naphthalene was the carbon source (Fig. 1, lanes 4, 5, 7 and 8

A second inducible NADP+-dependent dihydrodiol dehydrogenase activity was seen when *n*-decanol or *n-*pentane was the carbon source (iDD2) (Fig. 1, lanes 4 and 5; Table 3). The third inducible enzymes (iNDD) was seen only when aromatic hydrocarbons were used as sole carbon source in the growth media (Fig. 1, lanes 8 to 11, Table 3). A fourth inducible naphthalene-diol dehydrogenase (iDD3) was induced by some of the alcohols, alkanes and aromatic polycyclic compounds tested (Fig. 1, lanes 3, 4, 6, 8, 10 and 11; Table 3). When phenanthrene was used as the carbon source, two new bands with different relative motilities were revealed, iPDD1 and iPDD2, (Fig. 1, lane 10; Table 3). The iPDD1 was also

reproducible.

respectively, Table 3).

Polyacrylamide Gel Electrophoresis an Important Tool for the

C, lane 7) did not showed any enzymatic activity.

grown on glucose (Fig. 2C, lane 1).

Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms 255

When we used NAD+ as electron acceptor and ethanol as substrate the denominated ADH1, ADH3 and DD5 activity bands were revealed when the cytosolic fraction from mycelium

As a control, a sample of the culture media lacking carbon source (Fig. 2A to C, lane 6) and an assay lacking substrate in the reaction mixture for the activity in zymograms (Fig. 2A to

Fig. 2. Detection of dihydrodiol dehydrogenase activities in cytosolic fraction of *M.* 

at least three times. The activity patterns were always reproducible.

*circinelloides* YR-1 by activity-stained gels. All mycelia were obtained after grown for 22 h at 28ºC on the carbon source indicated. A. *Lanes 1,* 1.0% glucose; *2,* 0.5% naphthalene; *3,* 0.5% anthracene; *4,* 0.5% phenanthrene; *5,* 0.5% pyrene; *6,* a sample of the culture media without carbon source; *7,* without substrate in the activity-reaction mixture. The reaction was

performed with NAD+ and 100 mM cis-naphthalene-diol as substrate, described in Materials and Methods section. B. identical samples as A, but the activity was developed with NADP+ and 100 mM ethanol as substrate. C. identical samples as A, but the activity was developed with 100 mM ethanol as substrate. The amount of protein loaded per track was equalized to 300 g. These results are representative gels and mycelia were grown up and run on the gels

In order to compare the observed activities we performed a densitometric analysis to the bands intensity and the Table 5 shows the obtained values under the condition where the activity have its highest value, taking the iNDD activity as a 100%. As show, the denominated DD1 enzymes, a constitute one, has the highest activity of all, in contrast the activity denominated iDD3 and DD5 measured with ethanol and NAD+ are the lowest

the latest is the one that showed the highest induction value of all inducible enzymes (103.5%) (Table 4). The iPDD2 enzyme is the only one that it is induced by only one carbon source (Table 3). In the case of the iDD1, the highest induction value obtained was when naphthalene was used as a carbon source as iDD3 and iNDD enzymes (Table 4). The iDD2 showed its best induction value when n-pentane was used as a carbon source (Table 4). It is noticeable that glycerol, ethanol and *n*-decane do not induce any of the DD activities.


Table 4. Relative inducibility of NADP+-dependent dihydrodiol dehydrogenase activities in cytosolic fraction of *Mucor circinelloides* YR-1 grown in different carbon sources. aRelative units were obtained by densitometry, using the value from iNDD as 100% when the fungus was growth on naphthalene.

#### **3.4 Effect of ethanol as substrate and NAD+ as electron acceptor on induced dihydrodiol dehydrogenase activities in cytosolic fraction of** *M. circinelloides* **YR-1 grown on different carbon sources**

It is interesting to compare if the inducible DD enzymes are able to use NAD+ as electron acceptor and/or ethanol as substrate because some dehydrogenases are able to use both of them. In the presence of NAD+ as electron acceptor and *cis*-naphthalene-diol as substrate, there was not any staining in the region of either constitutive or inducible dihydrodiol dehydrogenase activities (Fig. 2A, lanes 1-5; Table 5). In the presence of ethanol as substrate and NADP+ as electron acceptor, two ADH activity in cytosolic fraction from mycelium grown on glucose were revealed (Fig. 2B, lane 1) one with a *Rm* of 0.42±0.008 (denominated ADH1) and the other with a *Rm* of 0.84±0.003 (denominated ADH2). Under these conditions we also observed the inducible dihydrodiol dehydrogenase enzymes denominated DD3, suggesting that this enzyme also possesses an ADH activity NADP+-dependent (Fig. 2B, lane 1). Also, under these assay conditions, two bands were observed when phenanthrene was used as a carbon source to growth the mycelia (Fig. 2B, lane 4). The bands correspond to the DD5 (*Rm* of 0.90±0.010) and a new ADH, denominated ADH3 with a *Rm* of 0.94±0.010 (Fig. 2B, lane 4).

the latest is the one that showed the highest induction value of all inducible enzymes (103.5%) (Table 4). The iPDD2 enzyme is the only one that it is induced by only one carbon source (Table 3). In the case of the iDD1, the highest induction value obtained was when naphthalene was used as a carbon source as iDD3 and iNDD enzymes (Table 4). The iDD2 showed its best induction value when n-pentane was used as a carbon source (Table 4). It is

> (relative units) iDD1 iDD2 iDD3 iPDD1 iPDD2 iNDD

> > **as electron acceptor on induced**

noticeable that glycerol, ethanol and *n*-decane do not induce any of the DD activities.

D-Glucose 0.0 0.0 0.0 0.0 0.0 0.0 Glycerol 0.0 0.0 0.0 0.0 0.0 0.0 Ethanol 0.0 0.0 0.0 0.0 0.0 0.0 *n*-Decanol 10.2 5.3 0.0 0.0 0.0 0.0 *n*-Pentane 8.7 18.2 14.6 0.0 0.0 0.0 *n*-Decane 0.0 0.0 0.0 0.0 0.0 0.0 *n*-Hexadecane 0.0 0.0 6.3 0.0 0.0 4.5 Naphthalene 56.8 0.0 16.3 0.0 0.0 100.0 Anthracene 0.0 0.0 0.0 0.0 0.0 90.5 Phenanthrene 0.0 0.0 8.1 82.9 103.5 84.5 Pyrene 0.0 0.0 10.2 8.0 0.0 38.4

Table 4. Relative inducibility of NADP+-dependent dihydrodiol dehydrogenase activities in

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

It is interesting to compare if the inducible DD enzymes are able to use NAD+ as electron acceptor and/or ethanol as substrate because some dehydrogenases are able to use both of them. In the presence of NAD+ as electron acceptor and *cis*-naphthalene-diol as substrate, there was not any staining in the region of either constitutive or inducible dihydrodiol dehydrogenase activities (Fig. 2A, lanes 1-5; Table 5). In the presence of ethanol as substrate and NADP+ as electron acceptor, two ADH activity in cytosolic fraction from mycelium grown on glucose were revealed (Fig. 2B, lane 1) one with a *Rm* of 0.42±0.008 (denominated ADH1) and the other with a *Rm* of 0.84±0.003 (denominated ADH2). Under these conditions we also observed the inducible dihydrodiol dehydrogenase enzymes denominated DD3, suggesting that this enzyme also possesses an ADH activity NADP+-dependent (Fig. 2B, lane 1). Also, under these assay conditions, two bands were observed when phenanthrene was used as a carbon source to growth the mycelia (Fig. 2B, lane 4). The bands correspond to the DD5 (*Rm* of 0.90±0.010) and a new ADH, denominated ADH3 with a *Rm* of 0.94±0.010

cytosolic fraction of *Mucor circinelloides* YR-1 grown in different carbon sources.

**dihydrodiol dehydrogenase activities in cytosolic fraction of** *M. circinelloides*

Carbon source Band intensitya

the fungus was growth on naphthalene.

**3.4 Effect of ethanol as substrate and NAD+**

**YR-1 grown on different carbon sources** 

(Fig. 2B, lane 4).

When we used NAD+ as electron acceptor and ethanol as substrate the denominated ADH1, ADH3 and DD5 activity bands were revealed when the cytosolic fraction from mycelium grown on glucose (Fig. 2C, lane 1).

As a control, a sample of the culture media lacking carbon source (Fig. 2A to C, lane 6) and an assay lacking substrate in the reaction mixture for the activity in zymograms (Fig. 2A to C, lane 7) did not showed any enzymatic activity.

Fig. 2. Detection of dihydrodiol dehydrogenase activities in cytosolic fraction of *M. circinelloides* YR-1 by activity-stained gels. All mycelia were obtained after grown for 22 h at 28ºC on the carbon source indicated. A. *Lanes 1,* 1.0% glucose; *2,* 0.5% naphthalene; *3,* 0.5% anthracene; *4,* 0.5% phenanthrene; *5,* 0.5% pyrene; *6,* a sample of the culture media without carbon source; *7,* without substrate in the activity-reaction mixture. The reaction was performed with NAD+ and 100 mM cis-naphthalene-diol as substrate, described in Materials and Methods section. B. identical samples as A, but the activity was developed with NADP+ and 100 mM ethanol as substrate. C. identical samples as A, but the activity was developed with 100 mM ethanol as substrate. The amount of protein loaded per track was equalized to 300 g. These results are representative gels and mycelia were grown up and run on the gels at least three times. The activity patterns were always reproducible.

In order to compare the observed activities we performed a densitometric analysis to the bands intensity and the Table 5 shows the obtained values under the condition where the activity have its highest value, taking the iNDD activity as a 100%. As show, the denominated DD1 enzymes, a constitute one, has the highest activity of all, in contrast the activity denominated iDD3 and DD5 measured with ethanol and NAD+ are the lowest

Polyacrylamide Gel Electrophoresis an Important Tool for the

for activity revealed at pH 9, was darken because of pH.

or 45 ºC were darker because of the incubation temperature.

**3.5.3 Requirement of different divalent ions** 

pH value.

temperature.

**3.5.2 Temperature** 

Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms 257

were showed at pH values below 8.5. It is important to say that the background in the lane

pH 3 5 7 8.5 9

Fig. 3. Effect of pH on dihydrodiol dehydrogenase activities present in cytosolic fraction of *M. circinelloides* YR-1grown in naphthalene. Each track was cut and stained at the indicated

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

T °C

4 25 28 37 45

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

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+.

iNDD

iNDD

(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 substrate (Table 5).
