Polyacrylamide Gel Electrophoresis an Important Tool for the Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms

Gel electrophoresis of enzymes is a very useful and powerful analytical method, which is at present widely used in many distinct fields of both biological and medical sciences and successfully applied in many different fields of human activity. The tremendous expansion of this methodology is mainly due to its simplicity and its high ability to separate both isoenzymes and alloenzymes, which have proven to be very useful genetic markers. The most important step of enzyme electrophoresis is the detection of native enzymes on electrophoretic gels; it means the procedure of obtaining electropherograms, or zymograms. Detection of enzymes on electrophoresis gels means the visualization of gel areas occupied by specific enzyme molecules after their electrophoretic separation. From this point of view, sometimes the testing of an enzyme-coding DNA sequence for expression of catalytically active enzyme is performed by zymograms, where the use of this technique for this purpose is very effective, cheap, and time saving. The number of applications of zymogram techniques for testing cloning enzyme-coding genes for their expression at the protein level is growing (Pfeiffeer-Guglielmi et al., 2000; Lim et al., 2001; Okwumabua et al., 2001). An absolute prerequisite for this is the specific and sensitive zymogram technique suitable for detection of the enzyme inside the gel and the use of the appropriate substrates.


Introduction
Gel electrophoresis of enzymes is a very useful and powerful analytical method, which is at present widely used in many distinct fields of both biological and medical sciences and successfully applied in many different fields of human activity.The tremendous expansion of this methodology is mainly due to its simplicity and its high ability to separate both isoenzymes and alloenzymes, which have proven to be very useful genetic markers.The most important step of enzyme electrophoresis is the detection of native enzymes on electrophoretic gels; it means the procedure of obtaining electropherograms, or zymograms.Detection of enzymes on electrophoresis gels means the visualization of gel areas occupied by specific enzyme molecules after their electrophoretic separation.From this point of view, sometimes the testing of an enzyme-coding DNA sequence for expression of catalytically active enzyme is performed by zymograms, where the use of this technique for this purpose is very effective, cheap, and time saving.The number of applications of zymogram techniques for testing cloning enzyme-coding genes for their expression at the protein level is growing (Pfeiffeer-Guglielmi et al., 2000;Lim et al., 2001;Okwumabua et al., 2001).An absolute prerequisite for this is the specific and sensitive zymogram technique suitable for detection of the enzyme inside the gel and the use of the appropriate substrates.
The zymograms has been used to detect a variety of oxidoreductases (Bergmeyer, 1983), including isoenzymes (Jeng & Wayman, 1987) and to classify various genera of yeast based upon the relative mobility of the activity bands produced by selected enzymes (Goto & Takami, 1986;Yamasaki & Komagata, 1983).Electrophorezed gels are placed in a staining solution containing a reduced substrate such as an alcohol, oxidized cofactor such as NAD + or NADP + , a dye such as nitroblue tetrazolium, and an electron acceptor-donor such as phenazine methosulphate.At the location of the appropriate enzyme catalyzing oxidation of substrate and reduction of cofactor, a dark-purple band appears as a result of the precipitate that forms upon reduction of the dye.
Previously we have investigated the use of zymogram staining of native electrophoretic gels as an initial approach to the identification of carbonyl reductase activities against both aliphatic (Silva et al., 2009;Zazueta et al., 2008) and aromatic hydrocarbons (Durón et al., 2005;Zazueta et al., 2003) in Mucor circinelloides YR1, an indigenous fungus isolated from petroleum contaminated soil.
Oil spills sometimes occur during routine operations associated with the exploration and production of crude oil.Crude oils vary widening in composition depending on factors such as source bed type and generation temperatures (Hunt, 1979).Biodegradation rates for crude oils will vary due to differences in composition, as reflected by hydrocarbon class distribution: saturates, aromatics, and polars, and the amount n-alkanes versus branched and cyclic alkanes within the saturated hydrocarbon class (Cook et al., 1974).In nature exist many types of microorganisms useful in the biodegradation processes of contaminant compounds (Atlas, 1995), such as the polycyclic aromatic hydrocarbons (PAH's) that are persistent soil contaminants and many of which have toxic and carcinogenic properties (Hyötyläinen and Oikari, 1999;Cerniglia, 1997).
In bacterial aerobic degradation of aromatic compounds, reactions of metabolic pathways generally lead to the formation of aromatic intermediates containing two hydroxyl constituents, which are subsequently ring-cleaved by excision dioxygenases (Neidle et al., 1992).In many catabolic pathways the formation of such intermediates is carried out by two successive enzymatic steps namely dihydroxylation of the polyaromatic substrate to produce cis-diols followed by dehydrogenation (Harayama & Timmis, 1989).The ring hydroxylation is catalyzed by multi-component dioxygenases, while the dehydrogenation is catalyzed by cisdiol-dehydrogenases.In mammalian tissues the enzyme dihydro-diol dehydrogenase (DD,EC 1.3.1.20)exists in multiple forms (Hara et al., 1990;Higaki et al., 2002) and catalyses the NADP + -linked oxidation of trans-dihydro-diols of aromatic hydrocarbons to the corresponding catechols (Penning et al., 1999).Studies on the metabolism of aromatic hydrocarbons by fungi are limited, nevertheless have been shown to posses the ability to metabolize aromatic compounds (Auret et al., 1971;Ferris et al., 1976) and the aryl oxidative enzymes of fungi appear to be similar to monooxygenases of hepatic microsomes (Cerniglia & Gibson, 1977;Ferris et al., 1976).Smith & Rosazza (1974) have also presented evidence that naphthalene is metabolized to 1-naphtol by six different genera of fungi.
In this work we analyze the cytosolic fraction of YR-1 strain by electrophoretic zymograms, methodology that there is not described in the literature for the NADP + -dependent dihydrodiol dehydrogenase (DD) activities.We analyze all the activity bands corresponding to proteins with DD activity present in an enzymatic extract in only one lane of the electrophoretic gel.Our results show eleven different DD activity bands, five of them are constitutive, DD1-5, since they appears when the strain is growth on glucose, and the others six are induced by different compound added to the culture media as a sole carbon source.Some biochemical-enzyme characteristics as pH, optimal temperature, cofactor dependence, substrate specificity and the effect of cations, EDTA and pyrazole were investigated for DD activities when YR-1 strain was grown in naphthalene as sole carbon source.

Organisms used and culture conditions
Mucor circinelloides strain YR-1 originally isolated from petroleum-contaminated soil in Salamanca, Guanajuato, Mexico was used as enzymatic source.A defined media containing Polyacrylamide Gel Electrophoresis an Important Tool for the Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms 249 yeast-peptone-glucose-agar (YPGA) (Bartnicki-García & Nickerson, 1962) was used for strain maintenance, spore collection and mycelium growth.Aerobic mycelium growth was also carried out on salt minimal medium (Alvarado et al. 2002) added with 0.1% (w/v) peptone (sMMP).As a carbon source we added D-glucose (1% w/v) or glycerol (1.0% v/v) or ethanol (2.0% v/v) or n-decanol (1.0% v/v) or n-pentane (1.0% v/v) or n-decane, (1.0% v/v) or n-hexadecane (1.0% w/v) or naphthalene (0.5% w/v) or anthracene (0.5% v/v) or phenanthrene (0.5% w/v) or pyrene (0.5% w/v).Liquid cultures (600 ml) inoculated with spores at a final cell density of 5 x 10 5 /ml were propagated in 2-l Erlenmeyer flasks and incubated in a reciprocating water bath at 28°C for 22 h at 125 rpm for all substrates except glucose that was incubated for 12 h at same other conditions.

Preparation of purified fractions
Mycelium of 22 h of incubation was harvested by filtration and exhaustively washed with cold sterile-distilled water; mycelial mass was suspended in 15 ml of 20 mM Tris-HCl pH 8.5 buffer containing 1 mM phenylmethanesulphonyl fluoride (previously dissolved in ethanol).Approximately 20 ml of cells was mixed with an equal volume of glass beads (0.45-0.50 mm diameter) and disrupted in a Braun model MSK cell homogenizer (Braun, Melsungen, Germany) for four periods of 30 sec each under a CO 2 stream.The homogenate (crude extract) was centrifuged at 4,300g for 10 min in a J2-21 Beckman rotor in a Beckman JA-20 centrifuge to remove cell walls and unbroken cells, a 1 ml sample of the supernatant was saved.The rest of the supernatant (low speed supernatant) was centrifuged at 31,000g for 20 min in a 70Ti Beckman rotor in a Beckman L8-80 ultracentrifuge and samples of 1 ml of the supernatant was saved; the resulting pellet (mitochondrion rich sample) was resuspended in 2 ml of buffer and saved.The rest of the supernatant was high-speed centrifuged at 164,500g for 45 min in a 70Ti Beckman rotor at 4ºC in a Beckman L8-80 ultracentrifuge; the supernatant (cytosolic fraction) was put aside, and the pellet, the mixed membrane fraction (MMF), was resuspended in 2 ml and saved.In all cases samples of different fractions were kept at -70 °C for further studies.

Gel electrophoresis
The slab gels were 1.5 mm-thick contained 6% (w/v) acrylamide/4% (w/v) bisacrilamide, loaded with the cytosolic fraction of each culture and run in the mini-gel system manufactured by Bio-Rad.The continuous buffer system described by Laemmli (1970) without SDS (native conditions) was used to run for 2.5 h at 80 V.The Rm values were calculated as the ratio of the distance migrated by the stained band divided by the distance migrated by tracking dye; standard deviation was calculated with Excel from three independent experiments and each experiment was made by triplicate on each substrate.

Enzymatic assays
All enzyme assays were carried out in a final volume of 1 ml and incubated for different times at 25 ºC.NAD + -dependent ADH activity was assayed in the oxidative direction according to Bergmeyer (1983).The enzymatic assays contained 25 mM Tris-HCl (pH 8.5), 2 mM NAD + or NADP + , cell-free extract (100-200 µg protein), and 100 mM of the substrate (1R, 2S)-cis-1,2-di-hydro-1,2-naphthalene-diol.The reaction was started by dihydrodiol addition, and reduction of NAD + or NADP + was monitored by the increase in absorbance at www.intechopen.comGel Electrophoresis -Advanced Techniques 250 340 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 electro-morphs were observed as blue-dark bands.
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 CaCl 2 , MgSO 4 , ZnSO 4 and FeSO 4 , 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 in the forward reaction dihydrodiol  diol.

Miscellaneous
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. Baker (Phillipsburg, NJ, USA).All reagents were analytical grade.
Densitometric analysis was performed in a Gene Genius Bio-Imaging System V. 6.05.01,SYNGENE, Synoptics Systems.Software used was Gene Tools V. 3.06.02,Syn. Ltd.

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 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 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.

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.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).a Rm; was calculated as described in Materials and Methods section as its standard deviation.a Rm; was calculated as described in Materials and Methods section as its standard deviation.

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 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.a Relative units were obtained by densitometry, using the value from iNDD as 100% when the fungus was growth on naphthalene.

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).
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 (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).

General properties of NDD activities
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 activity.
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 as enzyme substrate were used to reveal the zymograms.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 electron acceptor.a Relative units were obtained by densitometry, using the value from iNDD as 100% when naphthalene was the carbon source.

pH dependence
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 were showed at pH values below 8.5.It is important to say that the background in the lane for activity revealed at pH 9, was darken because of pH.
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 pH value.

Temperature
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.

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 Ca 2+ had an enhancing effect on DD activities meanwhile the other divalent metals tested and also EDTA were inhibitory Fe 2+ > Zn 2+ > EDTA> Mg 2+ .

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.

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 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, 2methyl 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).

Discussion and conclusion
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 + -dependent trans-dihydrodiol dehydrogenases, almost all investigations have been done in mammalian tissues (Carbone et al., 2008;Chang et al, 2009;Chen et al., 2008) but only a few reports about these important enzymes have been done in fungi (Bezalel et al., 1997;Hammel, 1995;Sutherland et al., 1993) particularly in Phanerochaete chrisosporium (Bogan & Lamar, 1996;Muheim et al., 1991).At date, there is no any report about the detection of dihydrodiol dehydrogenase activities by means of electrophoretic zymograms in any organism.This methodology represents an interesting approach because in this way it is possible to detect, study and compare the different isoenzymes present in the cell-free extracts of the organism used as enzymatic source.In our own work, YR-1 strain possesses extraordinary metabolic machinery that premises it to survive in a very dangerous place how is a petroleum-contaminated soil.
The results about the localization of DD activities in a differential centrifugation procedure from YR-1 grown in different carbon sources (Table 1), revealed that the activity measured with cis-naphthalene-diol as substrate and NADP + as electron acceptor was only present in the supernatant fractions of each centrifugation speed, suggesting that all DD activity observed must be a soluble enzyme.At date, we cannot discard the possibility that the DD activity could be located in the lumen of some kind of microsomal bodies, because of the drastic ballistic treatment used to homogenize the cells.Actually, we are conducting different experiments employing density gradients and electron transmission microscopy to resolve this question.
Complementary analysis of DD activities by electrophoretic zymograms led us to detect eleven different activities and all of them were NADP + -dependent (Fig. 2) this represents the first report about the detection of DD activities by electrophoretic zymograms, a nondenaturing gel electrophoresis stained with a colored product of the enzymatic reaction.
Of the eleven bands detected, we described five different constitutive DD activities, DD 1-5, since them were observed when D-glucose was the carbon source and only DD-2 was solely induced by this sugar, since the others are induced at least for another carbon source.When n-decanol was used as a carbon source, we observed four out of five of the constitutive bands, lacking only the DD-2 band.Its noticeable that only when glycerol, ethanol, npentane and n-hexadecane were the carbon source to grow the fungus, not a single constitutive band was observed, may be due to the fact that these compounds only can be metabolized specifically by the induced enzymes.In glucose grown mycelium, all inducible dihydrodiol dehydrogenase activities were absent suggesting that they could be subject to carbon-catabolite repression (Fig. 2).Surprisingly all the activities described here as DD are able to use cis-naphthalene-diol, since this substrate has been describes as bacterial specific (Cerniglia & Gibson 1977).The substrate reported for eukaryotic cells is the trans-naphthalene-diol (Cerniglia 1977).
Phenanthrene was the best inducer since when used as a carbon source four out of six inducible bands were observed, n-decanol and naphthalene were the second best inducers since each one led the induction of three different enzymes, sharing the bands denominated iDD1 and iDD2.Also in the case of the inducible enzymes glycerol, n-pentane and nhexadecane were unable to induce any activity.The specific induction of an activity must be due to substrate specificity.
We have shown that on naphthalene, anthracene, phenanthrene or pyrene used as sole carbon source, there exist three different inducible NADP + -dependent dihydrodiol dehydrogenase activities.One of them iNDD, was the only isoenzyme inducible by all aromatic hydrocarbons which presumably is involved in the aromatic hydrocarbon biodegradation pathway in YR-1 strain (Fig. 1).In particular iNDD was capable to use only cis-naphthalene-diol as substrate (Fig. 7) suggesting that this enzyme is specific part of the metabolic pathway of the naphthalene; all other activity bands are ADH substrate unspecific It is interesting that iPDD2 has broad substrate specificity when NADP + was the electron acceptor, suggesting that it could be one of the different dehydrogenases belonging to the microsomal system of alcohol (ethanol) oxidation [MEOS (Krauzova et al., 1985)].No one of all inducible DD's activities showed to be able to use NAD + as electron acceptor.
For iNDD, naphthalene was the best inducer and pyrene the worst.In the case of both iPDD1 and 2, phenanthrene was the only inducer of these enzymes, however pyrene shows a very low inducer effect on iPDD1 (Table 3).The finding of two inducible (iNDD and iPDD1) and one constitutive (DD1) enzymes that uses specifically a dihydrodiol as substrate is in agreement with the number of three possible DD's of predicted function, reported in database of Mucor circinelloides (Torres-Martínez et al., 2009).
Our above-mentioned findings with M. circinelloides YR-1 dihydrodiol dehydrogenase activities are indicative of developmental regulation of the different DD's enzymes; this interpretation is supported by following observations: in zymograms for DD's activities when YR-1 was grown in different carbon sources is showed a differential pattern of the activity bands depending of the carbon source used in the culture media.The present results suggest the existence of eleven enzymes with dihydrodiol dehydrogenase activity.Particularly important the DD1 that could be the constitutive DD, and iNDD iPDD1, that could be part of the aromatic hydrocarbon biodegradation pathway in YR-1 strain for naphthalene or the others aromatic hydrocarbon, respectively.Future genomic analysis after isolation of the respective genes should prove the existence of one gene for each constitutive or inducible activity in agreement with the M. circinelloides data base prediction.The details of the possible interaction between alcohols or hydrocarbons metabolism remain to be determined.

Acknowledgments
Support for this research by Universidad de Guanajuato (México), is gratefully acknowledged.

Fig. 1 .
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.

Fig. 4 .
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.
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.

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 cisnaphthalene-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.

Table 2 .
Polyacrylamide Gel Electrophoresis an Important Tool for the Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms Constitutive NADP + -dependent dihydrodiol dehydrogenase activities in cytosolic fraction of Mucor circinelloides YR-1 grown in different carbon sources. www.intechopen.com

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