**2.2 Preparation of purified fractions**

248 Gel Electrophoresis – Advanced Techniques

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

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

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 *cis*diol-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

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

*Mucor circinelloides* strain YR-1 originally isolated from petroleum-contaminated soil in Salamanca, Guanajuato, Mexico was used as enzymatic source. A defined media containing

naphthalene is metabolized to 1-naphtol by six different genera of fungi.

activities when YR-1 strain was grown in naphthalene as sole carbon source.

petroleum contaminated soil.

**2. Materials and methods**

**2.1 Organisms used and culture conditions**

(Hyötyläinen and Oikari, 1999; Cerniglia, 1997).

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 CO2 stream. The homogenate (crude extract) was centrifuged at 4,300*g* 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 31,000*g* 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 164,500*g* 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.

### **2.3 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.

#### **2.4 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

Polyacrylamide Gel Electrophoresis an Important Tool for the

Sample Carbon source

obtained with ethanol as a carbon source.

on different carbon sources.

with *cis*-naphtalen-diol as substrate.

**sources** 

acceptor (Table 1).

4,300 x *g*

31,000 x *g*

164,500 x *g*

Supernatant (Cytosol)

Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms 251

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

Supernatant 42 4.0 270 131 12 39

 Pellet 1.7 NDa ND 23 ND 12 Supernatant 4.5 1.3 91 57 23 61

Pellet (MMF) 0.7 0.1 0.1 5.2 1.2 ND

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

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

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

**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** 

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

means of three independent experiments with triplicate determinations. a ND, no detected.

DD activity (x 10-2) NADP+ NAD+

Glucose Ethanol Naphthalene Glucose Ethanol Naphthalene

21 0.5 178 59 2.8 1.4

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.

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 propane-1-ol*.* 

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 in the forward reaction dihydrodiol diol.
