Conclusion - An isolated Candida albicans TL3 capable of degrading phenol at large

Chapter 3 An isolated Candida albicans TL3 capable of degrading phenol at large

3.5 Conclusion

Here, we isolated a new phenol-degrading yeast strain ⎯ C. albicans TL3, which is obviously higher than other microorganisms in tolerance (up to 24 mM) and degradation rate of phenol. Exception phenol, interestingly, C. albicans TL3 can also degrade formaldehyde in waste water from a factory producing phenolic resin.

Based on the analyses of enzymatic, chromatography and mass-spectrometry, we confirmed that this strain via an ortho-fission pathway to perform the degradation of phenol. The production of phenol hydroxylase and catechol 1,2-dioxygenase activities of C. albicans TL3 depended on cell growth temperature, phenol

concentration and/or glucose. The maximum activity of phenol hydroxylase and catechol 1,2-dioxygenase were induced when this strain grew in phenol culture media at 22 mM and 10 mM, respectively. Alternatively, we suggest that synthesis of the phenol hydroxylase and catechol 1,2-dioxygenase would be modulated by one or more additional regulatory mechanisms such as catabolite repression.

3.6 References

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386-400.

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catechol-1,2-dioxygenase. Appl Environ Microbiol 39:1129-33.

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degradation of toxic organic chemicals”, eds Young, L.Y., and Cerniglia, C.E., Wiley-Liss, Inc., USA, 27-74.

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catechol-1,2-dioxygenase from Trichosporon cutaneum. Eur J Biochem 12:

427-34.

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833-40.

Table 3-1. Summary the capability of degradation of phenol by various yeasts.

Strain Limit of phenol

degradation (mM) Reference

Trichosporon cutaneum ＜ 5.3 Neujahr and Varga 1970

Rhodotorula glutinis 5 Hirayama et al. 1994

Trichosporon dulcitum 15 Margesin et al. 2005

Basidiomycetous yeast 15 Margesin et al. 2005

Candida tropicalis 16 Bastos et al. 2000

Trichosporon cutaneum sp. LE3 18 Santos et al. 2001

Candida maltosa 18 Fialova et al. 2004

Candida tropicalis mutant 22 Chang et al. 1995

andida albicans TL3 24 this work

Table 3-2. Identification of the phenol-degradation isolated strain. This strain was dentified as Candida albicans by CBS in the Netherland.

Table 3-3. Growth of Candida albicans TL3 on differents aromatic and related compounds（200ppm）after seven days in shake-flask.

Substrate Growth（mg cell dry wt/ hr.L）

Benzene ×

Toluene ×

Anisol ×

p-Cresol ×

Catechol 3.85

Benzoic acid ×

Benzaldehyde ×

o-Cholorophenol ×

p-Carboxyphenol ×

m-Nitrophenol ×

2,4-Dintrophenol ×

cis-cis-Muconic acid ×

×:no growth

Table 3-4. Comparison of enzyme specific activity of Candida albicans TL3^*

* Growth conditions: YNB medium (0.67 %) containing phenol and/or glucose, at 30 . Data are expressed as mean ℃ ± standard deviation (n=3).

Specific activity（unit/mg protein）

Source of carbon

phenol hydroxylase catechol-1,2-dioxygenase

glucose, 0.2 % 0 0

phenol, 5 mM 0.025±0.0008 0.270±0.01

phenol, 10 mM 0.044±0.0021 0.734±0.037

phenol, 15 mM 0.083±0.0046 0.217±0.019

phenol, 15 mM ＋glucose, 0.1％ 0.057±0.0027 0.174±0.008 phenol, 15 mM ＋glucose, 0.2％ 0.028±0.0022 0.149±0.012 phenol, 15 mM ＋glucose, 0.4％ 0.014±0.0018 0.128±0.007

phenol, 20 mM 0.115±0.0063 0.107±0.009

phenol, 22 mM 0.156±0.011 0.101±0.005

phenol, 24 mM 0.129±0.0045 0.087±0.006

Table 3-5. Effect of temperature on specific activity of phenol hydroxylase and catechol-1,2-dioxygenase from Candida albicans TL3.^*

specific activity（unit/mg protein）

Growth temperature

(℃) phenol hydroxylase catechol-1,2-dioxygenase

25 0.087±0.0041 0.275±0.012

30 0.083±0.0046 0.217±0.019

35 0.064±0.0037 0.158±0.007

40 0.061±0.002 0.100±0.003

* The microbe was grown with YNB medium (0.67 %) containing phenol (15 mM).

Data are expressed as mean ± standard deviation (n=3).

Figure 3-1. Time-course profiles of cell growth of Candida albicans TL3. The cells

were incubated at 30 with YNB medium (0.67 %) containing phenol at ℃ various concentrations: 5 mM ( ); 10 mM ( ); 15 mM ( ); 20 mM ( ); 22 mM ( ); 24mM (

•

), 0.2 % glucose ( ), and the mixture of 15-mM phenol + 0.2％ glucose ( ). Each data point represents the mean of triplicate independent measurements. Data shown are the mean of triplicate experiments with standard deviation within 10%.

Figure 3-2. Consumption of phenol and glucose of Candida albicans TL3. The cells were incubated at 30℃ with YNB medium (0.67 %) containing phenol at various concentrations: 5 mM ( ); 10 mM ( ); 15 mM ( ); 20 mM ( ); 22 mM ( ); 24 mM (

•

), 0.2 % glucose ( ), and the mixture of 15-mM phenol + 0.2％ glucose ( ), and the residual concentrations of phenol ( ) and glucose ( ) in the mixture medium. Each data point represents the mean of triplicate independent measurements. Data shown are the mean of triplicate experiments with standard deviation within 10%.

0 1 2 3 4 5 6 7 8

-1 -0.6 -0.2 0.2 0.6 1 1.4 1.8 2.2 2.6

1/ P he nol c onc e nt ra t i on (mM)^{- 1} 1/Phenol degradation rate (μmole/min● mg protein )-1

Figure 3-3. The kinetic parameters of phenol biodegradation catalyzed by C. albicans TL3. Each data point represents the mean of triplicate independent measurements.

0 0.5 1 1.5 2

16hr 32hr 48hr

Time OD

600

25℃

30℃

35℃

40℃

Figure 3-4. Temperature effect on the growth of Candida albicans TL3. The cell was grown on 15 mM phenol at the indicated temperatures.

Figure 3-5. Comparison of the growth of Candida albicans TL3 with different nitrogen bases. The cell was grown on phenol (15 mM) containing nitrogen base at 30℃.

Figure 3-6. HPLC analysis of the product of phenol is catalyzed by crude enzyme extract. Reactions were terminated at 10 or 25 min on addition of excess methanol. Samples taken from the mixtures were then

centrifuged to remove the precipitant before applying to a column. The resulting supernatants containing varied compositions were analyzed: (a) without addition of NADPH and phenol; (b) with phenol; (c) with

NADPH; (d) 10-min reaction with both NADPH and phenol; (e) with spiking 0.02-mM catechol in the sample (d); (f) 25-min reaction with both NADPH and phenol. The extract medium of crude enzyme as described in the text.

Figure 3-7. GC-mass analysis of the product of phenol is catalyzed by crude enzyme extract. The sample containing catechol, the anticipated product of phenol hydroxylase-catalyzed reaction, was analyzed and confirmed by (a) GC chromatography integrated with (b) electron ionization mass spectrometry (EI/MS). The feature at m/z 110 corresponds to the molecular ion of catechol (M⁺). All fragments shown in (b) are consistent with those of standard catechol.

Figure 3-8. Ion-chromatographic analysis of the product(s) of catechol is catalyzed by crude enzyme extract. Reactions were stopped at 2 min on addition of excess methanol. Samples taken from the mixtures were then

centrifuged to remove the precipitant before applying to a column. The upper figure shows the overlay of two chromatograms without addition of either the crude enzyme extract or catechol. The lower figure contains the overlay of chromatograms with sample from (a) 2-min reaction mixture; (b) 2-min reaction mixture with spiking of 0.015 mM cis, cis-muconic acid; (c) 10-min reaction mixture.

Figure 3-9. Electrospray ionization mass analysis (ESI) of the product of catechol is catalyzed by crude enzyme extract. The sample containing

monosodium muconic acid (mw.164), the anticipated product of catechol 1,2-dioxygenase-catalyzed reaction, was analyzed by ESI/MS (a) and confirmed by ESI/MS/MS (b). The feature at m/z 163 corresponds to monosodium muconate (M-). The pattern of fragments shown in (b) is identical to that of standard monosodium muconate.

Figure 3-10. Growth ( ) and phenol ( ) and formaldehyde ( ) consumption of Candida albicans TL3 was cultured on waste water as a sole carbon source. At the beginning, C. albicans TL3 culture (0.3 mL, OD600 = 1.5) at a stationary phase was added to waste water (49.7 mL)

containing phenol (14.5 mM) and formaldehyde (0.44 mM). Each data point represents the mean of triplicate independent measurements.

Data are expressed as mean ± standard deviation (n=3).

Chapter 4

Purification and characterization of a catechol 1,2-dioxygenase from a phenol degrading Candida albicans TL3

4.1 Abstract

A novel catechol 1,2-dioxygenase (1,2-CTD) was induced from a eukaryotic Candida albicans TL3 strain that possesses high tolerance for phenol and strong

phenol degrading activity. The 1,2-CTD was purified via ammonium sulfate precipitation, Sephadex G-75 gel filtration, and HiTrap Q Sepharose column chromatography. The enzyme was purified to homogeneity and found to be a homodimer with a subunit molecular weight of 32,000. Each subunit contained one iron. Optima for temperature and pH are 25 °C and pH 8.0, respectively. Substrate analysis showed that the purified enzyme is a type I catechol 1,2-dioxygenase and has higher catalytic activity toward catechol than 4-methylcatechol. The kcat and Km

Keywords: catechol 1,2-dioxygenase, Candida albicans TL3

4.2 Introduction

Catechols are formed during biodegradation of a variety of aromatic compounds by aerobic microorganisms. The aromatic rings of catechols may be cleaved by catechol 1,2-dioxygenases (1,2-CTDs), hydroxyquinol 1,2-dioxygenases(1,2-HQDs) (type I catechols 1,2-dioxygenase) (Nakai et al. 1990; Eck et al. 1991; Murakami et al.

1997; Briganti et al. 1997; Shen et al. 2004), chlorocatechol 1,2-dioxygenases (1,2-ClCTDs) (type II catechols 1,2-dioxygenase) (Broderick et al. 1991; Van der Meer et al. 1993; Maltseva et al. 1994) and protocatechuate 3,4-dioxygenases (3,4-PCDs) via an orth-cleavage pathway to form cis,cis-muconate or its derivates or by catechols 2,3-dioxygenases via a meta-cleavage pathway to form 2-hydroxymuconic semialdehyde or its derivates. These metabolites are degraded in the tricarboxylic acid cycle (Ngai, et al. 1990; Chen and Lovell 1990; Aoki et al.

1990). Among the catechols dioxygenases, 1,2-CTDs have been well characterized in terms of their biochemical properties (Aoki et al. 1984; Murakami et al. 1997;

Briganti et al. 1997; Strachan et al. 1998; Strachan et al. 1998; Ridder et al. 1998).

In general, catechol 1,2-CTDs are dimeric proteins with identical or similar subunits, which have molecular weights of 30,500–34,000 Da per subunit (Eck and Bettler 1991; Neidle et al. 1988) and possess one or two iron molecule in the dimeric form (Neidle and Ornston 1986, Briganti et al. 1997 ). 1,2-CTDs are specific for

catechols, alkylated catechols.

Although there are many reports concerning 1,2-CTDs, they are almost from bacteria. There are few reports on 1,2-CTDs from eukaryotic bacteria. Previously, we isolated a strain of yeast, Candida albicans TL3, which uses phenol and/or formaldehyde as its energy source, from soil at a petrochemical factory in Taiwan. It has high tolerance for phenol (up to 24 mM) and catalyzes phenol degradation through the ortho-cleavage pathway (Tsai et al. 2005). We thus attempted to purify the1,2-CTD of Candida albicans TL3 and characterize it compared with the 1,2-CTDs of bacteria strains. The present paper describes the purification and biochemical properties of the 1,2-CTD from C. albicans TL3 that grown in the presence of 10 mM phenol.

4.3 Experimental

4.3.1 Cell culture

Candida albicans TL3, which had previously been isolated from soil at a

petrochemical plant by our group, was cultured at 30 °C with an initial optical density at 600 nm (OD600) of 0.01 in a flask containing YNB medium (0.67% w/v) and 10 mM phenol (Tsai et al. 2005). Cells were harvested when the growth of C. albicans TL3 approached the stationary phase (OD600 of approximately 1.4). After centrifugation, the pelleted cells were washed twice with distilled water.

4.3.2 Preparation of crude extract and enzyme purification

Harvested cells were suspended in 50 mM Tris-HCl buffer (pH 8.3) containing 5 mM β-mercaptoethanol and then disrupted by sonication (60% amplitude and two pulses per second) with an ultrasonic processor (VCX-750, Sonics, USA). After centrifugation (13,700 × g for 30 min at 4 °C), the supernatant was collected and used as a crude enzyme extract. Enzyme purification was performed at 4 °C as follows:

Step 1: Ammonium sulfate fractionation

Ammonium sulfate was added to the crude extract to result in 50−70% saturation.

The precipitated protein was obtained by centrifugation at 22,860 × g for 30 min at 4

°C. The pellet was resuspended in 3 ml Tris-HCl buffer (50 mM, pH 8.3).

Step 2: Sephadex G-75 fractionation

A column (2 cm × 80 cm) packed with Sephadex G-75 (Sigma) was pre-equilibrated with 50 mM Tris-HCl buffer (pH 8.3). The crude protein solution was loaded onto the Sephadex G-75 column and eluted with 50 mM Tris-HCl buffer (pH 8.3) at a flow rate of 0.1 ml/min. Sequential 1 ml aliquots of eluent were collected and tested for 1,2-CTD activity. Fractions that exhibited enzyme activity were pooled and concentrated by ultrafiltration on an Amicon ultracentrifugal filter (MWCO:10000, Millipore Co., USA).

Step 3: Chromatography on Q Sepharose

The concentrated protein solution obtained after completion of Step 2 was purified further using a HiTrap Q Sepharose column (5 ml × 2; Pharmacia) and FPLC system. Proteins were eluted with a linear gradient from 0 M to 0.2 M of (NH4)2SO4

in Tris-HCl buffer (50 mM, pH 8.3) at a flow rate of 0.5 ml/min. Each 1 ml fraction was collected and used for the activity assay.

4.3.3 Determination of protein concentration

Protein concentration was determined using bovine serum albumin as a standard by the method of Bradford (Bradford 1976), and Scopes method (Scopes 1974) was also used to determine protein concentration in the analysis of iron content.

4.3.4 Determination of molecular mass

The molecular weight of the subunit of 1,2-CTD was determined using 12.5%

SDS-PAGE as described by Laemmli (Laemmli 1970). Coomassie Brilliant Blue R250 was used as a protein stain. The molecular weight of the native protein was determined using gel filtration on a Sephadex G-75 column and a series of standard proteins.

A more precise estimate of subunit molecular weight was obtained using electrospray ionization mass spectrometry (ESI-MS). Mass spectra were recorded with a quadrupole time-of-flight mass spectrometer (Q-TOF, Micromass, UK). The quadrupole mass analyzer scanned mass-to-charge ratios (m/z) from 100 to 2500 units with a scan step of two seconds and an interscan of 0.1 seconds per step. In the ESI-MS experiment, we used the quadrupole scan mode under a capillary needle at 3 kV, a source block temperature of 80 °C and a desolvation temperature of 150 °C.

Between five and 10 µg of protein in desalted form were used for MS.

4.3.5 Enzyme activity assays

1,2-CTD activity was determined from the rate of accumulation of cis,cis-muconic acid (increase in absorbance at 260 nm) (Varga and Neujahr 1970).

One unit of 1,2-CTD was defined as the amount of enzyme that catalyzes the formation of l µmol cis,cis-muconic acid or its derivatives per minute. The assay mixture contained 2 µl enzyme solution per milliliter and 998 µl 50 mM Tris-HCl buffer, pH 8.3, per ml. The Tris-HCl buffer contained 5 mM β-mercaptoethanol, 20

µM FeSO4, and 0.1 mM catechol.

The substrate specificity, optimal temperature, and optimal pH for enzyme activity of purified 1,2-CTD were determined using catechol analogues as substrate instead of catechol, at various temperatures and pH values. The increase in absorbance was measured at 260 nm for products from all substrates except hydroxyquinol and protocatechuate, for which wavelengths of 245 nm and 270 nm were used, respectively (Bull and Ballou 1981; Strachan et al. 1998).

To investigate the thermal and pH stabilities of the enzyme and the effects of various compounds on its enzyme activity, it was preincubated for 30 min at various temperatures, pH values, metal ions, and other compounds. The residual enzyme activities were measured by adding catechol and carrying out the enzyme assay at pH 8.3 and 25 °C.

4.3.6 Kinetic measurements

The initial velocity of catechol-1,2-dioxygenase from C. albicans TL3 was estimated at 25 °C and pH 8.3 and expressed as a function of catechol (1–150 µM) and 4-methylcatechol (2.5–300 µM) at enzyme concentrations of 0.072 µg/ml and 0.43 µg/ml, respectively. Estimates of kinetic parameters were determined graphically from double reciprocal plots.

4.3.7 Iron analysis

The purified enzyme was nitrated in 6N HNO3 and then the iron content of the protein was determined by inductively coupled plasma-mass spectrometry (ICP-MS) (Agilent 7500a, USA). All glassware was acid-washed to avoid contamination with iron.

4.4 Results and Discussion

4.4.1 Purification of 1,2-CTD

Purification of 1,2-CTD from cell-free extracts of C. albicans TL3 was performed using ammonium sulfate precipitation and Sephadex G-75 gel filtration (Fig. 4-1) followed by HiTrap Q Sepharose column chromatography (Fig. 4-2) as described in the experimental methods. Yields for each step of the purification process are summarized in Table 4-1. Purification enhanced the purity of 1,2-CTD 40-fold and produced a product with a specific activity of 63.0 units per mg protein and a yield of 33%. Gel-filtration analysis using a Sephadex G-75 column showed that the purified enzyme eluted at a position corresponding to a molecular mass of about 64 kDa (Fig.

4-3). After SDS-PAGE, the purified enzyme appeared as a single band with a

molecular mass of 31 kDa (Fig. 4-4), suggesting that the enzyme is a dimeric protein.

The molecular mass of the subunit of 1,2-CTD was estimated by ESI-MS to be 31,994

± 2 Da (Fig. 4-5), which is consistent with the results of the SDS-PAGE analysis.

The dimeric nature of this eukaryotic 1,2-CTD is similar to that of prokaryotic bacteria, which have molecular weights of 30,500–34,000 Da per subunit (Eck and Bettler 1991; Neidle et al. 1988).

4.4.2 Characterization of 1,2-CTD

The 1,2-CTD of C. albicans TL3 showed considerable activity towards catechol and 4-methylcatechol (18% of catechol), but no significant activity towards other catechol derivatives such as 3-methycatechol, 4-chlorocatechol, 4-carboxycatechol,

and hydroxyquinol (Table 4-2). ESI tandem mass spectrometric analysis showed that the products of the reaction between 1,2-CTD and catechol or 4-methylcatechol were disodium muconate and 3-methyl-disodium muconate, respectively (Fig. 4-6).

Because of its substrate specificity, we suggest that the 1,2-CTD of C. albicans TL3 possesses characteristics similar to those of Acinetobacter sp. (Caposio et al. 2002;

Kim et al. 2003), Pseudomonas arvilla C-1 (Nakai et al. 1990), and Frateuria sp.

ANA-18 (Aoki et al. 1984), which are classified as type I 1,2-CTDs. The kcat and Km

values of 1,2-CTD for catechol were 28 s^-1 and 9.3 µM, respectively. When 4-methylcatechol was used as substrate, the kcat value (5.6 s^-1) was only 20% of that of catechol and the Km value (21.5 µM) was about 2.3-fold larger than that of catechol (Fig. 4-7 and Fig. 4-8). This result showed that the enzyme has higher affinity and catalytic activity for catechol than 4-methylcatechol.

The optimal temperature and pH of 1,2-CTD from C. albicans TL3 was 25 °C

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