ویژگی‌ها و ارزیابی کاتکول اکسیژناز در دوازده باکتری‌ جدا شده از خاک‌های آلوده به نفت در مالزی

Introduction

Many bacteria have the ability to degrade aromatic hydrocarbons of contaminated sites. The soil microorganisms are potential catalytic sources for biodegradation of organic compounds. A number of bacteria are able to biodegrade toxic organic compounds that improved clean-up of different environments including water, soil and wastewater in aerobic and anaerobic conditions (1). Aromatic hydrocarbons could be degraded to less toxic compounds via conversion into dihydroxylated intermediates such as catechol or substituted catechols and then metabolized by intradiol or extradiol dioxygenases (2).

The aerobic degradation of hydrocarbon pathways consist three steps; in the first step, two hydroxyl groups are introduced into aromatic ring and catalyzed by mono-or dioxygenase to produce dehydroxy aromatic compounds which are usually catechols. Catechols are substrates for the next step of the catabolism by ring cleavage via catechol 1, 2 dioxygenase and catechol 2, 3 dioxygenase (Fig. 1) (3). Subsequently, catechol oxygenases have an important role in fundamental pathway in the carbon biochemical cycle and a high biotechnological potential in treatment of liquid wastes contaminated with aromatics compounds (4).

Fig. 1- Catechol degradation via ortho and meta pathway (7)

Catechol 1, 2 dioxygenase (ortho pathway) that also known as β-ketoadipate pathway, is Fe³⁺ enzymes and catechol 2, 3 dioxygenase (meta pathway) is Fe²⁺ enzymes. During the last step, the ring cleavage products convert to small aliphatic compounds which metabolized to carbon dioxide during tricarboxylic acid cycle (TCA cycle) (5). Catechol 1, 2 dioxygenase is an intradiol dioxygenase that incorporate two oxygen atoms between the vicinal hydroxyl groups, while an extradiol dioxygenase, such as catechol 2, 3 dioxygenase, cleaves the aromatic ring of the substrate with two hydroxyl groups and produces muconic semialdehyde (6). Catechol 2, 3 dioxygenase has an important role on biodegradation of alkyl substitution aromatic hydrocarbons such as xylenase and is usually encoded by plasmids (7). Catechol oxygenases are the common biomarker for evaluation of hydrocarbon biodegradation by bacteria. The objective of this study was assessment of catechol dioxygenases in cell-free extracts by 12 bacterial isolates in order to find the appropriate candidate for bioremediation in contaminated sites. All the used bacteria in this study were isolated from groundwater and oil contaminates soils samples which were collected from the Petronas crude oil refinery in Terengganu, Malaysia.

Materials and methods

Chemicals: Toluene and catechol were obtained from Merck Company.

Isolates and culture conditions: Twelve isolates had been identified from crude oil contaminated soils in Terengganu, Malaysia. The samples were obtained from culture collection of School of Bioscience and Biotechnology, University Kebangsaan Malaysia (UKM). All the isolates were identified using commercial kit Microbact 24E (Oxoid) according to the manufacture’s instruction. The isolates were more confirmed by polymerase chain reaction (PCR) assay targeting 16S rDNA gene. Several isolates (UKMP-5T, UKMP-7T, and UKMP-8T) were also analyzed morphologically and their biochemical and physiological properties were determined (8).

Preparation of standard inoculums: The bacterium was grown in nutrient broth and the culture was incubated at 37ºC in an orbital shaker (Infors Ht multitron Germany) at 150 rpm for 18 hours. The culture was harvested by centrifugation (Eppendorf centrifuge 5810 R) at 4000 rpm for 15 minutes at 4ºC. The pellet was washed and resuspended with 50 mM phosphate buffer to obtain an optical density (OD) ~ 0.5 at wavelength 550 nm using spectrophotometer (BiowaveII WPA England) (9). This served as the standard inoculums for the subsequent study.

Sample preparation for catechol oxygenase assay: The catechol oxygenase activity was performed according to Farrell and Quilty method (10). The activity was measured for two enzymes, catechol 1, 2 dioxygenase and catechol 2, 3 dioxygenase. Catechol 1, 2 dioxygenase activity was determined by increasing the absorbance at 260 nm by spectrophotometer due to the cleavage of catechol into cis, cis-muconate (11). Catechol 2, 3 dioxygenase was catalyzed the conversion of catechol to 2-hydroxymuconic semi-aldehyde which increased the absorbance at 375 nm. The culture preparation for catechol oxygenase assay was as follows: Ten percent culture from standard inoculums (v/v) and 0.25 mL of 100 mM toluene as an aromatic hydrocarbon source added into 50 mL Minimal salt medium (MSM) which containing (K₂HPO₄ 1.8 g, KH₂PO₄ 1.2 g, NH₄Cl 4.0 g, NaCl 0.1 g, MgSO₄.7H₂O 0.2 g, FeSO₄.7H₂O 0.01 g in 1 L distilled water (9) and pH adjusted to 6.5 in 250 mL conical flask. The culture was incubated at 30ºC, with shaking at 150 rpm for 5 days. At the end of the incubation, the bacterial cells were centrifuged at 4000 rpm for 20 minutes at 4°C. The pellet was washed twice and resuspended with 0.33 mM Tris-HCl buffer (pH 7.6). The OD _550nm of the cells was adjusted to 0.6. The cells were put in ice and sonicated (Sonics material vibra cell) for 20 seconds for 3 times to break the cells and centrifuged at 4°C, 20000 rpm for 20 minutes. The supernatant (cell free extract) was collected and kept in ice to use for catechol dioxygenase assay (10).

Catechol 1, 2 dioxygenase assay: The activity of catechol 1, 2 dioxygenase was prepared by mixing 2 mL of 50 mM Tris-HCl buffer (pH 8.0), 0.7 mL distilled water, 0.1 mL of 100 mM 2-mercaptoethanol, 0.1 mL cell free extract and 0.1 mL catechol from 1 mM (Sigma) in sterile tube. The sample was read in a quartz cuvette. A control was prepared by replacing the catechol with distilled water. The formation of cis, cis muconic acid from ortho-cleavage pathway was monitored using spectrophotometer at wavelength 260 nm for 10 minutes at 1 minute interval. The extinction coefficients for catechol at OD_260nm=16.800 l/mol/cm (10 and 12).

Catechol 2, 3 dioxygenase assay: For the assay, the mixture was prepared in a cuvette as follows: 2 mL (50 mM) Tris-HCl buffer (pH 8.0), 0.6 mL distilled water, 0.2 mL cell-free extract and 0.2 mL of 100 mM catechol. The formation of 2-hydroxymuconic semi-aldehyde from meta cleavage pathway was monitored for 10 minutes at 1 minute interval using spectrophotometer at wavelength 375 nm. The extinction coefficients for catechol at OD_375nm =36.000 l/mol/cm (10, 11).

The catechol oxygenase activity was calculated based on formula (13) as follows:

Enzyme unit/mL sample = Measured Value × Test Volume × Dilution Factor /

 (Time × Concentration Constant × Enzyme Volume)

One unit is defined as 1 µmol product(cis, cis-muconate or 2-hydroxymuconic semi-aldehyde by catechol 1, 2 dioxygenase or catechol 2, 3 dioxygenase) per minute in one milliliter assay (2 and 14).

Results

In this study, twelve isolates were used to determine the catechol oxygenase activities (Table 1).

Table 1- Bacteria used in this study

Catechol 1, 2 dioxygenase assay: The catechol 1, 2 dioxygenase activity for all 12 isolates was measured at 260 nm over a period of 10 minutes at one minute interval (Fig. 2).

Fig. 2- The catechol 1, 2 dioxygenase activity for all isolates using catechol as substrate

The highest enzyme activity for catechol 1, 2 dioxygenase was from B. cereus UKMP-6G (0.07 U/mL) at the first minute while the lowest enzyme activity was elicited in A. baumannii UKMP-12T (0.04 U/mL) after 10 minutes reaction. The optimum activity for each one of the isolates was different at range 5 to 9 minutes. For example, the highest catechol 1, 2 dioxygenase activity for P. aeruginosa UKMP-14T was 0.07 U/mL after 5 minutes incubation and for P. haemolytica UKMP-5T was 0.09 U/mL after 6 minutes. The highest catechol 1, 2 dioxygenase activity by Bacillus sp UKMP-10Twas 0.13 U/mL produced for 7 minutes and for B. cereus UKMP-6G was 0.14 U/mL after 8 minutes. The catechol 1, 2 dioxygenase activity by A. baumannii UKMP-2M was 0.12 U/mL after 9 minutes.

Catechol 2, 3 dioxygenase assay: The catechol 2, 3 dioxygenase activities for all 12 isolates was measured at 375 nm over a period of 10 minutes at one minute interval (Fig. 3).

Fig. 3- The catechol 2,3 dioxygenase activity for all isolates using catechol as substrate.