JCC0755

JOURNAL OF CULTURE COLLECTIONS

Volume 5, 2006-2007, pp. 35-37

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EXTRA- AND INTRACELLULAR ESTERASES INVOLVED IN DIMETHYLTEREPHTHALATE CATABOLISM BY= PSEUDOMONAS SP.<= /b>

Lilia Tserovska^1*, Raycho Dimkov², Tanya Rasheva³ and Tanya Yordanova¹

¹National Bank for Industrial Microorganisms and Ce= ll Cultures;1113 Sofia, P.O.Box 239, Bulgaria;
²Sofia University, Faculty of Biology, Department of Hydrobiology and Ichthyology,
8 Dragan Tzankov blvd., 1164 Sofia, Bulgaria;
³Sofia University, Faculty of Biology, Department of Microbiolog= y, 8 Dragan Tzankov blvd., 1164 Sofia, Bulgaria

*Corresponding author, e-mail: l_tserovska@abv.bg<= /p>
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Summary

The strain Pseudomonas sp. 054, isolated previously from polluted soil, possesses a high biodegrading activity towards the arom= atic ester dimethylterephthalate. The ester hydrolyzing activity of the strain w= as investigated. Two types of esterases, extra- and intracellular, were detect= ed after growth in liquid medium. The enzyme activities were proved in the cul= ture supernatant and in the crude cell extract by the use of a native electrophoresis. The extracellular protein with an esterase activity had a molecular weight of 67 kDa, while the intracellular fraction produced = four separate bands with ester hydrolyzing activity.

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Key words: <= /i>Pseudomonas, dimethylterephthalate, esterases.<= /o:p>

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Introduction

The ester hydrolyzing enzymes (esterases) are a roughly specified group of enzymes (EC 3.1.1.2) of a great importance for the primary attack of xenobiotics, inclu= ding phthalic derivatives [2]. Many insoluble or hardly soluble organic substrat= es become biodegradable only with the aid of esterases.

The diversit= y of the identified catabolic pathways for the aromatic ring cleavage suggests the existence of a great natural genetic pool [6, 14]. The initial stages of degradation, before the substrate enters the central metabolism, are poorly studied. Kurane et al. ha= ve isolated and purified a phthalate ester hydrolyzing enzyme from Nocardia erythropolis [7]. It exhi= bits a wide substrate specificity but is inactive towards the phthalic acid p-isomers.

Our investig= ations were focused on the p-isomer of the terephthalic acid – dimethylterephthalate (DMT). It is a main raw material and a hardly degrada= ble waste in the polyester fibre Yambolen production. In a previous study it was found that the bacterial strain 054 utilized DMT as a sole carbon and energy source. This strain, determined as Pseudomonas sp., degraded completely DMT in 72 hours. The degradation of= the compound included two-stage hydrolysis and cleavage of the aromatic ring at= m-position to protocatechuate [12]. Biodegradation and bio-transformation of xenobioti= cs with aromatic structure by Pseudomonas strains was recorded by many other authors [4, 11, 13].

In this work= we have paid attention to the initial microbial attack of DMT and describe the existence and localization of an ester hydrolyzing activity in the strain <= i>Pseudomonas sp. 054. =

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Materials and Methods

Microorganisms and growth conditions. The bacter= ial strain Pseudomonas sp. 054 was isolated from DMT polluted soil. It was adapted to growth in a minimal medium containing DMT as a single carbon and energy source by the use of enrichment cultures. The cultivation was performed periodically on a shaker= at 200 rpm, 28 ^oC, as described previously [12].

Preparation of extracts for electrophoretic analysis= . The extrace= llular extract was prepared from the supernatant of a 2-days liquid culture. The protein fraction was concentrated by precipitation with (NH₄)2SO₄and dialyzed overnight against distilled water. Crude cell extract was obtained from a culture in the late exponential phase. The cells were colle= cted by centrifugation and disrupted in ultrasonic disintegrator UD-20 [12]. All steps were done in the cold.

Protein determination. The protein concentrati= on in the cell extract was determined by the method of Lowry with bovine serum albumin as a standard [9].

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [8]. It was carried out in 12 % gel in 25 mM TRIS-gly-cine buffer, pH 8.3, containing 0.1 % SDS, at 35 V for 14 hours. Low molecular weight mar-kers (Pharmacia) were applied: phosphorylas= e b (94 kDa), bovine serum albu= min (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa) and a-lactalbumin (14,4 kDa). Gels were stained with Cooma= ssie blue.

Native PAGE. It was performed in 5&nbs= p;% gel at 100 V for 4 hours. The ester hydrolyzing activity was qualitatively proved by the method of Lund [10]. The gels were placed for 1 hour in 0.1 М Tris-maleic buffer, pH 6.4, a= nd then in 1 % <= span style=3D'mso-char-type:symbol;mso-symbol-font-family:Symbol'>a-naphthylace= tate in acetone-water containing Fast blue BB salt. The esterase activity led to the appearance of dark red bands on the gel which developed in 1 hour at r= oom temperature.

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Results and Discussion

In previous investigations with our isolates, utilizing DMT, high protein contents in t= he supernatant of the strain Pseudomonas sp. 054 was established (data not presented).

The concentr= ated protein from the supernatant showed a single band in SDS-PAGE (Fig. 1, lane 2). Other protein bands were not detected. Based on standard markers (Fig. 1, lane 1), the molecular weight of the observed protein was determined to be 67 kDa. The SDS-PAGE of the strain 054 crude cell extract produced a well distinguishable band of the same molecular weight (Fig.&nbs= p;1, lane 3), suggesting an extracellular secretion of the protein. The producti= on and secretion of microbial enzymes (esterases, lipases, laccases) in the cultural medium was also reported for bacteria, as well as for other microorganisms [1, 3, 5].

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The ester hydrolyzing activity of the studied protein was proved by native PAGE and <= /span>a-naphthylacetate as the indicator substrate. The extracellular protein exhibited a strong reaction = in a single band (Fig. 2, A<= /st1:metricconverter>). A similar extracellular protein from N. erythropolis was detected by disk gel electrophoresis and its molecular weight was determined as approximately 15 kDa [7]. The enzyme possessed wide substrate specificity and was defined as either a new lipase form or a newly characterized ester hydrolyzing enzyme. It, however, was inactive towards esters like DMT with p-position substitutes.

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The esterase activity of a crude intracellular fraction of Pseudomons sp. 054 was also studied. Four separate bands with ester hydrolyzing activity were identified by native PA= GE (Fig. 2, B). The patterns were active on a-naphthylacetate suggesting that these ester hydrolyzing enzymes also participate in the initial hydrolysis of the dimethylterephtha= late.

In conclusio= n, the strain Pseudomons sp. 054 produ= ces both extra- and intracellular esterase activities, which are important in t= he initial stage of biodegradation of aromatic substrates. The extracellular protein detected has an ester hydrolyzing activity and a molecular weight o= f 67 kDa.

Further investigations will be directed to-wards the biotechnological production of= an enzyme designed to catalyze the aromatic ester hydrolysis in the textile production waste waters.

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References

1.&n= bsp; Anguita, J.,= L. Aparacio, G. Naharro, 1993. Appl. Environ. Microbiol., 59, 8, 2411-2417.

2.&n= bsp; Cartwright, = C., S. Owen, I. Thompson, R. Burns, 2000. FEMS Microbiol. Lett., 186 (1), 27‑34.<= /span>

3.&n= bsp; Coll, P., J. Fernandes, J. Villanueva, P. Perez, 1993. Appl. Environ. Microbiol., 59, 8, 2607-2613.

4.&n= bsp; Heinaru, E.,= S. Viggor, E. Vedler, J. Truu, M. Merimaa, A. Heinaru, 2001. FEMS Microbiol. Lett., 37 (1), 79‑89.

5.&n= bsp; Jaeger, E., = K. Liebeton, A. Zonta, K. Schimossek, M. Reetz. 1996. Appl. Microbiol. Biotechnol., 46, 99‑105.

6.&n= bsp; Johnson, G.,= R. Olsen. 1997, Appl. Environ. Microbi= ol., 10, 4047‑4052.

7.&n= bsp; Kurane, R., T. Suzuki, S. Fukuoka, 1984. Appl. Microbiol. Biotechnol.,= 20, 378‑383.

8.&n= bsp; Laemmli, U., 1970. Nature, 227, 378-383.

9.&n= bsp; Lowry, O., N. Rosebrough, A. Farr, R. Randall, 1951. J. Biol. Chem., 193, 265‑275.

10.&= nbsp; Lund, B., 1965. = J. Gen. Microbiol., 40, 413‑419.

11.&= nbsp; Reich, T., S. Schmidt, P. Fortnagel, 1999. FEMS Microbiol. Lett., 176 (2), 477‑482.

12.&= nbsp; Tserovska, L., R. Dimkov, 2000‑2002. J. Culture Collections (Sofia), 3, 33‑37.

13.&= nbsp; Whiteley, A., S. Wiles, A. Lilley, J. Philp, M. Bailey, 20= 01. J. Microbiol. Methods, 44 (1), 79‑88.

14.&= nbsp; Widada, J., H. Nojiri, T. Omori, 2002. Appl. Microbiol. Biotechnol., 60 (1‑2), 45‑59.

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ИЗВЪН- И ВЪТРЕКЛЕТЪj= 3;НИ ЕСТЕРАЗИ, УЧАСТВАЩИ В КАТАБОЛИЗМh= 0; НА ДИМЕТИЛТЕРh= 5;ФТАЛАТ ПРИ PSEUDOMONAS SP.

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Лил = 80;я Церовска¹*, Райчо Dимков2, Таня Рашева3, Таня Йорданова¹=

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Резюме

Щам Pseudomonas sp. 054, изолиран от замърсена почва, прите= 078;ава ензимни активности за ефективн = 86; биологично разгражданk= 7; на ароматни= 03; естер димет = 80;лтерефтала&#= 1090;. Изследвана = 77; естер-хидро = 83;азната активност н = 72; щама. Устано= 074;ени са извън- и вътреклетъm= 5;ни естерази пр = 80; растеж в течна среда. Активностиm= 0;е са доказани = 074; хранителнаm= 0;а среда и в клетъчен ек = 89;тракт чрез нативн = 72; електрофорk= 7;за. Регистрираl= 5;ият извънклетъm= 5;ен белтък с естеразна активност има молекулно тегло 67 kDa. Вътреклетъm= 5;ната фракция показва 4 отделни субфракции = 89; естер-хидро = 83;азна активност.