Romanian Biotechnological Letters Vol.17, No.2, 2012 Copyright © 2012 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 17, No. 2, 2012 7043

Production, optimization and characterization of the lipase from Pseudomonas aeruginosa

Received for publication, July 20 2011 Accepted, April 20, 2012

BENATTOUCHE ZOUAOUI 1 ABBOUNI BOUZIANE 2 Benattouche Zouaoui (1) Department of biology, Faculty of Science, Mascara University, 29000-Algeria Tel :00213557172691 Fax : 0021348646312 e-mail : benattouche_22@ yahoo.fr Abbouni Bouziane (2) Department of biology, Faculty of Science, Sidi belabbes University, 22000-Algeria Tel : 00213 7712521 Fax : 0021348646312 e-mail : abbouni bouziane@ yahoo.de

Abstract

Six isolates of lipase producing ( Ps1, Ps2, Ps3, Ps4, Ps5 and Ps6 ) were secreened from wastewater on a selective medium agar that contained Tween 80 or olive oil as the only carbon source. The isolate showed highest lipase activity was Ps5 which was later identified as Pseudomonas sp. The effect of media composition was analyzed to maximize production of lipase. Pseudomonas aeruginosa had maximal activity at the pH range 6 to 8, with an optimum pH of 7, and the temperature range of 20 – 35°C, with optimum temperature for the hydrolysis of olive oil at 30°C. The lipase activity of the enzyme was enhanced by Ca +2 and Mg +2 but strongly inhibited by heavy metals Zn+2 and Cu+2 and Mn+2 .

Keywords: Pseudomonas, lipase, biomass, heavy metals.

Introduction

Lipases (glycerol ester hydrolases EC : 3.1.1.3) hydrolyse triacylglycerols to faty acids,

di-acylglycerols, mono acylglycerols and glycerol and under certain conditions, catalyze reverse reactions such as esterification and trans esterification [1].

Microbial lipases have already established their vast potential regarding to their usage in different industries. The interest in microbial lipase production has increased in the last decades. Due to the versatility of the molecular structure and catalytic properties, these enzymes have potential biotechnological application in different industrial sectors such as food, waste water treatment, cosmetics, oleo chemical, pharmaceutics, detergents and in the fuel sector, which applies lipase as catalyst for synthesis of esters, trans esterification of the oil and in the production of bio diesel [2].

Generally, the enzymes of industrial interest are produced in the presence of inducers in the case of lipases, the presence of triacyl glycerol, surfactants, vegetable oils, oil industry wastes or their hydrolysis products in the culture medium have, in most cases, an inducible effect on lipase production[3]. Most of the well studied microbial lipases are inducible extracellular enzymes [4] .They are synthesized with in the cell and exported to its external surface or environment. Many of them have been purified, characterized and their encoding genes cloned [5].

7187Romanian Biotechnological Letters, Vol. 17, No. 2, 2012

BENATTOUCHE ZOUAOUI 1 ABBOUNI BOUZIANE

Romanian Biotechnological Letters, Vol. 17, No. 2, 2012 7044

Several Pseudomonas species have been shown to produce lipases [6, 7]. Some of them have been sequenced, i.e.,Pseudomonas fragi [8], P. cepacia , and P. aeruginosa [9] .

The aim of the present work was to determine the optimum parameters favorable for the growth and lipase production of Pseudomonas aeruginosa using shake flasks studies.

Materials and methods Isolation of producer microorganism The bacterial strain Pseudomonas.sp used in this study was isolated from an urban

wastewater at Sidi Bel abbes, Algeria. The isolates were identified on the basis of various morphological, physico-chemical, and biochemical characteristics. Lipolytic bacteria are typically detected and screened through the appearance of clearing zones by using a selective medium [10], which was containing Tween 80 or olive oil as the only source of carbon. The diameter ratio of clear zone and colony was measured.

Optimization of lipase production Four different cultivation media were prepared to the production of lipase enzyme. The composition of the different fermentation media can be summarized as follows: Medium (A): 0.3 % yeast extract, 0.1 % bacto peptone, 1% olive oil, 0.07% K2HPO4, 0.03 KH2PO4, 0. 05 MgSO4, 0.01 MnCl2, 0.025 (NH4)2SO4 and 0.01 CaCl2 [11]. Medium (B): 1% Tween 80 which replaced olive oil in medium (A) Medium (C) : 2% dextrose added to medium (A) Medium (D): 2% dextrose added to medium (B) The pH of all the production media was adjusted to 7.2 using 0. 5 N NaOH, before

autoclaving at 121°C for 15 min. The enzyme assay was carried out in 500 ml Erlenmeyer flasks containing 94 ml of medium production inoculated with 6 ml of the overnight culture.

The flasks were incubated at 35 °C with a constant shaking at 125 rpm for 3 days. The cell was separated by centrifugation at 10000 rpm and 4 °C for 20 min. The

supernatants were collected and used to determine the lipase activity. The isolation expressed the highest activity was selected for further studied.

Biomass estimation Biomass concentration was estimated by measuring the optical density at 600 nm by using

a Spectronic instrument and standard curve previously determined (biomass g/l corresponding to 0.270x OD600) [12].

Protein determination Protein was measured photometrically by the method of Bradford with Coomassic protein Assay reagent [13]. Enzyme assay Determination of lipase activity in the crude enzyme extract was done using the titrimetric

method following the procedure described in previous work, with slight modifications [14]. The reaction mixture containing 5ml of the emulsion (50ml of water, 50ml of olive oil and

7g of gum Arabic prepared at room temperature during 3 min), 4 ml of 200 mM sodium phosphate buffer pH 7.2 and 1 ml enzyme extract was incubated for 30 min at 35°C. The analyses were carried out in duplicates or triplicates.

The reaction was stopped adding 10 ml of 1:1 acetone / ethanol solution and the titration performed with 0.05 N NaOH. Blank assays were conducted adding the enzyme just before titration. One unit (u) of lipase activity was defined as the amount of enzyme which produces 1µ mol of fatty acids per minute under assay conditions [15].

7188 Romanian Biotechnological Letters, Vol. 17, No. 2, 2012

Production, optimisation and characterization on the lipase. From Pseudomonas aeruginosa

Romanian Biotechnological Letters, Vol. 17, No. 2, 2012 7045

Determination of pH and temperature optimum The temperature and pH optimum of extra cellular lipase was determined at different

degrees ranging from 20-50°C for the first and from 3 to 10 for the second parameter. To determine the effect of temperature on lipase activity, extract enzyme and substrate were incubated at various reaction temperatures before starting the experiment and the enzyme assay was performed to determine the optimal temperature titrimetrically using olive oil as substrate.

The optimal pH was determined by incubating the enzyme-substrate at various pH from (3 to 10) and assayed for lipase activity. Thermo stability and pH stability of lipase The thermo stability of the lipase fraction was studied by incubating the enzyme extract at

various temperatures (20 -50°C) for 1 h. The residual lipolytic activities were then determined using olive oil substrate.

For pH stability, enzyme extract was incubated using different pH buffers for 1h. The reaction mixtures were incubated as per standard assay and the residual lipolytic activities were then determined using olive oil as substrate.

Effect of metal ions on lipase activity For determining the effect of metal ions on lipase activity, the enzyme extract were

preincubated with 1 mM of Ca+2, Mg+2, Mn+2, Cu+2 and Zn+2 for 1h at 30°C and the residual activity was determined using olive oil as substrate under standard assay conditions.

Results and discussion

Isolation of lipase –producing micro organisms Six isolates grown in the selective medium were found to produce lipase which were

identified as bacteria Ps1, Ps2, Ps3, Ps4, Ps5 and Ps6. Their growth showed that they can use olive oil and Tween 80 as carbon source and showed the lipase producing feasibility. Evaluation of the lipase producing efficiency, based on the clear zone around colony, indicated

that all of them could produce lipase enzyme. The result of ( Ps5 ) was outstanding (fig1).

A

B Figure 1: The clear zone displayed by lipase around the Pseudomonas aeruginosa colony on the emulsion Tween 80-agar (A) and olive oil-agar (B) at 30°C after 96 h of incubation.

When their produced lipases were applied on the emulsion olive oil- agar with Tween 80- agar and incubated at 35°C for 96h. Their abilities of lipase producing were evaluated with the diameter ratio of clear zone and colony (table 1).

7189Romanian Biotechnological Letters, Vol. 17, No. 2, 2012

BENATTOUCHE ZOUAOUI 1 ABBOUNI BOUZIANE

Romanian Biotechnological Letters, Vol. 17, No. 2, 2012 7046

Table 1 : The diameter ratio of clear zone and colony of the isolated microorganisms Bacterial Diameter ratio of clear zone and colony (mm) Olive oil Tween 80 Ps1 7 5 Ps2 8 6 Ps3 12 10 Ps4 16 13 Ps5 22 18 Ps6 14 12

Ps5 is the highest with 22mm with olive oil and 18mm with Tween 80, whereas of Ps4 is 16

mm with olive oil and13 mm with Tween 80, and Ps6 is 14mm with olive oil and 12 mm with Tween 80 (Table1).

The Ps5 was identified and found that it is a member in Pseudomonas genus. Therefore, the Ps5 was classified as Pseudomonas aeruginosa

Optimization of lipase production by Pseudomonas aeruginosa Four different media were used for lipase production by Pseudomonas aeruginosa. The

results in (fig 2) showed that medium (c) with dextrose as carbon, olive oil an inducer of lipase gave the maximum biomass and activity 18.36 g/l after 48 h. In the present study lipase production with different compositions were studied to maximize the production of lipase, the medium (c) presented the highest activity 37 U/ml with specific enzyme activity 37.75U/mg at 35°C after 24 h (fig 3).

0

2

4

6

8

10

12

14

16

18

20

2h 24h 48h 72h

Medium A Medium B

Medium C Medium D

0%

20%

40%

60%

80%

100%

120%

24h 48h 72h

Medium A Medium B

Medium C Medium D

Lipase activity (U/ml)

Biomass ( g/ l )

Time ( h )

Figure 2: Production of Pseudomonas aeruginosa biomass using different media

Figure 3: Production of Pseudomonas aeruginosa lipase using different media

Time(h)

Enzyme production %

7190 Romanian Biotechnological Letters, Vol. 17, No. 2, 2012

Production, optimisation and characterization on the lipase. From Pseudomonas aeruginosa

Romanian Biotechnological Letters, Vol. 17, No. 2, 2012 7047

Lipase production in medium (a) without dextrose was 16.7% less as can pored to the

medium (c). The medium (d) with Tween 80 and dextrose produced 50% less. Lipase production study on medium (b) shows a 75 % decrease in the production of lipase

Based on the obtained results, lipase production was greatly affected by nutrient conditions. Some Authors stated that lipase production greatly affected by the composition of the medium [16].

The present study indicated that olive oil was better inducer than Tween 80, under the test conditions whereas dextrose was the best carbon source for lipase production. In fact, dextrose has been considered as a growth medium for microorganisms providing a satisfactory supply of nutrients for growth.

In conclusion, the pseudomonas aeruginosa strain was found to be the best lipase producer. Wheat dextrose and olive oil were the best substrate and inducer, respectively for lipase production by this strain.

pH and temperature optimum Initial pH of the culture broth is one of the most critical environmental parameters

affecting both growth and lipase production. The results showed that the Pseudomonas aeruginosa was able to grow in the pH range from 6to 8 (Table 2) and reached the maximum lipase activity 38.5 U/ml at pH 7. These data are in agreement with that of Yuzo et al who reported that maximum lipase activity from Pseudomonas fluorescens HU 380 was detected at pH 7 [17].

The results showed that maximum lipase activity detected at 30°C (Table 3). A decrease in the lipolytic activity was observed above 35°C and completely ended at 40°C. Such results are similar to that reported for many bacterial specie [18].

Table 2: Effect of pH on Table 3: Effect of temperature Pseudomonas aeruginosa lipase Pseudomonas aeruginosa lipase

pH and temperature stability The pH stability of the lipase was determined by the activity retained at different pH from 4

to 10 after 1h of incubation. The pH stability curve showed that the lipase was stable at pH 6 to 8 (Table 4). The stability data showed a decline in lipase activity below 6 and above 8, however 60 and 70% relative activity was retained at these pH. Yuzo et al stated that the majority of bacterial lipase presents optimal pH stability in the range of 6 to 8 and was unstable at pH values above 8.

Maximal thermo stability of the lipase was observed in the temperature range of 25 to 35°C enzyme maintained 76 % of the initial activity after 1h.

pH Relative activity (%) Temperature Relative activity (%)

3 12 4 39 5 62 6 84 7 100 8 78 9 35 10 8

20 30 25 64

30 100 35 76 40 35 45 08

50 00

7191Romanian Biotechnological Letters, Vol. 17, No. 2, 2012

BENATTOUCHE ZOUAOUI 1 ABBOUNI BOUZIANE

Romanian Biotechnological Letters, Vol. 17, No. 2, 2012 7048

Table 4: Stability of lipase in different pH Table 5: Effect of temperature on lipase stability. pH Relative activity(%)

Temperature Relative activity (%)

3 11 4 24 5 38 6 62 7 100 8 74 9 32 10 8

20 30 25 64 30 100 35 76 40 35 45 08

50 00

Effect of metal ions on lipase activity

The results indicated that Ca+2, Mg+2 ions activated the lipase activity by different degree (table 6) these ions have been reported to play role as lipase cofactor [19]. Calcium ion, specially, is used in Ca+2 binding processing which impact to position specificity on active site [20].

However Zn+2, Mn+2 and Cu+2 reduced enzyme activity to less than 43% of its relative activity (Table 6). Table 6 : Effect of different metal ions on enzyme activity

Metal ions used Conc.used Remaining activity (Mm) (%)

Control none 100 Ca+2 1 109 Mg+2 1 104 Mn+2 1 43 Cu+2 1 31 Zn+2 1 29

Conclusion

Out of 36 bacterial isolates obtained from the wastewater at Sidi Bel abbes, Algeria, 6 exhibited lipase activity. Pseudomonas sp proved to be the best lipase producer. Various physicochemical parameters were studied to determine the optimum conditions for its lipase production. The obtained results showed that the medium composition for lipase production was ( 1 bacto peptone, 3 yeast extract, 20 dextrose, 10 olive oil, 0,7 k2HPO4, 0,3 KH2PO4, 0,5 MgSO4, 0,1 MnCl2 g/l ) at pH7, 30°C and 125 rpm in 100 ml in 500 ml flask. The hydrolytic

7192 Romanian Biotechnological Letters, Vol. 17, No. 2, 2012

Production, optimisation and characterization on the lipase. From Pseudomonas aeruginosa

Romanian Biotechnological Letters, Vol. 17, No. 2, 2012 7049

activity of the enzyme was enhanced by Ca+2and Mg+2but strongly inhibited by heavy metals Zn+2, Cu+2and Mn+2. Acknowledgements

We are grateful to the technical staff in the Department of Biology, Faculty of Science, Sidi Bel abbes and Mascara universities, Algeria. References 1. FERNANDES. M L M., SAAD. E B , MEIRA. J A , RAMOS. L P , MITCHEL. D A , KRIEGEIR. N,

Esterification and transesterification reactions catalysed by addition of fermented solids to organic reaction media.j.mol.catal.,B Enzym., 44, 8,13 (2007).

2. SHARMA. R, CHISTI. Y, BANERGEE. U C, Production, purification, characterization, and applications of lipases. Biotechnol. Adv., 19, 627,662 (2001).

3. DAMASO. M C T , PASSIANOTO. M.A , LORIDIO DIFREITAS. S , GUIMAREIS FREIRE. D M , CELIARAUJO LAGO. R , COURI. S, Utilization of agro industrial residues for lipase production by solid state fermentation. Brazilian. J. Microbial., 39(4),676,681(2008).

4. TAN. T , ZHANG. M , WANG. B , YING. C , DENG. L, Secreening of high lipase producing candida sp. And production of lipase by fermentation. Process biochem.,39,459, 465 (2003).

5. SAXENA. L , SHEORAN. A , GIRI. B , DANDSON. S W, Purification strategic for microbial lipase .J. Microbiological Methods., 52, 1,18 (2003).

6. MEACHER. J R , ALFORD. J A, Purification and characterization of the lipase of Pseudomonas fragi. J Gen. Microbiol., 48, 317,328 (1967).

7. STUER. W , JAEGER. K E, WLAKLER. U K, Purification of extracellular lipase from Pseudomonas aeruginosa. J Bacteriol.,168, 1070,1074 (1986).

8. AOYAMA. S , N . YASHIDA, S. Inouye, Cloning sequencing and expression of the lipase gene from Pseudomonas fragi IFO:12049 in E.coli. FEBS Lett. ,242, 36,40 (1988).

9. PRITA. S.BORKAN , RAGINI. G. BODATE , SRINIVASA. R. RAO, KHOBRAGADE. C N, Purification and characterization of extracellular lipase from a new strain-Pseudomonas Aeruginosa SRT9 . J Braz Microbiol. , 40 (2) ,358,366 (2009).

10. LOO. J L , LAI. O M , LONG. K , GHAZALI. H M, Identification and characterization of A locally Isolated lipolytic Microfungus Geotrichum candidum . Malaysian . J Microbiology., 2 (1), 22,29 (2006).

11. LI. C , CHENG. C ,CHEN. T, Fed- batch production of lipase by acineto bacter radioresistens using tween 80 as carbon source. Biochem. Eng. J., 19(1) , 25,31(2004).

12. COURI. S , FARIAS. A X, Genetic manipulation of aspergillus, niger for increased synthesis of pectinolytic enzymes. rev. Microbial., 26(4) , 314,317(1995).

13. BRADFORD. M M, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principe of protein-dye binding. Anal. Biochem., 72, 248,254 (1976).

14. CAMACHO. R M , MATEOS. J C , REYNOSO. O G , PRADO. L A , CODOVA . J, Production and characterization of esterase and lipase from Haloarcula marismortui.j.ind Microbio Biotechnol. , 36(7) ,901,909 (2009).

15. PEREIRA. E B, CASTRO. H F, MORAES. F F, ZANIN. G M, Kinetic studies of lipase from candida rugosa: a comparative study between free and enzyme immobilized onto porous chitosan beads.Appl. biochem. Biotechnol., 91(93) , 739,752 (2001).

16. GOMBERT . AK, PINTO. AL, CASTILLO. LR, FREIRE. DMG, Lipase production by penicillium restrictum in solid-state fermentation using babassu oil cake as substrate. Process Biochem., 35, 85,90(1999).

17. YUZO KOJIMA, SAKAYA SHIMIZU, Purification and characterization of the lipase from Pseudomonas fluorescens HU 380. Journal of bioscience and bioengineering., 96, (3), 211,226 (2003).

18. GILBERT. JE, CORNISH. A and JONES. CW, Purification and properties of extracellular ipase from Pseudomonas aeruginosa EF2. J. Gen. Microbiol., 137, 2223,2229 (1991)

19. HUAN. D, S. GAO, S. HAN and S. CAO, Purification and characterization of a Pseudomonas Sp. Lipase and its properties in non-aqueous media. Biotechnol. App., 30, 251,256(1999).

20. BIRUTE. S ,V. BENDIKIEN, B. JUODKA, I. BACHMATOVA and L. MARCINKEVICHIONE, Characterization and physicochemical properties of a lipase from Pseudomonas Mendocina 3121. Biotechnol. App. Biochem., 36, 47,55 (2002).

7193Romanian Biotechnological Letters, Vol. 17, No. 2, 2012