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Bioresource Technology
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An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis
Eltayib Hassan Ahmed, Tripti Raghavendra, Datta Madamwar *
BRD School of Biosciences, Sardar Patel Maidan, Vadtal Road, Satellite Campus, P.O. No. 39, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India

a r t i c l e

i n f o

a b s t r a c t
A mesophilic bacterium producing a thermostable alkaline lipase was isolated from oil rich soil sample and identied as Acinetobacter sp. EH28. The lipase was partially puried by ammonium sulphate precipitation followed by hydrophobic interaction chromatography with 24.2-fold purication and 57.1 U/ml specic activity. The partially puried enzyme exhibited maximum activity at pH 10.0 and at 50 C and was highly stable at 50 C retaining 100% of its activity up to 90 min. It was highly stable and retained more than 80% of its initial activity upon exposure to various organic solvents. The EH28 lipase was used for synthesis of the avor ester ethyl caprylate in organic solvents, thus providing a concept of application of Acinetobacter sp. lipase in non-aqueous catalysis. Reaction parameters best suited for this esterication reaction were 40 C reaction temperature, 1.3:1 ratio of caprylic acid to ethanol and cyclohexane as the medium. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 26 November 2009 Received in revised form 22 December 2009 Accepted 24 December 2009 Available online xxxx Keywords: Thermostable Partial purication Microemulsion based organogels Ethyl caprylate Non-aqueous

1. Introduction Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are ubiquitous enzymes that are activated when adsorbed to an oilwater interface (Schlatmann et al., 1991). Due to their widely diversied enzymatic properties, in particular, the ability to perform enantioselective hydrolytic reactions and catalyze the formation of wide range of ester and amide bonds, microbial lipases have become very attractive for industrial application (Hasan et al., 2006). Microbial lipases that can function as catalysts in non-aqueous solvents offer new possibilities such as shifting of the thermodynamic equilibria in favor of synthesis, enabling the use of hydrophobic substrates, controlling substrate specicity by solvent engineering and improving thermal stability of the enzymes (Lima et al., 2004). Apart from all the advantages, major limitation in carrying out the reaction under water-restricted environment is the tendency of organic solvents to strip water molecules from enzyme surface especially into the active site leaving the enzyme inactive. To overcome these limitations and for enhancing enzyme activity along with stability, several strategies like chemical modication of amino acids on enzyme surface (DeSantis and Jones, 1999), protein engineering (Magnusson et al., 2005), medium engineering (Laane, 1987), and colyophilization with non-buffer salts (Mine et al., 2003) have been demonstrated. Alternatively, it has been proposed that instead of modifying enzyme for increasing solvent
* Corresponding author. Tel.: +91 2692 229380; fax: +91 2692 231042/236475. E-mail addresses: eltayib1974@yahoo.com (E.H. Ahmed), tpt11@yahoo.co.uk (T. Raghavendra), datta_madamwar@yahoo.com (D. Madamwar). 0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.12.107

stability, it would be more desirable to screen for naturally evolved solvent tolerant enzymes for application in non-aqueous enzymatic synthesis. Among the numerous lipases described in the literature, only the enzymes belonging to few species have been demonstrated to have adequate stability and biosynthetic capabilities to allow routine use in organic reactions and hence they may be considered industrially relevant enzymes (Dandavate et al., 2009). One of the numerous applications of lipases in non-aqueous medium is production of avor esters. In recent times, avors represent over a quarter of the world market for food additives and it has been shown that consumer prefer foodstuff that can be labeled as natural (Cheetham, 1997). The tag natural and value added features of these biochemically produced esters excelling their chemical counterparts by having better odor and avor (Rizzi et al., 1992) have intensied research activities in this eld. The genus Acinetobacter is best known for its capacity for bioremediation of alkanes and aromatic hydrocarbons, as well as production of high molecular weight heteropolysaccharides that act as powerful emulsiers of high commercial potential, and many of them have been found to secrete esterolytic enzymes. Several authors have reported the purication, characterization, and optimization of lipase production from Acinetobacter sp. isolated from different sources (Hong and Chang, 1998; Snellman et al., 2002; Saisubramanian et al., 2008) but there are less reports on its application in non-aqueous media. Here, we have successfully isolated an alkaline thermostable solvent tolerant lipase producing bacteria identied as Acinetobacter sp. EH28. In this paper we report isolation of the bacterial culture,

Please cite this article in press as: Ahmed, E.H., et al. An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis. Bioresour. Technol. (2010), doi:10.1016/j.biortech.2009.12.107

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partial purication of the lipase and its characterization. We have demonstrated the potential application of this lipase as a catalyst for synthesis of ethyl caprylate. Furthermore, standardization of reaction parameters for esterication was carried out. 2. Methods Phenyl Sepharose 6 was procured from SigmaAldrich (Germany), tributyrin oil and bovine serum albumin were obtained from HiMedia (India), olive oil was procured from Figaro (Spain), gum arabic from Titan Biotech (India). Sodium bis-2-(ethylhexyl) sulfosuccinate (AOT) was obtained from Fluka (Switzerland). Ethyl caprylate and caprylic acid were purchased from Fluka (Switzerland). All other organic solvents used were of HPLC/GC grade. 2.1. Bacterial strain 2.1.1. Screening and isolation of organic solvent lipase producing bacteria Soil samples collected from various oil spilling sites of Vallabah Vidyanagar, Gujarat, India were subjected to enrichment culture technique by inoculating 1 g of soil sample into 100 mL tributyrin broth (1% (v/v) tributyrin oil, 0.5% (w/v) tryptone and 0.3% (w/v) yeast extract) in Erlenmeyer asks. The asks were incubated at 37 C on orbital shaker (150 rpm) for 10 days. The samples were serially diluted and plated onto 1% tributyrin agar plates. The colonies showing zone of clearance after incubation for 48 h at 37 C were selected and lipase assay was performed for each isolate. The isolates showing high lipase activity were selected for further experimentation. Selected cultures were subjected to screening for potential organic solvent tolerant bacteria. The isolated bacteria were streaked on tributyrin agar plates upon which each plate was ooded with 7 mL of different solvent, and incubated at 37 C for 48 h. The organic solvents used were 1-butanol, benzene, toluene, n-hexane and n-heptane. The ability of the isolates to grow and produce lipase was observed and diameter of clearance zone of colony showing hydrolysis was measured. 2.1.2. Identication of isolated bacterium using 16S rRNA gene sequence The isolate was identied using 16S rRNA gene sequence. Genomic DNA of isolate was extracted as described by Ausubel (Ausubel et al., 1997). The genomic DNA was diluted properly to (2050 ng) and used as template (30 ll) in PCR reaction using universal eubacteria primers 8F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 GGTTACCTTGTTACGACTT-30 ) custom synthesized (BIORON, GmbH, Germany). The amplication of 16S rRNA gene was done in BioRad PCR cycler (U.S.A). Each PCR cycle (35 cycles in total) consisted of a 2 min denaturation step at 94 C, followed by a 1 min annealing step at 55 C, and a 1.5 min elongation step at 72 C, with an initial denaturation step at 94 C for 5 min and a nal extension step at 72 C for 15 min. PCR products were resolved on a 1.2% (w/v) low-melting-point agarose gel in 1 TAE buffer, with a 1 Kb ladder (BIORON, GmbH, Germany) and visualized with ethidium bromide staining in Gel documentation (Alpha-Inotech. U.S.A). The amplied PCR product was subjected to sequencing by automated DNA Analyzer 3730 using ABI PRISM BigDye cycle sequencing kit (Applied Biosystems, USA). The nearly complete sequence (>95%) was submitted to Genbank at NCBI. BLAST (n) program at NCBI server was used to identify and download nearest neighbor sequence from BLAST database (Ausubel et al., 1997). 2.1.3. Growth and lipase production prole The 500 mL Erlenmeyer asks containing 200 ml tributyrin broth were inoculated with an overnight grown culture of EH28

to obtain an initial culture density (OD 600 nm) 0.05 and incubated on orbital shaker (150 rpm) at 37 C. The samples were withdrawn at regular time interval of 24 h and analyzed for cell growth and enzyme activity. The enzyme activity was estimated from the supernatant obtained upon centrifugation of 10 ml medium. The cell pellet obtained upon centrifugation was resuspended in 10 ml of distilled water and its absorbance measured at 600 nm and reported as growth of the culture. 2.2. Partial purication of lipase 2.2.1. Ammonium sulphate precipitation The calculated amount of solid ammonium sulphate was added to cell free supernatant with constant stirring at 4 C to achieve 80% saturation. The precipates thus obtained were harvested by centrifugation at 13,000 xg for 30 min, and resuspended in minimum volume of 25 mM sodium phosphate buffer (pH 7.2). This enzyme solution was subjected to dialysis for 24 h at 4 C against the same buffer, with three intermittent changes of the buffer. Further, the ltrate was concentrated using 30 KDa centricon lter at 4 C for 60 min at 800 xg. Lipase activity and protein content were determined for both, the dialyzed and the concentrated ltrate sample. 2.2.2. Hydrophobic interaction chromatography (HIC) The concentrated lipase solution was applied on a HIC column using phenyl Sepharose 6 (1.5 6 cm), which was pre-equilibrated with 0.6 M ammonium sulphate dissolved in 25 mM phosphate buffer (pH 7.2). The bound enzyme was eluted by ammonium sulphate dissolved in 25 mM phosphate buffer (pH 7.2) at a ow rate of 1 mL/min through negative linear gradient. The resultant fractions were subjected for determination of lipase activity and protein content. The lipase preparations (crude and puried fractions) were electrophoresed on native PAGE (10%). The protein bands on the gel were visualized upon silver staining. 2.3. Characterization 2.3.1. Effect of pH on lipase activity and stability Optimum pH of the extracellular lipase was determined by measuring the enzyme activity over a pH value ranging from 4.0 to 12.0 at 30 C. Buffers (50 mM) used were acetate buffer for pH 4.05.0, sodium phosphate buffer for pH 6.08.0, glycine NaOH buffer for pH 9.010.0, and phosphate NaOH buffer for pH 11.0 12.0. 2.3.2. Effect of temperature on lipase activity and stability The effect of temperature on the activity of partially puried lipase was determined by monitoring the enzyme activity at different temperatures in the range of 30 to 80 C at pH 10.0. The thermal stability of lipase was studied from 50 to 70 C at pH 10.0 and the residual activity was measured at every 15 min interval for a total period of 2 h. 2.3.3. Effect of organic solvent on lipase activity To study the effect of organic solvents, 0.1 ml enzyme($410 U/ ml activity) was preincubated in substrate free reaction buffer (pH 10) substituted with the organic solvents (15% and 30% v/v) for 1 h and subjected to enzyme assay at 50 C. The organic solvents used were ethanol, methanol, isopropyl alcohol, dimethyl formamide, dimethyl sulfoxide, n-hexane and acetone. 2.4. Immobilization of partially puried EH28 lipase in w/o microemulsion based organogels (MBGs) The partially puried lipase was immobilized in AOT based organogels following the method described by Dandavate and

Please cite this article in press as: Ahmed, E.H., et al. An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis. Bioresour. Technol. (2010), doi:10.1016/j.biortech.2009.12.107

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Madamwar (2007). The Wo value (ratio of concentration of water to that of concentration of surfactant), which relates to the water activity in MBGs was adjusted to an optimized value of 60. The MBGs thus prepared were stored at 4 C prior to use. 2.5. Synthesis of ethyl caprylate under water-restricted environment using free and immobilized EH28 lipase The reaction mixture consisted of 20 mL isooctane and equimolar concentration (100 mM) of ethanol and caprylic acid in 250 mL glass stoppered conical ask. The esterication reaction was initiated by addition of partially puried free/ immobilized lipase (57.1 units) to the reaction mixture and asks were incubated in orbital shaker at 30 C and 150 rpm. 100 lL of the reaction mixture was withdrawn every 24 h and analyzed by gas chromatography. Ester identication and quantication was done by comparing retention time and the peak area of the sample with a standard (0.1 M ethyl caprylate). 2.6. Standardization of reaction parameters for esterication reaction 2.6.1. Effect of solvents on esterication To study the interactive effect of solvents as medium for reaction on esterication, AOT based organogels were used to carry out esterication in various organic solvents. Solvents used were isooctane, n-hexane, n-heptane and cyclohexane. 2.6.2. Effect of temperature on esterication Effect of temperature was studied by carrying out the reaction at different temperatures (20, 30, 40 and 50 C). The reaction system consisted of MBGs of AOT/isooctane in cyclohexane at 150 rpm. A control was kept at each temperature with free lipase as the catalyst. 2.6.3. Effect of substrate on esterication For determination of effect of substrates on esterication, the concentration of one substrate was kept constant while the other was varied. The concentration of the varied substrate was then kept constant and the other (kept constant in rst set) was varied. 2.6.4. Reusability of the MBG preparation After completion of a reaction cycle, the MBGs were washed with solvent 34 times for complete removal of substrates and product. They were air dried and reused for ester production. After 34 runs, when a sharp decline in percentage esterication was observed, the MBGs were treated with dry reverse micellar solution of 1 M AOT overnight for removal of excess water (byproduct of esterication reaction); given 23 solvent washes for complete removal of AOT and used for esterication. 2.7. Analytical procedures 2.7.1. Lipase assay The pH stat method: Lipase activity was measured under controlled temperature using a pH 718 STAT Titrino Titrator (Metrohm, Switzerland). The buffer used for the activity determination comprised of 10% (w/v) gum arabic, 0.2% (w/v) bile salt, and 20% (w/v) CaCl2, 5% (v/v) olive oil as substrate and 0.1 M NaOH as the titrant. The insoluble triglycerides were dispersed by vigorous stirring. One unit of enzyme activity was dened as the amount of enzyme that liberates 1 lmol of fatty acid per min at 50 C. 2.7.2. Protein estimation Soluble protein was estimated by Lowrys method using bovine serum albumin as standard.

2.7.3. Quantication of ester Ethyl caprylate was estimated by Gas Chromatography (PerkinElmer, Model Clarus 500, USA) equipped with ame ionization detector and 30 m Rtx-R-20 (Cross bond 80% dimethyl-20% dipenhyl polysiloxane) capillary column. Nitrogen served as carrier gas at a split ow rate of 90 ml/min and column temperature ranging from 40 to 280 C. The temperature was programmed with a rise of 40210 C at the rate of 6/min and from 210 to 280 C at the rate of 15/min. The temperatures of injector and detector were 250 and 280 C, respectively. 3. Results and discussion 3.1. Isolation and selection of organic solvent lipase producing organism Enzymes are often denatured and inactivated by organic solvents. Therefore, we hypothesized that organic solvent tolerant bacteria should produce enzyme that can tolerate organic solvent to a certain degree and will be very useful in industry. Solvent tolerance of the isolates was investigated by their ability to grow on tributyrin agar plates ooded with different solvents (n-heptane (log P = 4), n-hexane (log P = 3.5), toluene (log P = 2.5) and benzene (log P = 2.0)) during incubation. Here, log P is the logarithm of the partition coefcient, P, of the solvent between n-octanol and water and is used as an index of the solvent polarity. The EH28 (3.8 U/ml lipase activity) culture exhibited growth as well as lipase production in presence of all of the above solvents tested with maximum zone of hydrolysis on plates ooded with n-hexane (3 mm) and hence it was selected for further studies. Organic solvents with a log P between 1.5 and 4.0 are extremely toxic to microorganisms and other living cells because they partition preferentially in the cytoplasmic membrane, disorganizing its structure and impairing vital functions. Solvent toxicity depends not only on the inherent toxicity of the compound but also on the intrinsic tolerance of the bacterial species and strains. The solvent tolerance in certain strains has been attributed to the change in the composition of the membrane fatty acids and/or presence of active efux pumps which maintain minimal sub toxic concentration of toxic organic solvents within cells (Ramos et al., 2002). Several authors have reported solvent tolerant lipases produced by Pseudomonas sp. and Burkholderia sp. (Kim et al., 1998; Dandavate et al., 2009). However, description of Acinetobacter lipases and their development as industrial biocatalysts has lagged behind that of Pseudomonas/ Burkholderia lipases, despite having been isolated from the same environment. This may be because the latter were the rst to be studied (Snellman and Colwell, 2004). 3.2. Identication of lipase producing bacterium Microscopic examination indicated that the isolate was gram negative rod and biochemical analysis (data not shown) indicated it as Acinetobacter. Also, the DNA sequencing and BLAST analysis of 16S rRNA gene sequence of the strain EH28 showed maximum sequence homology (100%) with the complete sequence of Acinetobacter sp. and hence in the present study it is referred as Acinetobacter sp. EH28. The sequence has been deposited in Gene Bank with accession Number EU703817. Phylogenetic afliation of isolate EH28 with related Acinetobacter sp. is shown in Fig. 1. 3.3. Growth and lipase production prole Maximum lipase activity of 3.8 U/ml was observed on the second day of production with optical density 1.750 at 600 nm. On third day, increase in lipase activity was negligible and after third

Please cite this article in press as: Ahmed, E.H., et al. An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis. Bioresour. Technol. (2010), doi:10.1016/j.biortech.2009.12.107

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97 87

EH28 (EU703817) Uncultured bacterium (AM183113) Acinetobacter sp. phenon 2 (AJ275041.2) Acinetobacter sp. phenon 2 (AJ275040.2) Uncultured gamma proteobacter (DQ463728.2) Uncultured gamma proteobacter (DQ463701.2) Uncultured Acinetobacter sp. (EF371492.1)

44 99

52 98 100

Uncultured Acinetobacter sp. (EU407207.1) Uncultured bacterium clone KS (DQ532284.1) Uncultured bacterium clone Pa (EF632913.1) Escherichia coli K12 U00096 /1

0.01

Fig. 1. Phylogenetic tree based on 16S rRNA gene sequence of Acinetobacter sp. EH28. DMAX (GenBank accession No. EU703817) and sequence of closest phylogenetic neighbors obtained by NCBI BLAST (n) analysis, numbers in the parenthesis indicate accession numbers of corresponding sequence. The tree was constructed using neighbor joining algorithm with Kimura 2 parameter distances in MEGA 4.0 software. E. coli K12 has been taken as an out group. Number at nodes indicates percent bootstrap value above 50 supported by more than 1000 replicates. The bar indicates the Jukes-Cantor evolutionary distance.

day, decline in lipase activity was observed. In a study to determine optimum time to harvest the lipase for purication, Snellman et al. (2002) reported maximum lipase production of 0.25 U/ml activity from Acinetobacter sp. RAG-1 after 50 h. The decrease of lipase production at the later stage could be possibly due to pH inactivation, proteolysis or both (Bayoumi et al., 2007). It was observed that EH28 exhibited maximum lipase production at 37 C, 100 rpm and initial medium pH 7.0. Therefore, these conditions were employed for cultures used in future experimentation. Response surface methodology involving central composite design (CCD) was applied for optimizing lipase production and 4fold increase in lipase production was achieved (data not shown). 3.4. Partial purication of lipase A high state of purity is generally not required in areas such as food processing, detergent, paper and pulp industry. However, it may be necessary to exclude certain other unwanted impurities such as proteins and enzymes that may exert an antagonistic affect

on the desired enzymes activity (Hasan et al., 2006). The extracellular lipase from free fraction of liquid culture was subjected to ammonium sulphate precipitation (80% saturation), ultraltration and phenyl Sepharose 6 column chromatography in sequence resulting in partial purication (Fig. 2) by 24.2-fold with specic activity of 57.1 U/mg (Table 1). Compared to other reports, good yield and purication was achieved; Lee et al. (2006) reported 9fold purication of lipase from Acinetobacter ES-1 with 28.9 U/mg specic activity, whereas Saisubramanian et al. (2008) puried lipase from Acinetobacter sp. to homogeneity with 14.9-fold and 88.8 U/mg specic activity. 3.5. Characterization 3.5.1. Effect of pH on lipase activity and stability The activities of lipases are highly pH dependent and any alteration in the pH of reaction mixture is likely to affect their catalytic potential. The pH inuences the structure of proteins and hence governs their catalytic activity (Fullbrook, 1996). The optimum pH for lipase activity of Acinetobacter sp. EH28 was found to be 10.0, and it retained 90% of its maximum activity at pH 9.0 while a remarkable drop in enzyme activity was observed below pH 8.0 and above 11.0. Several workers have found optimal lipase activity in highly alkaline pH range (910.5), from Acinetobacter sp. RAG-1 (Snellman et al., 2002) and from Acinetobacter radioresistens CMC-1 (Hong and Chang, 1998) while Saisubramanian et al. (2008) reported 8.5 as optimum pH for alkaline lipase from Acinetobacter sp. The partially puried lipase EH28 showed maximal stability at pH 9 as it retained 100% of its activity even after 24 h incubation and 97% of its activity at pH 10 upon 24 h of incubation at 20 C. The pH stability curve of EH28 showed that the enzyme is stable

Table 1 Summary of purication steps. Purication method Protein (mg) 359.6 65 7 Activity (U/ml) 850 680 400 Specic activity (U/mg) 2.36 10.5 57.1 Fold Yield (%) 100 80 47

Fig. 2. Native PAGE (10%) of partially puried lipase from Acinetobacter sp. EH28, lane 1, ammonium sulphate precipitation sample; lane 2, ultraltration sample; lane 3, phenyl Sepharose 6 column chromatography sample.

Crude extract Ammonium sulphate precipitation HIC

1 4.4 24.2

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under pH range 811. These data are in agreement with those for alkaline lipases reported for strains of Acinetobacter sp. (Hong and Chang, 1998; Snellman et al., 2002). However, Saisubramanian et al. (2008) studied the lipase from Acinetobacter sp. and reported that the enzyme was stable between pH 39. 3.5.2. Effect of temperature on lipase activity and stability Since most of the industrial processes involving enzymes operate at temperatures above 50 C, the importance of thermostable enzymes is of great signicance. The stability of thermophilic proteins is intrinsic and resides in their primary structure. Thermostabilization of proteins can be achieved through optimization of intramolecular interactions, packing densities, internalization of hydrophobic residues and surface exposure of hydrophilic residues (Jaenicke and Zavodszky, 1990). In this study, initial reaction rate was determined in temperature range of 3080 C. The optimum temperature for lipase EH28 was found to be 50 C and the lipase activity signicantly increased with increase in temperature from 30 to 50 C but further increase in temperature adversely affected the lipase activity. Higher temperature optima have been reported for several lipases of Acinetobacter sp. (Hong and Chang 1998; Snellman et al., 2002). The distinguishing feature of lipase from EH28 is that it was highly stable at 50 C, retaining 100% of its activity up to 90 min whereas at 60 C the enzyme retained about 100% activity up to 60 min (Fig. 3). The known lipases produced by the genus Acinetobacter are active between 35 and 50 C but are not stable above 45 C (Sullivan et al., 1999; Park et al. 2009). 3.5.3. Effect of organic solvents on lipase activity Lipases are diverse in their sensitivity to solvents, but there is general agreement that polar (water miscible) solvents are more destabilizing than non-polar solvents (Cardenas et al., 2001; Castro-Ochoa et al., 2005; Dandavate et al., 2009). Table 2 shows the effect of different organic solvents on lipase activity. In this study lipase from Acinetobacter EH28 exhibited high stability in the presence of 15% and 30% of different organic solvents, as the decrease in activity of lipase was negligible in presence of most of the organic solvents. Except for n-hexane, dimethyl sulfoxide and acetone the enzyme showed higher activity in higher concentration (30%) than in lower (15%). Similar phenomenon was observed by Hong and Chang (1998) for an alkaline lipase from A. radioresistens CMC-1, and they reported that several possibilities could be considered to explain the stimulatory effect of solvents on enzyme activity. Firstly, the solvent may modify the oilwater interface to make enzymatic action easier without causing protein denaturation. Secondly, enhancement in enzyme activity could be due to disaggregation of lipase and solvents may induce some structural change in enzyme.

Table 2 Effect of different solvents on activity of partially puried lipase of Acinetobacter sp. EH28 at pH 10 and 50 C. Solvents Ethanol Methanol Isopropyl alcohol Dimethylformamide Dimethylsulfoxide n-Hexane Acetone Concentration (mM) 15 30 15 30 15 30 15 30 15 30 15 30 15 30 Residual activity (%) 96.00 90.00 85.00 74.00 96.00 89.00 93.80 81.00 100.0 130.0 102.0 112.0 104.0 118.5

The standard deviation never exceeded 5.0%.

3.6. Synthesis of ethyl caprylate under water-restricted environment using free and immobilized EH28 lipase Synthesis of avor compounds by biotechnological methods plays an important role in food industry nowadays. Various avor esters have been synthesized till date by esterication reaction using microbial lipases. Few of the numerous esters synthesized enzymatic esterication or transesterication reactions are ethyl butyrate and butyl butyrate by Welsh and Williams (1990), ethyl hexanoate by Chowdary and Prapulla (2003a) and ethyl isovalerate by Chowdary and Prapulla (2003b) and Dandavate and Madamwar (2007). The EH28 lipase was employed for synthesis of avor esters such as ethyl butyrate, ethyl valerate and ethyl caprylate in organic solvents. The enzyme showed preference for the medium chain fatty acids (valeric and caprylic acids) than short chain fatty acid (butyric acid) which may be attributed to its specicity for medium chain acids (data not shown). Hence, further studies were carried out for synthesis of ethyl caprylate. Ethyl caprylate, which has a fruity-owery fragrance, is used as a constituent in various fruity avors such as peach, apple, banana and pineapple aroma. It has been discovered as a avor enhancing compound in fermentation industry and commonly associated with wines and whiskey (Teevic et al., 2005). The partially puried lipase from EH28 exhibited signicant esterication efciency with 40% for synthesis of ethyl caprylate. When immobilized in AOT based organogels, it exhibited 1.45-fold ($58%) higher esterication efciency for synthesis of ethyl caprylate when compared to free enzyme (Fig. 4). Hence, EH28 enzyme may prove to be promising in esterication and standardization of reaction parameters may increase its efciency as a biocatalyst. The immobilization of lipase in MBGs has been reported to improve the catalytic of enzyme by providing protection against inhibitory effect of organic solvents as well as aid in reusability of lipase facilitating easy recovery by simple ltration (Dandavate and Madamwar, 2007).

120 Residual (%) activity 100 80 60 40 20 0 0 20 40 50C 60 80 Time (min) 60C 100 120 70C 140

3.7. Effect of reaction parameters on synthesis reaction 3.7.1. Effect of organic solvents It is crucial to introduce organic solvents for bioconversions of lipophilic compounds for improving the poor solubility of the substrates in water. Organic solvents produce various physicochemical effects on enzyme molecules, and the effects differ depending on the kinds of organic solvents and enzymes used. It has been reported that suspension of enzymes in organic solvents results in

Fig. 3. Thermostability of partially puried lipase of Acinetobacter sp. EH28 at pH 10.

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80 70
Esterification (%)

60 50 40 30 20 10 0 0 1
n-hexane n-hexane

crease in ester production with increase in temperature (Schlatmann et al., 1991). Similar results have been observed by Dave and Madamwar (2005) and Kantaria et al. (2003). Also, free lipase used as control showed similar behavior of increasing conversion rate with increase of temperature from 20 C (27%) to 40 C (60%) (Fig. 5). The free lipase reached its maximum conversion on fth day while immobilized enzyme revealed better stability for longer time which conveyed the effective protection to lipase offered by immobilization process against thermal denaturation.
2 3 4 Time (days) 5
isooctane isooctane

n-heptane n-heptane

cyclohexane cyclohexane

Fig. 4. The effect of solvents (n-hexane, n-heptane and cyclohexane) on synthesis of ethyl caprylate using MBGs at 37 C and 150 rpm. The solid lines represent lipase entrapped in MBGs and the dotted lines represent free lipase.

conformational changes and thereby the specicity of substrates (Fitzpatrick et al., 1993). Different solvents such as n-hexane, n-heptane, isooctane and cyclohexane were used as reaction medium. Fig. 4 shows the effect of different organic solvents on esterication reaction. In this study free and immobilized lipase from Acinetobacter sp. EH28 exhibited highest esterication of 46% and 70%, respectively in the presence of cyclohexane, followed by, isooctane showing 40% and 58%, whereas n-hexane and n-heptane showed less efciency. Hence, cyclohexane as reaction medium was chosen for further experimentation. 3.7.2. Effect of temperature on esterication The activity of lipase EH28 towards the esterication reaction was studied as a function of incubation temperature at 20, 30, 40 and 50 C. The activity of lipase signicantly increased with increase in temperature from 20 to 40 C, further increase in temperature to 50 C resulted in marginally decrease of conversion. Optimum temperature was found to be 40 C, as it showed the highest conversion of 83.0% product on seventh day whereas the reactions proceeded till days 89 at other temperatures. At lower temperatures (20 C) low conversions (35%) were observed (Fig. 5). This suggests that at higher temperatures, the conversion rate is controlled by reaction steps and hence strongly increases with increase in temperature. However, at lower temperatures, the reaction rate is limited by mass transport phenomena and not by the reaction steps which can be conferred to the low in-

3.7.3. Effect of substrate concentration on esterication As substrate concentration is a very signicant parameter in any enzyme catalyzed reaction, it is important to determine their effects on the enzyme and reaction kinetics. It is also very essential to set up reactions in such a way that result in maximum production with minimal wastage/usage of substrates. To determine the optimum ratio of the substrates, the concentrations of ethanol and caprylic acid were varied one at a time keeping the other constant and carrying out esterication reaction. The ethanol concentration was kept constant at 0.1 M and caprylic acid concentration was varied as 0.04, 0.07, 0.1, 0.13 and 0.16 M, the conversions of caprylic acid were 55%, 68%, 83%, 90% and 62%, respectively on seventh day (Table 3). It was found that there an increase in the esterication rate with an increase of acid concentration up to 0.13 M and in those with more than 0.13 M caprylic acid i.e. (0.16 M) adversely affect was observed. In another set of experiments caprylic acid was kept constant at 0.13 M (obtained from the previous study) and ethanol concentration was varied via. 0.05, 0.1, 0.15, 0.2 and 0.25, the conversions were 50%, 90%, 73%, 55% and 37%, respectively (Table 3). The reactions proceeded very quickly at low alcohol content (up to 0.1 M) and reached a maximum in 6 days while higher concentrations of alcohol exhibited an inhibitory effect on reaction slowing it down drastically. Similar phenomena have been observed by Yadav and Lathi (2003), Somashekar and Divakar (2006), Gogoi et al. (2008) and which could be attributed to the formation of dead end complex between enzyme and excesses of ethanol (Table 3). Thus, the molar ratio of caprylic acid to alcohol was found to be 1.3:1.0. 3.7.4. Reusability of the MBG preparation The enzyme preparation was given solvent washes after every cycle and reused in fresh media supplemented with substrates. It was observed that the MBGs catalyzed the reaction appreciably for rst four cycles after which their activity started declining slowly (Fig. 6). This may be due to accumulation of excess water formed as a byproduct of esterication reaction. For removal of this excess water, 1 M AOT/isooctane dry reverse micellar treatment was given to the MBGs for 24 h. Signicant increase or maintenance in esterication (no further decrease) was observed in MBGs

100 90 80 Esterification (%) 70 60 50 40 30 20 10 0

Table 3 Effect of substrate concentration on ethyl caprylate production using AOT based organogels containing lipase at 37 C and 150 rpm. Substrate Caprylic acid Concentration (M) 0.04 0.07 0.1 0.13 0.16 0.05 0.1 0.15 0.2 0.25 Esterication (%) 55 68 83 90 62 50 90 73 55 37

2
20C 30C

3
30C 20C

4
Time (days)

5
40C 40C

6
50C 50C

8
Ethanol

Fig. 5. Effect of temperature on ethyl caprylate synthesis using MBGs at 150 rpm. The solid lines represent lipase entrapped in MBGs and the dotted lines represent free lipase.

The standard deviation never exceeded 5.0%.

Please cite this article in press as: Ahmed, E.H., et al. An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis. Bioresour. Technol. (2010), doi:10.1016/j.biortech.2009.12.107

ARTICLE IN PRESS
E.H. Ahmed et al. / Bioresource Technology xxx (2010) xxxxxx 7

110 100 Esterification (%) 90 80 70 60 1 2 3 4 5 6 Number of runs 7 8 9

AOT/isooctane-EH28
Fig. 6. Reusability of organogels in cyclohexane. The up arrows indicate intermittent treatment of the organogels with 1 M AOT/isooctane dry reverse micellar solution for removal of excess water. The reactions were carried out at 37 C and 150 rpm.

demonstrating partial recovery of the lost activity (Fig. 6). The EH28 lipase exhibited 70% residual esterication even at ninth run. The stabilization of activity after intermittent treatments of AOT/isooctane dry reverse micelles imply maintenance of water activity by removal of excess water from the organogels which had resulted in decrease of ester production in the previous runs. These results indicate the increase in efciency and protection offered by immobilization to the enzyme for repeated used without much loss of activity. 4. Conclusion A mesophilic organism Acinetobacter sp. EH28 producing an alkaline thermostable lipase was isolated from oil rich soil. It was puried 24.2-fold by ammonium sulphate precipitation and hydrophobic interaction chromatography to give a nal specic activity of 57.1 U/ml. The distinguishing features included its stability towards organic solvents and high temperatures. The partially puried lipase exhibited signicant esterication activity (63%) for synthesis the ester ethyl caprylate while its efciency increased by 1.43-fold (90%) upon immobilization in MBGs. The distinguishing characteristics and its applicability in organic synthesis undoubtedly prove EH28 lipase as strong candidate as a green catalyst. Acknowledgements The authors are grateful to Indian Council for Culture relation ship (ICCR), for nancial assistance to one of the authors Mr. Eltayib Hassan and to Dr. Yogesh Souche and Mr. Rasesh Parikh of NCCS, Pune, India for 16S rDNA sequencing. The authors would also extend their acknowledgment to University Grants Commission, New Delhi, for nancial assistance. References
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Please cite this article in press as: Ahmed, E.H., et al. An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis. Bioresour. Technol. (2010), doi:10.1016/j.biortech.2009.12.107