Purification and characterization of thermostable and alcohol tolerant lipase from Pseudoxanthomonas sp .

An extracellular lipase from Psedoxanthomonas sp. was purified 47.3 folds with an overall yield of 27% through purification procedure of acetone precipitation, ion exchange and gel filtration chromatography. Protein precipitation using acetone fractionation showed that the enzyme was precipitated at the fraction of 0 to 40%. Further purification of the enzyme by ion exchange followed by gel filtration chromatography showed that there were two types of lipases with similar molecular size at around 50 kDa. Characterization of optimum pH, temperature, and substrate specificity were carried out against the isolated lipase. Lip1 showed specific substrate preferences towards p-nitro phenyl myristate meanwhile Lip2 exhibited hydrolytic activity towards short and medium acyl chain of the substrate. Further analysis of both enzyme activities on variation of pH and temperature showed that the optimum pH and temperature for Lip1 were at pH 10.0 and 70°C, respectively while pH 8.0 and 50°C were the optimum pH and temperature for Lip2 respectively. Lyophilized lipase isolated from acetone fraction showed lipolytic activity under variation of methanol concentration up to 30%.


INTRODUCTION
Lipase is one of the hydrolase enzymes used as biocatalysts in industry.The ability of lipase in catalyzing reactions in nonpolar environments makes lipase as the preferable catalyst in organic reactions.Lipase is generally used as biocatalyst in synthesis of flavor compounds, food industry, modification of the physicochemical properties of triglycerides in the oil and fat industry, and the synthesis of biopolymers and biodiesel (Gupta et al., 2014;Houde et al., 2004;Jaeger and Eggert, 2002;Salihu and Alam, 2014).Thermostable and solvent tolerant lipases play important roles in industrial processes, since the enzymes are applicable in the enzymatic processing of lipids, even at high temperature and alkaline condition (Lotti et al., 2015).The enzyme has been used in many fields of industries such as lipid and oil hydrolysis, detergent, peptide synthesis, and pharmacy (Hasan et al., 2006).
Application of lipases for chemical synthesis has *Corresponding author: E-mail: loka@chem.itb.ac.id.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License numerous advantages such as general ease of handling, broad substrate tolerance, high stability towards temperatures and convenient commercial availability.
Most of the synthetic reactions on industrial scale are carried out in organic solvents due to the easy solubility of non-polar compounds (Kumar et al., 2016).This approach requires an enzyme which is stable in the organic solvents.General enzymes show very low rate of reactions or inactive in non-aqueous media; therefore, search for solvent stable enzymes has been an extensive area of research (Yilmaz and Sayar, 2015;Bisht et al., 2013;Rahman et al., 2005).Application of lipase as biocatalyst in the biotechnological industries like production of biodiesel, preparation of food emulsifiers, personal care and cosmetic products, flavours and pharmaceuticals require alcohol stable enzyme (Uphues et al., 2001;Ozyilmaz and Gezer, 2010).Methanol however, is reported to hamper the activity of several lipases when used at concentrations that would be optimal for the alcoholysis reaction (Lotti et al., 2015).Enzyme stability against methanol may be improved by many techniques, like directed evolution or random mutagenesis.Korman et al. (2013) applied directed evolution to a Proteus mirabilis lipase that is relatively tolerant to short chain alcohols but is irreversibly inactivated when incubated at over 50% methanol.Random mutagenesis by error prone PCR was used to increased methanol stability in the lipase from Geobacillus stearothermophilus T6, an enzyme able to resist high temperature but which is poorly stable in polar organic solvents (Dror et al., 2014).Other researches also focused in search of novel lipase from new bacterial isolate.Enzymes secreted from organic solvent tolerant microorganisms tend to be stable under solvent rich environment.A few solvent stable lipases have been reported from solvent tolerant Pseudomonas and Bacillus sp.(Isken and de Bont, 1998;Baharum et al., 2003).Madayanti et al. (2008) cultivated and collected 10 isolates from thermogenic phases (50-70°C) during composting process which showed lipolytic activity.
In previous report (Syihab et al., 2015), a methanol tolerant Pseudoxanthomonas strain was isolated from domestic compost.The isolate able to survive in medium containing 3% of methanol and showed better lipolytic activity among other isolates tested.The objective of this research is to purify and characterized lipase from compost isolate.This present paper describes purification of lipase from Pseudoxanthomonas taiwanensis using ion exchange chromatography, followed by gel filtration chromatography.The purified lipases were characterized to determine optimum pH, temperature and substrate specificity.

Inoculum preparation
Inoculum was prepared by transferring loopful of stock culture to the medium consisting 0.5% meat extract, 0.5% yeast extract, 0.1% NaCl and 0.1% CaCl2.2H2O.The cultivation was performed at 55°C with shaking at 150 rpm for 20 h.

Cultivation for lipase production
Medium for lipase production (100 mL) was prepared based on the same composition with media using 500 ml Erlenmeyer flask.The media was added by 1 mL of inoculum and incubated at 150 rpm in a shaker maintained at 55°C.After 17 h, cells were harvested by centrifugation at 8,000 g at 4°C for 30 min.The cell-free supernatant was used as crude extract for lipase purification.

Lipase assay
Lipolytic activity was measured by spectrophotometer based on assay with p-nitrophenyl fatty acids dissolved in acetonitrile at concentration of 0.01 M as substrate (Lee et al., 1999).Subsequently, ethanol and sodium phosphate buffer (0.05 M; pH 8.0) were added to final composition of 1:4:95 (v/v/v) of acetonitrile/ ethanol/ and buffer respectively.The enzyme was added to the substrate (1:3, v/v), then incubated at 55°C for 15 min.Enzyme activity was measured by monitoring the absorbance at 405 nm, representing the amount of p-nitrophenol (PNP) released.One unit of lipase activity is defined as the amount of enzyme producing 1 mmol PNP per minutes under the assay conditions.

Protein concentration
The protein concentration was determined based on Bradford dye method, and standard protein was made using bovine serum albumin.The enzyme was mixed with Bradford reagent (1:1, v/v), then incubated at room temperature for 10 min.Protein concentration was measured by monitoring the absorbance at 595 nm wavelength (Nurhasanah et al., 2017).

Methanol tolerance lipase assay
To determine the lipase tolerance against methanol, enzyme activity was assayed at various methanol concentrations (3-30%).The assay was conducted based on lipase activity assay with the replacement of ethanol with methanol (Lee et al., 1999).The varying amount of methanol was prepared under similar conditions.

Lipase purification
The cell free supernatant was fractionated using two types of precipitation, that is, ammonium sulfate and acetone.All subsequent steps were carried out at 4°C.The protein pellet from ammonium sulfate precipitation was resuspended in 0.05 M sodium phosphate buffer pH 8.0 and dialyzed against the same buffer to remove the residue of ammonium sulfate.Lyophilization was conducted against the pellet from acetone fraction to evaporate the remaining solvent, and continued by resuspending the protein using the same buffer.
The precipitated enzyme was loaded onto a column of DEAEsepharose fast flow (1.2 × 50 cm), previously pre-equilibrated with 0.02 M sodium phosphate buffer pH 6.0.The column equilibrated with 0.02 M sodium phosphate buffer pH 6.0 which contained 0.1 M NaCl.Subsequently, 10 mL of enzyme was loaded into the column.The column was eluted with 0.02 M sodium phosphate buffer pH 6.0.It was subsequently eluted with step gradient of 0.2 to 0.8 M NaCl (0.2, 0.4, 0.6 and 0.8 M NaCl) in 0.02 M sodium phosphate buffer pH 6.0 at flow rate of 0.5 mL/minute.The fractions which showed higher protein concentration (A 280) were collected.
The enzymes collected from DEAE sepharose FF were dialyzed using 0.02 M sodium phosphate buffer pH 6.0 containing 0.15 M NaCl.Subsequently, the enzymes were loaded to a Sephadex G-75 column (0.6 × 5 cm), and elution conducted using the same buffer at flow rate of 0.2 mL/minute.Enzyme activity and protein concentration of pooled fraction was determined.Specific activity of enzyme was calculated in every step of purification.

Polyacrylamide gel electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli (1970), using 12% crosslinked polyacrylamide gel to separate and determine the molecular mass of the purified enzymes.
Zymography analysis was carried out for detection of lipase activity.The enzyme was assayed and visualized on zymograms using 12% polyacrylamide gels after renaturation using 0.05 M phosphate buffer pH 8.0, containing 0.1% Triton X for 4 h at 4°C.The gels were finally incubated for 1 h at 65°C in developing solution consisting of 0.003 M α-naphthyl acetate, 0.001 M Fast Red TR (Sigma), and 0.05 M sodium phosphate buffer, pH 8.0 (Soliman et al., 2007).

Optimum pH and temperature assay
To determine the optimum pH of purified lipase, enzyme activity was assayed at various pH values (5.0-12.0).Lipase activity was examined at 55°C using 0.05 M buffer with pH range from 5.0 to 12.0 were used in this experiment.To determine the effect of temperature on lipase activity, the assay was carried out at various temperatures (30 to 80°C) under standard conditions (0.05 M Tris-HCl, pH 8.0).

Substrate specificity assay
Substrates of p-nitrophenyl fatty acid esters at varying chain length (C2, C4, C10, C12, C14 and C16) were used at 0.001 M concentration and the enzyme activity was measured based on optimum pH and temperature.

Production and purification of lipase
An extracellular lipase was produced on the media consisting of 0.5% meat extract, 0.5% yeast extract, 0.1% NaCl and 0.1% CaCl 2 .2H 2 O.The lipase was produced optimally at 55°C and pH 7.0 on the late of stationary phase.From 2000 mL of culture, extracellular lipase was recovered at 9.62 mg of crude enzyme with specific activity of 0.19 U/ mg enzyme (Table 1).
The crude extract of lipase was purified using 2 methods, that is, ammonium sulfate and acetone precipitation.Either precipitation using ammonium sulfate or acetone was carried out at 0-40, 40-60, and 60-80% saturation, respectively.Specific activity among ammonium sulfate fractions showed no differences, indicating that the lipase was precipitated in all fractions.The acetone fractions showed higher specific activity compared to ammonium sulfate fraction, whereas the 0-40% acetone fraction has the highest specific activity among other fractions (Figure 1).
The SDS-PAGE and zymogram analysis revealed that there were two lipase band with the size of 30 and 50 kDa from 0-40% ammonium sulfate fraction.Meanwhile in 40-60 and 60-80% fraction, the 50 kDa lipase appeared with less intensity (Figure 2A).Another result based on SDS-PAGE and zymogram towards the acetone fractions showed that 0-40% fraction has several lipase bands with the size of 70, 50, and 40 kDa (Figure 2B), while other fraction showed no lipase band.The hydrophobicity of amino acid residue in lipase may lead to more protein precipitated in acetone fractionation.Acetone fractionation method is widely used to precipitate proteins with many hydrophobic amino acids residue such as lipases.Bihst et al. (2013) reported that the maximum lipase activity of Pseudomonas aeruginosa was obtained on the addition of acetone with a ratio of 1: 1 to the crude extract of the enzyme.In this study, application of acetone fractionation managed to precipitate lipase in one fraction.Based on the results, the acetone fraction was purified further.Purification of lipase from acetone fractionation was followed by ion exchange chromatography using DEAE Sepharose Fast Flow (FF) as the matrix.The pattern of ion exchange chromatography showed 2 peaks of protein (Figure 3).The 1 st peak was eluted with 0.4 M NaCl, meanwhile the 2 nd peak was eluted with 0.6 M NaCl.The SDS-PAGE and zymogram analysis showed that both peaks have the same molecular mass of 50 kDa.Further purification using Sephadex G-75 showed that the lipases were successfully purified into one single band with the size at 50 kDa (Figure 4).
Purification of lipase from DEAE Sepharose FF and Sephadex G-75 managed to isolate 2 lipases with the same size of 50 kDa.Acetone fraction from AL17 showed 2 peaks from anion exchange chromatography.The 1 st peak has protein with the size of 50 kDa with specific activity of 1.7 U/mg, 3.1 purification fold, and 55% yield, while 2 nd peak has specific activity of 2.9 U/mg, 5.2  purification fold, and 46% yield.Further purification towards 1 st peak by gel filtration chromatography managed to isolate 50 kDa lipase with specific activity of 15.6 U/mg, 28.1 purification fold, and 33% yield, meanwhile 2 nd peak also has 50 kDa lipase with specific activity of 26.3 U/mg, 47.3 purification fold, and 27% yield (Table 1).
Lipase purification from Pseudomonas S5 was successfully carried out with high recovery by using affinity chromatography in combination with ion exchange chromatography (Rahman et al., 2005).Other lipase from Trichoderma viride was purified using ammonium sulfate precipitation, ion exchange and gel permeation chromatography resulting in lipase with 134-folds purification with an overall yield of 46% (Kashmiri et al., 2006).Lipase from local thermophilic microorganism was successfully isolated using DEAE Sepharose FF and Sephacryl S-200, resulting in 4 bands of protein exhibiting lipase activity (Febriani et al., 2010).

Methanol tolerance of the enzyme
Effect of methanol at various concentrations towards lipase activity was determined using lyophilized lipase from acetone fraction.Lipase is capable to maintain its activity in various methanol concentrations after incubation for 15 min at 60°C.The enzyme showed activity of 0.84 U in 30% methanol (Figure 5).Employing lipase as a biocatalyst in biodiesel synthesis requires a stable and active enzyme, hence lipase activity in methanol is considered as an important parameter.Most of microbial lipases rarely showed high stability in hydrophilic solvents (Doukyu and Ogino, 2010;Zhao et al., 2008).The isolated lipase from P. taiwanensis showed stability in hydrophilic solvents, therefore appear promising for catalysis in low water medium.This property is present only in several cases viz.Pseudomonas aeruginosa (97% after 24 h in methanol), Bacillus megaterium CCOC-P263 (97% after 1 h in isopropanol), and Serratia marcescens (108% after 24 h at 10% DMSO) (Gaur et al., 2008;Zhao et al., 2008).

Optimum pH, temperature, and substrate specificity
Characterization of lipases from previous purification carried out against optimum pH, temperature and substrate specificity.The activity of lipase in various condition of pH was examined from pH 5.0 to 12.0.The 1 st peak exhibited high hydrolytic activity at the pH range of 9 to 12, with the maximum activity at pH 10, suggesting that the enzyme was an alkaline lipase.In contrast, 2 nd peak showed high hydrolytic activity in a neutral environment at around pH 7 to 8, with the maximum activity at pH 8 (Figure 6A).The high activity of isolated lipases over a wide alkaline pH, suggests its application in a range of industrial applications, such as synthesis of biodiesel, biopolymers, and other industries such as pharmacy, cosmetics, and flavor (Abdelkafi et al., 2009).
Optimum temperature for 1 st and 2 nd peak was tested based on their activity on various temperatures ranged from 30 to 75°C.The 1 st peak showed high activity at 60 to 75°C, with optimum activity at 65°C (Figure 6B).Activity of 1 st peak showed no significant decrease even when it was incubated at 75°C.Optimum temperature of 2 nd peak was at 50°C, and significantly dropped at 55°C.Overall, activity of 1 st peak was higher than 2 nd peak at temperature range of 30 to 60°C.However, 1 st peak has higher optimum temperature compared to 2 nd peak.Activity of lipase at high temperature could be due to the optimum temperature needed to trigger the lid opening of the lipase (Masomian et al., 2013).The stability of enzyme at high temperature was also found in lipase from Bacillus thermoleovorans ID-1 and Geobacillus sp.(Dong-Woo et al., 1999;Abdel-Fattah, 2002).
Substrate specificity of 1 st peak and 2 nd peak was determined using p-nitrophenyl esters with varying acyl chain lengths at 60°C and pH 8.0. 1 st peak demonstrated a single substrate specificity towards p-nitrophenyl myristate while 2 nd peak showed broader substrate specificity, from p-nitrophenyl butyrate to p-nitrophenyl myristate  caprate (Figure 7).Based on the results, it was suggested that 1 st peak is a different enzyme from 2 nd peak because it has different optimum pH, temperature, and substrate specificity.Since, the enzymes were different type of lipase, therefore 1 st peak was namely as Lip1 and 2 nd peak as Lip2.The specificity of Lip1 towards p-nitrophenyl myristate is favorable in enzymatic synthesis of myristyl myristate which is commonly used in cosmetic industries (Garcia et al., 2009).True lipases were defined as enzymes that able to hydrolyze ester substrates with long chain fatty acids (Glogauer et al., 2011;Reyes-Duarte et al., 2005).Lip1 is most likely to be true lipase, since it showed high hydrolytic activity towards long chain fatty acid.Meanwhile, Lip2 were classified as esterase group, since it has activity towards short and medium acyl chain length.Substrate specificity of lipases may be due to differences in the geometry and size of their active sites (Pleiss et al., 1998).This type of lipase is more suitable for short chain ester synthesis such as synthesis of flavour esters (Langrand et al., 1990).

Figure 1 .
Figure 1.Specific activity of enzyme fraction; specific activity of ammonium sulfate fraction (■) and acetone fraction (□).The ammonium sulfate fractions showed higher specific activity compared to acetone fractions.

Figure 3 .
Figure 3. Purification of Pseudoxanthomonas lipase from DEAE sepharose fast flow.Protein concentration was measured using spectrophotometer at 280 nm wave length.The first peak (A) was eluted in sodium phosphate buffer containing 0.4 M NaCl, while the second peak (B) eluted in sodium phosphate buffer containing 0.6 M NaCl.

Figure 5 .
Figure 5. Methanol tolerance of lipase.Activity of lipase in various concentration of methanol range from 3 to 30%.

Figure 7 .
Figure 7. Substrate specificity of purified lipase.The lipase activities of 1 st peak (■) and 2 nd peak (□) towards various pNP-esters were determined based on each optimum pH and temperature for their activities.