Introduction

Lipases are widely known enzymes, which able to catalyze triglyceride into diacyl glyceride, glycerol, and free fatty acid in hydrolysis process. The enzyme commonly works on interface of water-lipid micellar or emulsion formed by non-polar substrate (long chain triacyl glycerol) and water solvent^1-2. It is also recognized that lipases perform reversible reaction so that the enzyme is widely used in hydrolysis process, glyceride trans-esterification, enantioselective synthesis and also hydrolysis of various ester groups³. Within the condition of insufficient water, lipases catalyze the reverse reactions that are esterification, alcoholysis, and acidolysis. Even though lipases have various substrates some lipases shows very specific catalytic properties such as chemo, region, and enantionselective reaction⁴.

Lipase is one of important enzymes and used in industries, such as detergent, pharmaceutical, food, textile and waste treatment industries^5-6. The detergent industry has emerged as one of the major consumers of hydrolytic enzymes especially their work at alkaline pH. At the moment lipases are used more than a quarter of global enzyme production. The use of alkaline lipase is increased remarkably after the availability of lipase source, derived from thermophilic bacteria.⁷Alkaline lipase is an important enzyme since it has activity at high pH.  Now days, alkaline lipases are mostly isolated from Geobacillus sp^8-9. The application of lipases in detergent industries require characteristics factors such as stable on high temperature, active in alkaline condition and tolerant to additive materials such as surfactants and  denaturation by proteolysis¹⁰.

Many novel lipases are still to be explored and identified. The enzymes with new and exciting properties could be discovered from thermophilic microorganisme¹¹.  Indonesia is one of the most tectonically active area in the world, has highly potential as habitat of thermophiles¹². A few thermophilic microorganisms were isolated from various hot springs around West Java, such as, Domas ¹³, Papandayan ¹⁴ and Kawah Hujan (Kamojang) ^12-15 hot springs. Some of these bacteria are identified as thermostable alkaline lipase producer strains^16-17.

In this report, we describe the isolation and purification of thermostable alkaline lipase from local isolate of thermophilic bacterium obtained from Domas hot spring in West Java, namely DMS-3. The enzymes have been purified by chromatography of DEAE sepharose fast flow and Sephacryl S-200.

Material and Methods

Material

p-nitropheny palmitate, pepton, yeast extract, NaCl, bacto agar, aceto nitril, etanol ammonium sulfate (NH₄)₂SO₄ were purchases from Sigma. DEAE-Sepharose Fast Flow and Sephacryl S-200 were purchased from Pharmacia.

Morpholological Analysis

A local isolate of thermophilic bacteria which was previously isolated from hot spring in Domas, West Java Indonesia was regenerated on solid medium of ½ T containing 0.8 % polypepton, 0.4 % yeast extract, 0.2 % NaCl, and 3 % bacto agar. The media was then incubated for 18 hours at 70 ^oC. For scanning electron microscope analysis the samples were fixed for 18 h in 0.25% glutaraldehyde (in Na-phospate pH 7.2). It was subsequently dehydrated in a graded series of ethanol, critical point dried, gold coated by sputtering (ion Sputter CE102 Hitachi) and observed with S-4700 scanning electron microscope. Colony morphology was determined with microscope with 400 x zoom using cultures grown on agar plates for 24 h at 70 °C. Gram staining was performed according to Dussault¹⁸.

Isolation of Lipase 

A single colony from the solid medium was transferred onto a 25 mL liquid medium contaning 0.5% peptone, 0.5 % yeast extract, 0.05 % NaCL, 0.05 % CaCl₂ as starter media. The pH was adjusted up to 9.0 by Glysin-NaOH buffer. The inoculated media was incubated at 70 ^oC on shaker incubator, 150 rpm for 24 hours. 100 µL of inoculums bacteria was transferred to 100 mL of production starter media. The supernatant of the culture after centrifugation (12000 g, 10 min) at  4 ^oC was used to determine extracellular lipase activity as crude enzyme.

Enzyme Purification

The cell free supernatant was fractionated by precipitation with ammonium sulfate at 20 – 60% of saturation. All subsequent steps were carried out at 4 °C. The protein pellet was resuspended in 0.02M Tris-HCl buffer, pH 8.0 and dialyzed against the same buffer to remove the residual ammonium sulfate.

The precipitated enzyme was load onto a column of DEAE-sepharose fast flow (2.2 x 20 cm), previously pre-equilibrated with 20 mM Tris-HCl buffer pH 8.0. The column equilibrated with 20 mM Tris-HCl buffer (pH 8.0 0.15 M NaCl). The column was eluted with 20 mM Tris-HCl buffer pH 8.0. It was subsequently eluted with step gradient of 0.15 M-0.8 M NaCl (0.15; 0.3; 0.6; 0.8 M NaCl) in 20 mM Tris-HCl buffer pH 8.0 at flow rate of 2 mL/minute. The fractions exhibiting higher lipase activity were collected.

The pooled enzyme from DEAE sepharose fast flow was concentrated by membrane filters through 10 kDa-cut off (Amicon) and loaded to a Sephacryl S-200 column (1,2 X100 cm), equilibrated with 20 mM tris-HCl buffer (pH 8.0 0.15 M NaCl) at 1 mL/minute. Enzyme activity and protein concentration of pooled fraction was determined. Specific activity of enzyme was calculated in every step of purification.

Native and SDS PAGE  

Sample proteins were analyzed on native and sodium dedocyl sulfate-polyacrylamide gel electrophoresis based on Laemmli ¹⁹ to check the purity and determine the molecular mass of the purified enzyme. Zymography analysis was carried out for detection of lipase activity. The enzyme was assayed and visualized on zymograms using 12 % Native-polyacrylamide gels. The protein samples containing lipase were washed in 100 mM Tris–HCl buffer pH 7.5 for 15 minute at 4 ^oC. The gels were finally incubated for 1 hour at 65 ^oC in developing solution consisting of 3 mM  α-naphthyl acetate, 1 mM Fast Red TR (Sigma), and 100 mM sodium phosphate buffer, pH 8.0.

Enzyme Assay

Lipase activity was determined using spectrophotometric assay as described by Lee et al ²⁰ with p-nitropheny palmitate as a substrate. The enzyme activity was measured by monitoring the change in absorbance at 405 nm that represents the amount of released p-nitrophenol (PNP). One unit of lipase is defined as the amounts of enzyme releasing 1 umol PNP per min under the assay condition.  Protein concentration was determined by the method of Bradford using Bovine Serum Albumin as the standard ²¹.

Results and Discussion

Cell Morphology

Morphology of the isolate was analyzed using gram Staining and Electron Microscopy (SEM) methods. Based on the gram staining analysis, the isolate showed as a gram positive bacterium. In addition the isolate show rod shape form with specific clear zone within the cell (Figure 1 A). Furthermore, SEM analysis of the isolate showed that the bacterium appear as long and slim rod shape form (Figure 1B). SEM analysis (Figure 1 B) shows that the cell has uniform. This is suggested that the cell is single strain. The result of morphological analysis is in agreement with the ribotyping analysis result¹⁵.

Figure 1a: Microscopical characteristics of the isolated from Domas hot spring: (A), gram positive staining on light microscopy (B) Scanning electron micrograph, 10 um.   Click here to View figure

Production of Thermostable Alkaline Lipase

An extracellular alkaline lipase was produced on the media consisting of 0.5% peptone, 0.5 % yeast extract, 0.05 % NaCL, and 0.05 % CaCl₂. The lipase was produced optimally at 70^oC, pH 9.0 on the late of exponential or the early of stationary phase. 5.115 g of lipase with specific activity of 22.07 U/g are isolated from 1500 mL of culture (Tabel 1). The crude enzyme was then purified.

Purification of Thermostable Alkaline Lipase 

Purification of the enzyme was carried out by using ammonium sulfate fractionation followed by DEAE sepharose fast flow and Sephacryl S-200. Ammonium sulfate fractionation was carried out at 0-20, 20-40, 60-80% saturation respectively. Based on fractionation result, we collected ammonium sulfate fraction at 20-60%. Following purification by Sephacryl S-200, the specific activity of purified enzyme still showed 54 times higher compared to that the crude extract, with the yield of 4.9 % (Table 1). The result suggested that have good quality of purified lipase ^7-20.

Tabel 1: Summary of thermostable alkaline lipase purification.

SDS-PAGE of crude and purified enzyme (Figure 2) showed that the purified enzyme still exhibited few band of proteins, this is suggested that either the enzyme is poorly purified or there are more than one protein showing lipase activity. Following separation by native gel and zymogram analysis (Figure 3 A,B) showed that there were four bands exhibiting lipase activity. This is suggested that there is more than one type of lipase produced by the isolate. There were few reports showing that lipase or esterase from microorganism could form as single or multiple protein^22-23. In addition, some there are reports showed that single microorganisms expressed more than one type of lipases ^24-25.

Figure 2: SDS-PAGE of crude and purified enzyme lane 1, marker protein; lane 2, crude extract enzyme; lane 3, ammonium sulfate fractionation (fraction 20-60%); lane 4, DEAE sepharose fast flow; lane 4, Sephacryl S-200 results.   Click here to View figure

Figure 3 a: Electrophoregram of Native Gel. lane 1, marker protein ; lane 2, DEAE fast flow; lane 3, Sephacryl S-200 results. (B) Zymogram of the gel with α-naphthyl acetate and fast red TR for detection of hydrolase activity. lane 1, protein Marker, lane 2, DEAE fast flow;  lane 3. Sephacryl S-200 results.   Click here to View figure

Figure 4: SDS-PAGE from electro elution result. lane 1, marker protein; lane 2, first band, lane 3, second and third bands, lane 4, the fourth band from native gel respectively.     Click here to View figure

Further analysis on the bands which have lipase activity were carried out by eluting each band from the native gel, the bands were then exposed to SDS-PAGE. The result showed that the fourth band exhibited single protein on the SDS-PAGE. Meanwhile, the other bands showed few protein bands but in similar pattern. This is suggested that the isolate expressed more than one type of lipases.

Acknowledgements

This research was partial supported by Graduate Research Grant to Fida Madayanti, and BPPS Scholarship and sandwich program to Febriani, Directorat General for Higher Education, Ministry of National Education Republic of Indonesia, and ITB Excelent Research Grant to Akhmaloka.

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