Original Article

Identification Of Lipase – Producing Psychrophilic Yeast, Leucosporidium Sp.

F Rashid, R Rahim, D Ibrahim

Keywords

leucosporidium sp., lipase, psychrophilic

Citation

F Rashid, R Rahim, D Ibrahim. Identification Of Lipase – Producing Psychrophilic Yeast, Leucosporidium Sp.. The Internet Journal of Microbiology. 2009 Volume 9 Number 1.

Abstract

Cold-active enzymes have recently received great attention due to their potential applications in a broad range of industrial, agricultural and medical processes. One of the enzymes is lipase (triacyglycerol acylhydrolases E.C 3.1.1.3) which is unique in catalyzing the hydrolysis of triacylglycerols into free fatty acids and glycerol. In this particular research, an obligate psychrophilic microorganism was isolated from Casey Station, Antarctica. The growth of this microorganism has been tested at different temperatures, 4oC, 27oC and 37oC. At 4oC, the microorganism was able to grow whereas at 27oC and 37oC, there was no growth at all. The presence of lipase enzyme in this microorganism was detected by halo zone on palm oil (substrate) agar plates. Identification of this microorganism was done based on its morphological, biochemical and molecular characteristics. For the morphology analysis, two types of microscopy observation were carried out:

phase contrast microscopy and Scanning Electron Microscopy (SEM). Both observations showed budding structures. This suggested that this particular microorganism is psychrophilic yeast. Biochemical tests were done based on its capability to ferment and assimilate sugar. In addition, assimilation of nitrate was also tested. In molecular approach, the genomic DNA (gDNA) of this microorganism was successfully extracted and the extracted gDNA was used for amplification via polymerase chain reaction (PCR) technique using Internal Transcribed Spacer (ITS) primers. The PCR product obtained was sequenced and submitted for Basic Local Alignment Search Tool (BLAST) at National Center for Biotechnological Information (NCBI). The analysis showed that this microorganism contained ITS sequences (highest identity with Leocosporidium sp.).

 

Introduction

Nowadays, the demand for industrial enzymes, particularly of microbial origin is greatly increased owing to their applications in a wide range of processes (Saxena et al., 1999). Microbial enzymes are often more useful than enzymes derived from plants or animals because of their inclusive significance such as great variety of catalytic activities available, high yields possible, ease of genetic manipulation, regular supply due to absence of seasonal fluctuations and rapid growth of microorganisms on inexpensive media (Hasan et al., 2006).

Cold-adapted microorganisms are potential sources of cold-active enzymes that can exhibit higher catalytic activity at low temperature (Lo Giudice et al., 2006; Gerday et al., 2000). Psychrophilic enzymes function effectively at cold temperatures with high rates of catalysis in comparison to the enzymes from mesophiles or thermophiles (Joseph et al., 2008). These enzymes might offer novel opportunities for biotechnological exploitation based on their high catalytic activity at low temperatures, low thermostability and unusual properties (Russell, 1998).

Lipases (triacylglycerol acylhydrolases, E.C 3.1.1.3) are one of the enzymes that have found a great number of biotechnological applications. These enzymes catalyze the hydrolysis of triacylglycerol to glycerol and free fatty acids at oil water interface (Kamini et al., 2000). The aim of this study was to screen the lipase activity and identify the psychrophilic microorganism isolated from Casey Station, Antarctica.

Methodology

Microorganism

Microorganism used in this research was selected from the laboratory collection, previously isolated from Casey Station, Antarctica. The particular microorganism was initially named as Isolate S.

Growth media and culture condition

Isolate S were grown in 100ml media in 250ml conical flask. The media contains 0.45% (w/v) NaCl, 1.25% (w/v) yeast extract, 1.00% (w/v) glucose and 0.10% (v/v) olive oil. The cultivation was carried out at 15°C with the agitation rate of 150rpm for 5 days.

Temperature dependence of the isolate

At the initial stage of this research, the growth of isolate S has been tested at various temperatures. The temperatures were 4°C, 27°C and 37°C.

Screening of lipase activity on plates

In order to detect the activity of lipase, isolate S was cultivated at 4°C on palm oil agar plates. The presence of lipase enzyme has been detected quantitatively based on the halo zone observed.

Identification of lipase-producing microorganism

Morphology analysis

Phase-contrast microscopy and Scanning Electron Microscopy (SEM) was carried out to characterize the morphology of isolate S.

Biochemical approach

Biochemical tests that have been done are based on its capability to ferment and assimilate sugar (glucose, lactose, galactose and mannose). In addition, assimilation of nitrate also has been tested.

Molecular approach

DNA extraction for Polymerase Chain Reaction (PCR)

Isolate S was cultivated in Nutrient Broth (NB). The cultivation was carried out at 15°C with the agitation rate of 150rpm for 5 days. Culture was centrifuged at 4000rpm for 20 minutes to get the pellet. Supernatant was discarded and 4ml 2x CTAB buffer (containing 100ml 1M Tris pH 8, 280ml 5M NaCl, 40ml 0.5M EDTA pH 8, 20g CTAB, 1g polyvinylpyrrolidone-40, ddH2O up to 1000ml and 0.2% of β-mercaptoethanol) was added. Sample was incubated at 55°C for 1 hour. Within incubation period, it was shaken for 2 times. After that, 50µl lysozyme (50mg/ml) was added and incubated at 37°C for 1 hour. Then, 50µl 10% SDS was added and incubated again at 37°C for 2 hours. 5ul RNase A (10mg/ml) was added and incubated at 37°C for 1 hours. Next, 2ml chloroform : isoamyl alcohol (24:1) was added and rotated with 200rpm at 37°C for 1 hour. It was followed by centrifugation at 4000rpm for 10 minutes. Then, the uppermost layer (supernatant) was transferred into new tube. 2ml chloroform : isoamyl alcohol (24:1) and 1/10 volume 10% CTAB were added and rotated overnight with 200rpm at 37°C. Again, the sample was centrifuged at 4000rpm for 10 minutes and the uppermost layer (supernatant) was transferred into a new tube. 1 volume of phenol : chloroform : isoamyl alcohol (25:24:1) were added and mixed by inverting for 20 times. The emulsion formed was centrifuged at 12000rpm for 1 minute. The upper layer was transferred into a new tube and 1 volume of phenol : chloroform : isoamyl alcohol (25:24:1) was added again. This step was repeated for 3 times. After that, 1 volume of chloroform was added and mixed by inverting for 20 times. The emulsion formed was centrifuged at 12000rpm for 1 minute. The upper layer was transferred into a new tube and 1 volume of chloroform was added again. This step was repeated for 3 times as well. 2 volume of 95% ethanol was added and mixed by inverting until the DNA precipitate. The emulsion formed was centrifuged at 12000rpm for 20 minutes. The supernatant was discarded and the pellet was air dried for 15 minutes. Finally, the DNA was resuspend in 30µl ddH2O. This extraction method was done according to Sambrook and Russell, 2001.

Internal Transcribed Spacer (ITS) – Polymerase Chain Reaction (PCR)

1µl of the template DNA solution was amplified in a 24µl reaction volume containing 5µl 5x colorless Go Taq flexi buffer, 2µl 25mM MgCl2, 0.5µl 25mM forward primer, 0.5µl 25mM reverse primer, 1µl 10mM dNTP, 13.5µl ddH2O and 0.5µl Go Taq DNA polymerase (5U/µl). The template was amplified in the thermo cycler by using the following steps: an initial denaturation at 94°C for 5 minutes, followed by 1 minute denaturation at 94°C, 1 minute and 30 seconds of annealing at 55°C and elongation at 72°C for 1 minute 30 seconds. After that, 35 cycles of denaturation at 94°C was carried out and followed by a final elongation for 10 minutes at 72°C. The cooling step was done at 10°C. The primers used were ITS1 (5´-TCCGTAGGTGAACCTGCGG-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) (White et al., 1990).

Agarose Gel Electrophoresis

The extracted DNA was checked by electrophoresis on 1% agarose gel with 0.5x TBE buffer. Lambda HindIII was used as DNA marker. After the electrophoresis, gel was stained with ethidium bromide and documented by the Gel Doc 2000 (Bio-Rad). Purification of PCR product was done by using QiAquick Gel Extraction Kit. Then, the purified PCR product obtained was sequenced and submitted for BLAST (Basic Local Alignment Search Tools) at NCBI (National Center for Biotechnological Information).

Lipase assay by titration

Activity of lipase was assayed by titration using oil emulsion (polyvinyl alcohol: olive oil; 3:1) as substrate (Arima et al., 1972). The assay medium consists of 5ml oil emulsion, 4ml phosphate buffer (0.2M, pH7) was incubated at 15°C for 10 minutes. After that, 1ml of enzyme was added and incubated for 30 minutes in 4°C with the agitation rate of 150rpm. The reaction was terminated by the addition of 20ml acetone:ethanol (1:1). Titration with 0.05M NaOH has been carried out to the end point of pH10. For the comparison, a control using heat-inactivated lipase enzyme has been used as control. One unit of enzyme activity was defined as µmoles of free fatty acids released per ml per minutes under the assay condition.

Protein determination

Protein content was determined by Bradford method (Bradford, 1976). 2 ml of Bradford reagent was added to the tubes containing 0.1 ml sample. Then, it was incubated for 10 minutes. Finally, absorptions were measured at 595nm. Standard curve by using bovine serum albumin (BSA) was previously prepared.

Results

Temperature dependence of the isolate

Growth of isolate S was observed only at 4°C, whereas no growths detected at 27°C and 37°C.

Figure 1
Figure 1: Growth of isolate S on Nutrient Agar (NA) at different temperatures after 14 days of incubation. (a) 4°C. (b) 27°C. (c) 37°C.

Figure 2

Figure 3

Screening of lipase activity on plates

Halo zone has been detected on palm oil agar plates (Figure 2). This result confirmed that this particular microorganism; isolate S could produce lipase.

Figure 4
Figure 2: The halo zone on the plate of palm oil.

Lipase assay by titration and protein determination by Bardford method

The activity of lipase investigated was 1.33 U/ml with the protein amount was 0.494mg/ml.

Identification of lipase-producing microorganism

Morphology analysis

Figure 5
Figure 3: Phase contrast microscopy observation (magnification: 40000).

Figure 6
Figure 4: Scanning Electron Microscopy (SEM) observation of isolate S.

Biochemical approach

Figure 7
Table 1: Biochemical tests for the identification of isolate S (Notes: (+) positive result; (─) negative result.

Molecular approach

The analysis showed that isolate S contained ITS sequences (100% identity to Leucosporidium sp.). Figure 5 shows the extracted gDNA while Figure 6 shows the PCR and purified PCR product.

Figure 8
Figure 5: gDNA of the particular psychrophlic microorganism on the agarose gel (0.7%). The exact size of the genomic was unknown but the large band in the 23kb region. Legends: M: Lambda HindIII / DNA Marker; 1: gDNA of isolate S.

Figure 9
Figure 6: The PCR and purified PCR product using ITS forward and reverse primers on the agarose gel (1.0%). Legends: M: 1kb DNA Ladder; 1: PCR product; 2: Purified PCR product.

Discussion

This study was conducted initially with the temperature study. At 4°C, isolate S was growing whereas at 27°C and 37°C, there was no growth at all. These results suggested the confirmation that isolate S is a psychrophilic microorganism. To detect the lipase enzyme, agar media that contain substrate (palm oil) has been prepared. The halo zone observed confirmed the production of lipase enzyme by isolate S.

At the first stage of identification, morphology analysis has been done. Under microscopic observation, both phase-contrast and Scanning Electron Microscope (SEM) showed rod cocci with budding structure. This observation suggested that isolate S is a psychrophilic yeast. The identification was then continued by biochemical approach. In addition to morphology analysis, biochemical tests become most important part to identify yeast. These tests showed the capability of isolate S to ferment and assimilate sugar as a source of carbon. Assimilation of nitrate also has been carried out. All the results of the biochemical tests have been tabulated in Table 1. For the molecular approach, analysis for Internal Transcribed Spacer (ITS) of isolate S revealed 100% sequence identity to Leucosporidium sp. Lipase activity determined by titration method 1.33U/ml with 0.494mg/ml of protein content.

Conclusion

In conclusion, activity of lipase enzyme from psychrophilic microorganism has been detected and screened in this study. The morphology characteristics of this particular microorganism were successfully analyzed.

Acknowledgement

This research was supported by Research University Postgraduate Research Grant Scheme, Universiti Sains Malaysia, Universiti Pendidikan Sultan Idris and Ministry of Higher Education, Malaysia.

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Author Information

Fatimah Azzahra Ahmad Rashid
School of Biological Sciences, Universiti Sains Malaysia

Rashidah Abdul Rahim
School of Biological Sciences, Universiti Sains Malaysia

Darah Ibrahim
Industrial Biotechnology Research Laboratory, Universiti Sains Malaysia

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