CLONING AND EXPRESSION OF A LIPASE GENE FROM PSEUDOMONAS AERUGINOSA INTO E.coli

768  CLONING AND EXPRESSION OF A LIPASE GENE FROM PSEUDOMONAS AERUGINOSA INTO E.coli J. M. Auda M. I. Khalifa Assist. Prof. Researcher Food sciences Dept., College of Agriculture, University of Baghdad, Iraq Mustafa.11m11@gmail.com ABSTRACT Fifteen local isolates of Pseudomonas were obtained from several sources such as soil, water and some high-fat foods (Meat, olives, coconuts, etc.). The ability of isolates to produce lipase was measured by the size of clear zone on Tween 20 solid medium and by measuring the enzymatic activity and specific activity. Isolate M3 (as named in this study) was found to be the most efficient for the production of the lipase with enzymatic activity reached 56.6 U/ml and specific activity of 305.94 U/mg. This isolate was identified through genetic analysis of the 16S rRNA gene. and it was shown that the isolate M3 belongs to Pseudomonas aeruginosa with 99% similarity. The DNA of isolate M3 was extracted and lipase gene was amplified through PCR technique, then purified and cloned into E.coli DH5α cells first using pTG19-T plasmid, and expressed in E.coli Bl21 with expression vector pet-28a. The activity of lipase from transformed E.coli Bl21 was 196.6 U/ml and the specific activity 618.2 U/mg.


‫العراقية‬ ‫الزراعية‬ ‫العلوم‬ ‫مجلة‬
. Lipases are a large group of industrial enzymes, which used in various industrial like food pharmaceuticals, detergents, beverages, cosmetics, degreasing formulations, biofuel, and paper (14). In the field of food industry, Lipases has been widely used in improving the flavor by producing esters of short fatty acids with alcohols. It has been used in the development of flavor in the dairy industry such as cheese, butter, margarine and others. Lipases were also used to produce low-fat meat (lean meat) by removing fat from meat and fish products. Moreover, in bakery lipases can improve dough rheology, increase in volume, dough strength and stability (2, 3. 12) Lipases are produced by many organisms like fungi, bacteria, yeast, animal, and plant (19). Microbes are an excellent source of lipases compared to other organisms because of their fast growth, small space for cultivation, with standing various temperatures and easy genetic manipulation to generate high yields desirable for diverse applications (18). In the past few years, recombinant DNA technology has allowed scientists to produce a large number of varied proteins in microorganisms, that were unavailable, relatively expensive, or difficult to produce in large quantity (5). The production of proteins using recombinant techniques is exponentially increasing as the demand increased (1). Escherichia coli is the most expression system that used for the production of recombinant proteins due to its inexpensive medium, short generation time, well-known genetics, easy genetic manipulation and the availability of a large number of cloning vectors . All these advantages enable E. coli to offer a rapid, high yield, and economical production of recombinant proteins (6,15,16). The present study aimed to find high lipase production Pseudomonas isolate and cloning the gene into E.coli.

MATERIALS AND METHODS Collection of samples and identification for lipase producing isolates:
Bacterial isolates were obtained from various sources such as soil, water and some fatty foods (meat, olive fruits, coconut fruits, etc.) and placed in sterilized plastic tubes. After the serial dilution were done, 0.1 ml was transferred to Pseudomonas agar plates and incubated at 37C for 24h and lipase producer colonies were picked up to a new culture.

Primary screening for lipase producing isolates
The ability of isolates to lipase production was measured by grown the bacterial colonies on the Tween 20 agar and incubated at 37C˚ for 24h. Tween 20 was used in the medium as a sole source of carbon (10). The ability of the bacterial isolates to produced lipase was scanned by measuring the diameters of the clear zones formed around the grown colonies. Lipase production Lipase production medium was prepared from (Arabic gum, 1% olive oil 1%, 0.5% sodium chloride, 1% pipton) according to (2). The pH of the medium was adjusted to 7.0. 100 mL of medium was added to 250 mL Erlenmeyer flasks incubated with a volume of (1×10 8 cell) of inoculum culture. The inoculated flasks were incubated at 37C on a incubator shaker at 200 rpm for 48 h. The supernatant was used as crude enzyme Lipase assay (Secondary screening) Lipase activity was measured by using modified titrimetric method as described by (10). 1 ml of crude enzyme solution was added to the reaction mixture containing 10 ml of 10% homogenized olive oil in 10% arabic gum, 2 ml of 0.6% CaCl2 solution and 5 ml of 0.2 mol/L Phosphate buffer, pH 7.0. The enzyme -substrate was incubated on an orbital shaker at 150 rpm at 37ºC for 30min. The reaction was stopped by adding 20 ml ethanolacetone (1:1). The free fatty acids were titrated with 0.1 mol/L NaOH using phenolphthalein as indicator. The reaction mixture without the enzyme was titrated in the same way and used as a blank. The lipase activity was calculated using a particular formula Lipase activity = Vol. of NaOH (mL) × Molarity of NaoH × 1000 × df/ Vol. of Lipase (mL) × Reaction Time (min).
One unit of lipase activity was defined as the amount of enzyme that liberated 1µmol fatty acid per minute under assay conditions Isolate identification The isolate that produced the highest enzym was identified by analysis of the 16S rRNA gene. The genetic identification was performed by extract the bacterial DNA using Geneaid DNA extraction kit according to the manufacturer's recommendation, after that, the 16S rRNA was amplified by PCR technique using primers designed for this purpose, Forward (5′-GACGGGTGAGTAATGCCTA-3′), Reverse (5′-CACTGGTGTTCCTTCCTATA-3′), as described by (6) and PCR premix. The reaction mixture (Table1) was added to the eppendorf tube and placed in the PCR thermocycler, which was programmed as shown in Table2. The PCR product was migrated in 1% agarose gel along with 2 µl of 2000 bp DNA ladder for 45 min at 90V, stained with ethidium bromide, then agarose gel electrophoresis was visualized by UVtransilluminator. After that, the amplified produt was send to Macrogen Korean Company to analyze of the 16S rRNA nucleotides sequence.  (Table.3) was used for amplified. The PCR machine was programmed as shown in (Table.4

Insertion of lipase gene into pTG19-T vector
The lipase gene was extracted and purified from agarose gel using Gel/PCR DNA extraction kit from Geneaid. The gene was ligated with pTG19-T vector using T4 DNA ligase. the ligated mix was prepared according to the manufacturer's instructions as shown in Table.5

Transformation of E.coli DH5α cells
The cloned pTG19-T vectors were inserted into E.coli DH5α competent cells using TA cloning kit. The transformed cells were spread on LB (Luria Bertani) plates containing 50μg/ml of ampicillin and 80μg of each X-Gal 20mg/ml and 100mM IPTG and spread on surface, and then incubated overnight at 37 °C.

Lipase gene ligation to pET-28a(+) Expression Vector
The pTG19-T cloned vectors were extracted from positive E.coli DH5α cells (white colonies) by BiONEER Plasmid Extraction kit and according to the kit instructions. Lipase gene was restricted from the pTG19-T vector using Bam HI restriction endonucleases and purified by migrated in 1% Agarose gel and extracted with gel DNA extraction kit. The extracted DNA fragments were ligated with Bam HI restriction site in pET-28a (+) expression vector usingT4 DNA ligase with the same ligated mix that mentioned in (Table.5).

RESULTS AND DISSCUSSION Isolating and identifying of lipase production isolate
From 15 isolates obtained in this study, it was found that the isolate M3 had highest activity 56.6 U/ml .This one identified by genetic analysis using the 16S rRNA gene, and the results of electrophoresis showed one band with ( fig.1), which is confirmed the amplification of the 16S rRNA gene. The results of sequencing was analyzed using Blast program from NCBI, and it was found that the gene has size of 637 bp and the isolate belongs to Pseudomonas aeruginosa with 99% similarity (Table.6).  .2). The pTG19-T vector was extracted from transformed E. coli DH5ɑ, digested with the restriction endonuclease BamHI, and run on 1% agarose gel. The result showed two bands one with size of ≈2900 bp, which represent the vector and the other ≈1000 bp, which belongs to the lipase gene. These result confirmed insertion of lipase gene to E. coli DH5ɑ (fig.3).