Lipase production from Enterobacter Sp.

 

R. Kamalambigeswari*, S. Sharmila, E. Kowsalya, S. Selva Janani, V. Deva, L. Jeyanthi Rebecca

Department of Industrial Biotechnology, Bharath Institute of Higher Education and Research, Selaiyur,

Chennai-600073, India.

 

ABSTRACT:

Lipase (triacylglycerol acyl hydrolase) hydrolases the triglycerides to fatty acids and glycerol and have the capacity of catalyzes the reverse reaction forming glycerides from glycerol and fatty acids. These are the group of water-soluble enzymes, which exhibit the ability of acting at the interface between aqueous and organic phases. These enzymes find numerous applications in the industrial sector to provide regio selective molecules of commercial interest. Enterobacter is a genus of a common gram-negative, facultative anaerobic, rod-shaped, non-spore-forming bacteria belonging to the family Enterobacteriaceae. Bacteria was isolated from wet soil through serial dilution method and grown on Luria Broth medium. The pure culture was isolated through streak plating. The bacterium was characterized and presumed to be Enterobacter after several biochemical tests. The seed culture was prepared using the isolated pure bacterium. 10% of seed culture is inoculated in the production medium containing olive oil (which acts as an inducer for lipase). Quantitative assay of an enzyme is as to obtain its enzymatic activity, was performed through titrimetric analysis. The activity of crude enzyme was identified as 250 units/ml. The partial purification of crude enzyme was performed using ammonium sulphate. The partially purified enzyme found to have 380 units/ml after dialysis at 4°C.

 

 

INTRODUCTION:

The demand for microbial industrial enzymes has attracted much interest due to their novel and multifold applications in a wide variety of processes. Until the 1960s, the world market of microbial enzymes was only a few million dollars business; but later, the market was broadened up enormously. At present, more than 200 microbial enzymes are used commercially and approximately 20 types are produced on truly industrial scale^1,2. Lipase (triacylglycerol acyl hydrolase) hydrolases the triglycerides to fatty acids and glycerol and have the capacity of catalyses’ the reverse reaction forming glycerides from glycerol and fatty acids. These are the group of water-soluble enzymes, which exhibit the ability of acting at the interface between aqueous and organic phases.

 

The interest in lipase has grown over last few years due to their excellent catalytic properties such as esterification, interesterification, transesterification, acetolysis, amino lysis, and may show some enantioselective properties. Unlike other enzymes, oil-water or air-water interfaces activate the lipases³.

 

Lipases have cosmic presence in nature such as soil, industrial effluents, oil contaminated areas etc. and originated mostly from plants, animals, fungi, yeast and bacteria⁴. Microbial lipases attracted more attention owing its easy isolation, ease of genetic manipulation, high yield possible, systematic amount due to its absence in seasonal variations and quick growth of micro-organisms from low-priced media. Microbial lipases have biotechnological applications. This research was carried out for the production of lipase from bacteria through partial purification.

 

 

 

MATERIALS AND METHODS:

Screening and isolation of bacterial isolate:

10.0gm of soil sample was suspended in 90ml sterile saline water, serial 10-fold dilutions of that suspensions were made and then 0.1ml of saline water from the 10̵ ² dilutions was spread evenly on the surface of NA (Nutrient agar) medium. The plate was then incubated at RT⁵. After 24-48hrs of incubation, the microbial colonies, which appeared on nutrient agar plates, were purified and subjected to qualitative screening for identification of lipase producing micro-organism on tributyrin agar (TBA). These plates were incubated for 24 to 48 hours at 37℃. The selective bacterial white colonies were transferred to fresh Luria Bertani agar and incubated at room temperature (28°C±1) for sub culturing. Then the absolute cultures were identified, maintained on the LB broth plate or slants at 4°C. The identified cultures were gone through gram staining and used for the production. Pure culture of the above mentioned sp., was prepared by quadrant streaking.

 

BIOCHEMICAL TEST OF BACTERIA:

The bacterium was characterized and presumed to be Enterobacter after several biochemical test such as indole production test, methyl red test, citrate utilization test, catalase test and starch hydrolysis⁶.

 

CITRATE UTILIZATION TEST:

Streaked the slant back and forth with a light inoculum picked from the center of a well-isolated colony.

Incubated aerobically at 35 to 37°C for up to 7 days. Observed a color change from green to blue along the slant.

 

CATALASE TEST:

Used a loop or sterile wooden stick to transfer a small amount of colony growth in the surface of a clean, dry glass slide. Placed a drop of 3% H₂O₂ in the glass slide. Observed for the evolution of oxygen bubbles.

 

STARCH HYDROLYSIS:

Prepared agar media with 1% starch. Inoculate the starch agar plate with the given organism (E. coli and Bacillus in separate plate) using inoculating loop making small central line or streak in the plate. Incubated the plates at 37 °C for 24 hours. Flooded the plates with gram’s iodine solution. Observed the plates after few minutes. A clear zone around the inoculated area indicates starch hydrolysis; positive test.

 

Production medium for lipase production:

Seed culture was prepared in 50 ml medium containing (g/L) peptone 10g; yeast extract 5g; NaCl 10g; the pH was adjusted to 6.0 and autoclaved at 121°C for 20 mins. 10% inoculum was added and the flasks were incubated for 48 hrs at 28°C⁷. About 10% of seed culture has been transferred to the fermentation medium containing 3% yeast extract; 3% sucrose, Na₂SO₄ 0.1g; KH₂PO₄ 0.5g; MgSO₄ 0.1g; olive oil 1%. The final pH was adjusted to 6.5 and sterilized at 121°C for 15min. The culture medium was then incubated for 5 days at RT on a rotary shaker at 120rpm.

 

TITRATION METHOD:

Reagents:

A.   200 mM Tris HCl Buffer, pH 7.2 at 37°C (Prepared 100 ml in deionized water using Trizma Base. Adjusted to pH 7.2.)

B.   Olive Oil Substrate Solution (Olive Oil) (Used Sigma Lipase Substrate.)

C.   95% Ethanol (Nondenatured) (Prepared 50 ml in deionized water using 200 Proof USP Ethyl Alcohol, available from Quantum Chemical Company.)

D.   0.9% (w/v) phenolphthalein Indicator Solution (PH Indic)

E.    50 mM Sodium Hydroxide Solution-Standardized (NaOH) (Prepared 100 ml in deionized water using Sodium Hydroxide, Anhydrous)

F.    Lipase Enzyme Solution (Immediately before use, prepared a solution containing 1000 units/ml of Lipase in cold deionized water.)

 

Mix by swirling and incubate at 37°C for exactly 30 minutes. Immediately after starting the incubation, pipette (in milliliters) 1.00 ml of Reagent F (Enzyme Solution) into a 50 ml Erlenmeyer flask marked "Blank" and store at 0 - 4°C.

 

After 30 minutes transfer the Test solution to a 50 ml Erlenmeyer flask and the Blank solution to the 50 ml Erlenmeyer flask labeled "Blank

 

Mix by swirling and then add 4 drops of Reagent D (PH Indic) to both the Test and Blank solutions. Titrate each solution with Reagent E (NaOH) to a light blue color. Use a 25 ml burette with 0.1 ml graduations for the titration.

 

CALCULATIONS:

Units/ml enzyme = (v) (NaOH) (Molarity of NaOH) (1000) (2) (df) (1)

1000 = Conversion factor from milliequivalent to micro equivalent

2 = Time conversion factor from 30 minutes to 1 hour (Unit Definition)

df = Dilution factor

v = Volume (in milliliter) of enzyme used

Units/mg solid =   units/ml enzyme mg solid/ml enzyme

 

Units/mg protein =  units/ml enzyme

          mg protein/ml enzyme

 

 

PARTIAL PURIFICATION OF LIPASE FOLLOWED BY DIALYSIS:

Lipase purification was carried out at 4°C. The culture medium was centrifuged at 10000 rpm for 20 min to obtain crude enzyme, the supernatant fluid was subjected to precipitation with Ammonium Sulphate to 70%, 80%, 90% saturation and stirred for 2h. The precipitate was removed by centrifugation. Lipase activity both in the precipitate and supernatant was determined.

 

The precipitate was collected, dissolved in PBS buffer PH 7.2 and dialyzed against same buffer. The enzyme mixture was dialyzed using cellulose acetate membrane against PBS buffer. The enzyme was eluted with a linear gradient of 0.1M NaCl in the same buffer at a flow rate of 1 ml/min. The active fractions that contained lipase enzyme were dialyzed and assessed for enzyme activity content by titrimetric method. The resulting enzyme was utilized for the characterization of the extracellular lipase.

 

RESULTS AND DISCUSSION:

Screening and isolation of Enterobacter sp.:

1g of soil sample was taken from Bharath university, Selaiyur, Chennai and was screened for bacterial lipase enzyme producer. 5 different strains of bacteria were obtained and 2 isolates were found to be Enterobacter based on gram staining and confirmed by biochemical test (citrate utilization test, catalase test, starch hydrolysis test)⁸. These strains were screened for the lipase enzyme production using TBA, and BS1 was found to be the maximum producer of lipase and proceeded for further studies. Fig1 depicts the inoculum used and Fig 2 indicates the pure culture Enterobacter which produces lipase. Fig 3 and 4 depicts the seed and production medium employed for the study.

 

  

Fig.1  Inoculum

 

Fig.2 Pure culture

Fig.3 Seed medium                            Fig.4 Production culture

 

BIOCHEMICAL TEST:

CITRATE UTILIZATION TEST:

The citrate utilization test was performed to observe the presence of Enterobacter sp., Fig 5 indicates the medium before inoculation and Fig 6 depicts the medium after inoculation. Growth with color change from red to intense blue along the slant indicates presence of Enterobacter.

 

Positive :

Growth on the medium even without colour change will be considered as positive⁹. A colour change in the medium would be observed if the test organism produces acid or alkali during its growth. The usual colour change observed is blue (alkaline)

 

Fig.5 Before inoculation                    Fig.6 After inoculation

 

CATALASE TEST:

Catalase test can be used as an aid to the identification of. members of Enterobacteriaceae family. Catalase is a common enzyme found in nearly all living organisms exposed to oxygen (such as bacteria, plants, and animals). It catalyzes the decomposition of hydrogen peroxide to water and oxygen. Enterobacter sp., are catalase positive. Fig 7 indicates the presence of bubbles in the slide¹⁰.

 

 

 

 

 

POSITIVE REACTION:

Copious bubbles produce active bubbling

 

Fig.7 Positive reaction

 

STARCH HYDROLYSIS TEST:

The starch hydrolysis test was performed. After inoculation the plates were incubated for 24 hours at 37°C. Iodine, which changes color from a yellow-brown to blue-black in the presence of starch, was applied to the agar surface and allowed to stand for 10 minutes. The inoculated agar plate was observed with clear zone around the inoculated area. Fig 8 shows control plate and Fig 9 shows the inoculated plate with colour change¹¹.

    

    

Fig 8 Control plate                            Fig 9 Sample plate

 

TITRIMETRIC ASSAY OF LIPASE:

Titrimetric method is used for the enzymatic assay of lipase¹². From the medium 2ml is taken in eppendorf tube and centrifuged for 5mins in 5000rpm and the crude supernatant served as enzyme source. Fig 10 shows the end point of titrimetric method. Fig 11 depicts the dialysis set up used for partial purification. Fig 12 depicts the lipase assay end point after dialysis. After dialysis about 0.5 fold increase in enzyme activity was observed.

 

CALCULATION:

Blank value = 10

V= volume (in millimeter) of enzyme used= 12.5

Units/ml enzyme =

(v) (NaOH) (Molarity of NaOH) (1000) (2) (df) (1)

      

= (12.5-10) (0.05) (1000) (2)(1)                             (1)

                            = 250 units/ml.

 

Fig.10 End point of tritrimetric          Fig.11 Dialysis method

Assay

 

Fig.12 Assay of lipase after dialysis

 

CONCLUSION:

Optimization and downstream processing has to be carried out so as to commercially employ this enzyme for many of its application in various sector.

 

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Accepted on 23.05.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(9):4417-4420.