Biotechnological important lipase producing bacterium strain LP-II was isolated from local oil mill soil at Gangakhed (India). Lipase is an important industrially employed enzyme and used in trans-esterification of algae oil to biodiesel. LP-II is Gram positive log rods have showed growth at 40oC and pH 6.7. LP-II has utilized dextrose and fructose as carbon source and produced acid in the medium. LP-II lipase produced lipases 1.5 U/mL at 400C and 8.0 PH after 48 hours of incubation at 150 rpm. In addition, LP-II have showed positive result for gelatinase, caseinase oxidase and catalase tests at its optimum temperature and pH. Strain LP-II was identified as Aneurinibacillus aneurinilyticus using morphological, and biochemical methods. Identification made in former step was confirmed by 16SrRNAgene sequencing method as Aneurinibacillus aneurinilyticus strain LP-II (Accession no.: MF696161). LP-II lipases have an ability to work at moderate temperature and slightly high pH. Thus, lipase produced by LP-II strain may find application in various industrial processes and household chemicals
Keywords: 16S rRNA; Algae oil; Biodisel; Lipase; Tributyrin medium; Transesterification
Lipases (triacylglycerol acyl-hydrolases, EC3.1.1.3) are the hydrolysing enzyme that catalyses hydrolysis and synthesis of ester [1]. Lipases have applications in industries such as in dairy for hydrolysis of milk and fats, in detergent industry as an additive in washing powder and textile industries to increase fabric absorbency [2-5].
Additionally, Lipases are uses in transesterification reaction [6, 7], in pulp and paper industry [8], in the synthesis of biodiesel [9]. Commercially significant microbial lipases are produced by varieties of organisms [10-12] include bacteria, fungi, plants and animals.
However, microbial lipases are more interesting because their high yield using cost effective raw materials [13-15].
In the present study lipase producing bacterium was isolated from oil mill soil sample by algae oil enrichment technique and lipase production was estimated using qualitative and quantitative methods.
All laboratory aids, chemicals, media and solvent required for the experiments were purchased from S. D.
Fine Chemicals Ltd and HiMedia Pvt. Ltd. (India).
Soil sample was collected from oil mill at Gagakhed (MS), India using random composite sampling method [16, 17]. Soil was cleaned in laboratory. One gram of soil was measured and added to 250 mL Erlenmeyer flask containing 50 mL basal medium (ingredients/L: peptone 1.5 g, yeast extracts 0.5 g, algae oil 1 mL (v/v), pH 7.0.
o The medium was incubated at 30 C on cooling shaking incubator (HiMedia Pvt. Ltd.) at 150 rpm for 72hours [18].
Enriched medium was used to isolate liapse producing microorganism. Spread plate method was used to isolate lipase producer. 100 L enrichment broth culture was spread tributyrin agar plates containing 1% tributyrin. Inoculated plates were incubated at variable temperature o from 30 to 60 C for 24 to 48hours. Colonies showing zone of clearance was isolated on same agar medium and obtained as pure culture for further experiments. Lipase producers showing highest zone of clearance were chosen for further work [19, 20].
Isolated lipase producing bacterium was identified using morphological and biochemical methods. 16S rRNA gene sequencing methods was used for confirmation of identification made in former steps. Gram nature was determined. Isolated species were observed for utilization of carbon source (glucose, fructose, mannitol, xylose & mannose and their ability to produce lipase, oxidase, catalyse, urease, gelatinise, starch, nitrate reduction, IMViC [21, 22].
Lipolytic activity was estimated by a spectrophotometric assay using pNPP (para nitro phenyl palmitate) as a substrate. The reaction mixture consisted of 0.1 mL enzyme extract, 0.8 mL of 0.05 M Tris buffer (pH 7) and 0.1 mL of 0.01M of p-NPP dissolved in isopropanol. The reaction mixture was kept at 40°C in a water bath for 30 min. After 30 min. 0.25 mL of 0.1M Na CO was added 2 3 to terminate the reaction. The reaction mixture was centrifuged at 10000 rpm for 10 min. Optical density (O.D.) was determined at 410 nm. One unit of lipase activity was defined as the amount of enzyme which liberated 1 μmol of p-nitro phenol per min from p-nitro phenyl palmitate [23-25].
The optimum temperature for lipase was evaluated by using lipase activity with 2, 4- dinitrophenolpalmitate at o temperature ranged from 30-60 C at pH 7. The effect of pH (6-10) on enzyme activity was analysed by the spectroscopic assay using pNPP as substrate. pH was optimized at optimum temperature recorded in former step. Optimum enzyme activity was measured under standardized enzyme test conditions [26, 27].
Lipase producing bacterium was isolated form soil collected at oil mill in Gangakhed (India). Isolated species were obtained as pure culture and stored at low o temperature (4 C). Isolated species were observed for production of lipase enzyme. Bacterial species LP-II showed maximum production of lipase amongst isolated species. Therefore, it was selected for further experiment.
Morphological and biochemical methods (Table 1) were used to identify bacterium LP-II. As per the Baergy’s Manual of Systematic Bacteriology isolated bacterium LP-II was identified as Aneurinibacillus aneurinilyticus. 16S rRNA gene sequencing methods was used to confirm identification of bacteria made in former steps. The identified strain LP-II showed 99.45% similarity type strain Aneurinibacillus aneurinilyticus ATCC12856(T).
16S rRNA gene sequence was deposited in NCBI Gene Bank database under the accession number MF696161.
Qualitative plate test showed that algae oil hydrolysing lipase producing strain LP-II showed zone of clearance 30 mm (Figure 1). Similar types of results were reported by
Effect of temperature on lipase activity was carried out at o temperatures 30 to 60 C. Crude lipase showed maximum o activity at 40 C (1.82 U/mL). LP-II lipase showed o remarkable activity at 60 C (Figure 2). Many research groups worldwide have reported that lipase produced by Bacillus and Pseudomonas have optimum temperature o o 37 C to 45 C. Mutants of Bacillus species showed 1.358 U/mL lipase production [7, 14].
The effect of pH (6-10) on lipase was determined. It was recorded that lipase produced by LP-II showed maximum activity 1.93U/mL at pH 7.0. LP-II lipase showed considerable activity at pH 9 and pH 10 (Figure 3). Similar types of results were reported by in the case Bacillus sp. FH5h [14, 28]. It was recorded that Bacillus sp. FH5h showed activity up to pH 8-9 but there was significant drop in activity at pH 10 [29]. Therefore, LP-II lipase produced by Aneurinibacillus aneurinilyticus strain LP-II have advantages over the previously reported strains.
Lipase produced by LP-II showed considerable and maximum lipolytic activity than earlier reported strains such as Aneurinibacillus danicu NBRC 102444, Aneurinibacillus thermoaerophilus DSM 10154T (AB112726) produced lipase about 0.512U/mL. These reports confirms that Aneurinibacillus aneurinilyticus strain LP-II isolated by us have considerable industrial potential than the previously reported lipase producing bacteria.
Aneurinibacillus aneurinilyticus strain LP-II is capable of producing industrially significant lipase enzyme. LP-II o lipase can selectively hydrolyse algal lipids at 40 C and more stable at pH 8. This is prerequisite for the best enzyme to be used in the transesterification reaction, o which mostly carried above 40 C. LP-II produced by Aneurinibacillus aneurinilyticus strain LP-II may find applications many industries.
Authors have declared that there is no any conflicts of interest exist.
The author (s) are grateful to the Director, School of Life Sciences, Swami Ramanand Teerth Marathwada University (Nanded) and Head, Department of Microbiology Arts, commerce and Science College (Gangakhed) for their providing all necessarily facilities to carry out this research work.
This work was carried out by DC as a part of his Ph.D. thesis. CK and DC both the authors are involves in designing protocol, laboratory analysis and writing the manuscript. DC and CK have equally contributed in writing and proof reading of MS in this final form.