International Journal of Modern Engineering Research (IJMER)                 Vol.2, Issue.6, Nov-De...
International Journal of Modern Engineering Research (IJMER)             Vol.2, Issue.6, Nov-Dec. 2...
International Journal of Modern Engineering Research (IJMER)              Vol.2, Issue.6, Nov-Dec. ...
International Journal of Modern Engineering Research (IJMER)            Vol.2, Issue.6, Nov-Dec. 20...
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Optimization of Lipase Enzyme Activity Produced By Bacillus Amyloliquefaciens Isolated From Rock Lobster Panlirus Homarus


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Optimization of Lipase Enzyme Activity Produced By Bacillus Amyloliquefaciens Isolated From Rock Lobster Panlirus Homarus

  1. 1. International Journal of Modern Engineering Research (IJMER) Vol.2, Issue.6, Nov-Dec. 2012 pp-4231-4234 ISSN: 2249-6645 Optimization of Lipase Enzyme Activity Produced By Bacillus Amyloliquefaciens Isolated From Rock Lobster Panlirus Homarus T. Selvamohan1, V. Ramadas2 and T. A. Sathya3 1 P.G & Research Department of Zoology, R.D.Govt Arts College, Sivagangai – 630 561, Tamil Nadu, India 2,3 Department of Microbiology, Sivanthi Aditanar College, Pillayarpuram - 629 501, Nagercoil, Tamilnadu, IndiaAbstract: The production of extracellular lipase pharmaceuticals, and agrochemical industries. They are(triacylglycerol hydrolase) is a widely distributed also employed in organic chemical processing,phenomenon among eukaryotic and prokaryotic biosurfactant synthesis, nutrition and biomedical sciencesmicroorganisms. Microbial lipases have mostly been [5].employed in food and detergent industry for purposes such Most of the commercial lipases produced areas the ripening of cheese and an additive to laundry utilized for flavour development in dairy products anddetergent. Lipase producing organisms were isolated from processing of other foods, such as meat, vegetables, fruit,Rock Lobster (Panlirus homarus). Bacillus baked foods, milk products and beer. Lipases areamyloliquefaciens was found to be the highly active strain extensively used in dairy industry for the hydrolysis offor lipase enzyme production. The most suitable for lipase milk fat. The dairy industry uses lipases to modify theproduction is fish bone and the maximum amount of fatty acid and chain lengths, to enhance the flavours ofparanitrophenyl palmitate released is found to be 0.84 various cheeses. Lipases enzyme also accelerates cheeseµg/ml/mints at 48hrs of incubation. The optimum ripening and the lipolysis of butter, fat and cream [6]. Thetemperature and pH for the organism was found to be 47oC objective of this study was the production of lipase andand 9, the amount of paranitrophenyl palmitate released characterization of the enzyme with regards to its stabilitywas recorded as 0.89 µg/ml/min and 0.74 µg/ml/min for in relation to temperature, PH and the optimization of thevarious substrates such as Glycerol, Olive Oil, Coconut Oil temperature and PH conditions for obtaining higher lipaseand Fishbone. Lipase production was obtained at different activity.substrate concentration and the bacterial species yieldmaximum production of lipase and it was analysed by II. MATERIAL AND METHODSspectrophotometric method. 2.1 Collection of Sample: Lobster sample was collected in a sterile bagKeywords: Rock lobster (Panlirus homarus) – Bacillus from the fish market near Kanyakumari, Tamil Nadu,amyloliquefaciens , Optimization of Lipase enzyme with India and brought to the laboratory within 15 minutes.different substrates.. 2.2 Isolation of Intestinal Flora of Lobster: I. INTRODUCTION The intestine of the lobster was dissected out Lipases are enzymes capable of hydrolyzing the aseptically and homogenized with 0.089% of NaClester bonds of water-insoluble substrates at the interface solution. The homogenate was serially diluted and spreadbetween substrate and water. It is well known that the on sterilized nutrient agar plates and incubated at 370C forreaction is reversible and this enzyme can catalyse ester 24 hours. The colonies were counted for TVC (Totalsynthesis and transesterification since lipases can catalyse viable Count). The dominant colonies were isolated andnumerous different reactions they have been widely used in streaked in nutrient agar slants as master culture.industrial applications, such as in food, chemical,pharmaceutical and detergent industries [1]. Many 2.3 The Composition of Production Medium Used Formicroorganisms are known as good producers of Lipase Production:extracellular lipases [2]. The production medium was prepared for lipase Lipases occur widely in bacteria, yeast and fungi production by the Bacillus amyloliquefaciens in the[3]. Most of the lipase research focuses on the production of following composition.extracellular lipases through a wide variety ofmicroorganisms. Studies on the production of extracellular  Peptone - 3% (w/v)lipases with Bacillus have shown variations among  Yeast Extract - 1% (w/v)different strains. However, the requirement for lipid carbon  Sodium Chloride - 0.5% (w/v)source remains essential for enzyme production. Lipase  Olive Oil - 1% (w/v)producing organisms are isolated from Lobster (Panlirus  PH - 7homarus) and that the wild and cultured lobsters harbours a 2.4. Assay of Lipase Enzymesdiverse bacterial flora which includes the dominant generaslike Aeromonas, Pseudomonas, Bacillus, E.coli, Salmonella 2.4.1. Optimization of Fermentation Media:and Vibrio [4]. These organisms are having the ability to The fermentation media prepared was optimizedproduce enzymes. These enzymes are produced during the with following factors such as Incubation period, PH ,utilization of certain nutrients such as proteins, lipid and Temperature and Lipid Substrates.carbohydrate. Lipases have potential applications indetergent, oleo chemical, paper manufacturing, cosmetics, 4231 | Page
  2. 2. International Journal of Modern Engineering Research (IJMER) Vol.2, Issue.6, Nov-Dec. 2012 pp-4231-4234 ISSN: 2249-66452.4.1.1. Optimization by Incubation Period: The effect of lipase production by the Bacillus The organisms were subjected to different amyloliquefaciens (k1) with various substrates (glycerol,incubation periods such as 24, 48 and 72 hours using olive oil, coconut oil and fish bone) at differentproduction media. incubation periods (24hrs, 48hrs and 72hrs) were carried out and the results were indicated in Fig.1. The most2.4.1.2. Optimization by different PH suitable substrate for lipase production was identified as The microorganisms isolated from the rock lobster fish bone, and the maximum amount of paranitrophenylwere incubated in the production media containing different palmitate released was recorded as 0.84 µg/ml/min at 48PH varied from 5, 7 & 9. hrs of incubation period. The minimum amount of paranitrophenyl palmitate was released with olive oil was2.4.1.3. Optimization by different temperature recorded as 0.10 µg/ml/min at 48hrs of incubation time. The mircroorganism in the production media wasincubated at different temperature 270C, 370C, and 470C. Optimization by Lipid Substrate In order to optimize the enzyme lipase differentlipid substrates were used, such as, Glycerol, Olive oil,Coconut oil, Fish Bone and chicken intestine. Enzyme Assay Microbial Culture was assayed for lipase enzymeactivity using Spectrophotometric method and results wererecorded.2.5. Principle In the present investigation one unit (U) of lipaseactivity was defined as the amount of enzyme solutionliberating 1µmol of p-nitrophenol per minute under Fig.1 Effect of different substrates on lipase productionstandard assay conditions. by Bacillus amyloliquefaciens (K1)2.6. Reagents Required The effect of pH on lipase production at various To carry out the study following reagents were incubation time intervals have been carried out and therequired such as, Reaction Buffer (500µl) (It contains the results were indicated in Fig. 2. At 48hrs of incubation atmixture of 50 mM Tris Hcl (PH 9.0) and Triton X-100), pH 9, the Bacillus amyloliquefaciens (k1) showed the2-Propanol (20µl) p-nitrophenylpalmitate (PNPP) maximum production lipase and the amount of The spectrophotometric method, using P- paranitrophenyl palmitate released was recorded as 0.89nitrophenyl Palmitate as substrate was applied for rapid and µg/ml/min, whereas the minimum production was notedroutine measurement of the lipase activity. Enzyme or at pH 5 at 24hrs of incubation period and the amount ofblank solution (480µl) was added to the reaction buffer paranitrophenyl palmitate released was recorded as 0.31(500µl) which has 50mM Tris-HCI (PH 9.0) with variable µg/ml/min.concentrations of Triton x-100. The content was incubatedat 250C for 5 minute and ten millimolar PNPP in 2-propanol(20µl) was added to the enzyme buffer solution and shakenwell. The mixture was emulsified for 2 minutes at 500C.The progress of the reaction was followed by monitoringthe change in the absorbance at 400nm over a period of 5minutes at 500C using Perkin-Ealmer Spectrophotometer.The molar extinction coefficient of P-nitrophenol(E=16.900M-1 Cm-1) was estimated from the absorbancemeasured at 400 nm of standard solutions of PNP. III. RESULTS The enzymatic activity was assayed with differentsubstrates like Glycerol, Olive oil, Coconut oil and Fishbone. The production of lipase was observed at varying pH(5,7 & 9), temperature (270C, 370C & 470C), substrateconcentration (0.1, 0.2, 0.3, and 0.4) and at different timeintervals (24, 48, and 72 hrs). Lipase producing organismswas isolated from rock lobster and was noted as k1.It wasidentified as lipase positive organism by comparing withBergey’s Manual of Determinative Bacteriology and this Fig.2 Effect of different pH on lipas production by Bacillus amyloliquefaciens (K1)organism was the highly active strain for lipase production. 4232 | Page
  3. 3. International Journal of Modern Engineering Research (IJMER) Vol.2, Issue.6, Nov-Dec. 2012 pp-4231-4234 ISSN: 2249-6645 The effect of different temperatures on lipase palmitate released was recorded as 0.43 µg/ml/min.Inproduction at various time intervals was shown in Fig.3. At contrast, in the present study, the PNPP-hydrolysing72hrs of incubation at 470C, the Bacillus amyloliquefaciens activity had a clear temperature which is “optimum” at(k1) showed a maximum production and the amount of 37oC even though the enzyme was fully stable up to 80oCparanitrophenyl palmitate released recorded wsa 0.74 for at least 15 min, which is much longer than theµg/ml/min, whereas the minimum production observed at duration of the activity assay [9].370C at 24hrs and the amount of paranitrophenyl palmitate The overall properties showed by Bacillusreleased was recorded as 0.43 µg/ml/min. amyloliquefaciens (k1) indicated that the cloned lipase is a mesophilic enzyme acting on a broad range of pH that becomes inactivated only by a limited number of lipase inhibitors. In the present study, pH was varied using different substrates concentration [10]. In connection with their biochemical properties, both lipolytic enzymes display different substrate specificities [11]. In the present study the substrate specificities are done using Glycerol, Coconut Oil, Olive Oil, and Fish Bone as substrates. The cell produces lipases in order to obtain the energy required to form new cells from the available carbon sources and at the same time it does a normal metabolic activity irrespective of growth [12]. Comparing results of lipolytic activities with those of other research studies is difficult owing to the different methodologies used by different groups. However, comparing the results obtained in this work with those reported using the same methodology and reaction substrate. Glucose, Olive Oil, Peptone and Fecl3 6H2O were found to have more significance on lipase production byFig.3 Effect of different temperature on lipase produnction Candida rugosa.[13]. In the present study glucose, olive by Bacillus amyloliquefaciens (K1) oil, coconut oil and fish bone are used as substrates and among that fish bone showed the highest enzymatic production. IV. DISCUSSION Lobsters harbors a diverse bacterial flora and mostof the microorganisms produce enzymes.Lipase producing Referencesmicroorganism isolated from the lobster was identified as 1. Harwood, J. 1989. The vessatility of lipase forBacillus amyloliquefaciens (k1). The lipase enzyme activity Industrial uses. Trends Biochem.S11.14: 12-126.was assayed with varying pH , temperature and susbstrates 2. Ratledge, C., Tan, K.M. 1990. Oils and fatsat different time intervals In the present study the effect of Production degradation and utilizationby yeasts. In:lipase enzyme activity with the fish bone as a substrate Verachter, H., Mot, D. (Eds), Yeasts, Biotechnologyshowed the maximum production (0.84 µg/ml/min) at 48hrs and Biocatalysis, Marcel Dekker, New York.of incubation and the minimum amount of paranitrophenyl PP.223-253.palmitate released in olive oil as a substrate was recorded as 3. [3] Jaeger, K.E., B.W.Dijkstra, M.T. Reetz. 2000.0.10 µg/ml/min at 48hrs of incubation. Among the various Bacterial Biocatalysts Molecular biology, three –substrates, gingili oil cake and wheat bran were found to be dimensional structures, and biotechnologicalthe best substrates [7]. Whereas in the present investigation, application. Annu-Rev-Microbiol. 53: 315-351.the fish bone used as substrate was found to be the best with 4. Oxley, A.P.A., W.Shipton, Owen. 2002. Bactericala high enzyme production. flora from the gut of wild and cultured banasa At 48hrs of incubation with pH 9, the Bacillus prawn. Applied Microbiology. 93: 214-218.amyloliquefaciens (k1) showed the maximum production 5. Pandey, A., S. Benjamin, P. Nigam, C.R. Soccoland the amount of paranitrophenyl palmitate released was and N. Krieger 1999. Realm of microbial lipases inrecorded was 0.89 µg/ml/min and the minimum production biotechnology. Biotechnol Appl. 29: pH 5 at 24hrs the amount of paranitrophenyl palmitate 6. Sharma, R., Y.Chisti and U.Banerjie. 2001.released was 0.31 µg/ml/min.The lipase enzyme was active Production, Purification, Characterization andin the range of pH 7.5-9.5, and the maximal activity was applications of lipase. Biotechnol. Adv., 19: 627-662observed at pH 9.0. It is a characteristics that the most 7. Kamini, N.R.., J.G.S. Mala and R. Puvanakrishnan.microbial lipases, the optimum pH falls on the alkaline side. 1998. Lipase production from Aspergillus niger byIn present study the similar trend was observed that pH was solid-state fermentation using gingelly oil cake.optimum and it falls on the alkaline side [8]. Process Biohemistry. 33: 505-511. At 72hrs of incubation at 470C the Bacillus 8. Kambourova, M.N., R. Kirilova, A. Mandiva, A.amyloliquefaciens (k1) showed the maximum production Derekora 2003. Purification and properties ofand the amount of paranitrophenyl palmitate released was thermostable lipase from a thermophilic Bacillusobserved as 0.74 µg/ml/min and the minimum production stearothermophilus MCF. Journal of Molecularnoted at 370C at 24hrs, and the amount of paranitrophenyl Catalysis B. Enzymatic. 22: 307-313. 4233 | Page
  4. 4. International Journal of Modern Engineering Research (IJMER) Vol.2, Issue.6, Nov-Dec. 2012 pp-4231-4234 ISSN: 2249-66459. Fitter N, R.Hermann, N.A. Dencher, A. Blume, expression, biochemical properties and three T.Hauss. 2001. Preliminary characterisation of a dimensional structure. FEBS Lett. 502: 89-92. lipolytic enzymes. Journal of Molecular catlysis. 40: 12. Shu, and Lin. 2006. Purification and Partial 10723 characterization of a lipase from Antrodia10. Prim, N., Blanco, A. Martinez, J., Pastor, F.I.J and cinnamomea. Process Biochem. 41: 734-738. Diaz P. 2000. A gene coding for a cell- bound 13. Aravindan Rajendran., Anbumathi Palanisamy and esterase from Paenibacillus sp.Dpt of Microbiology. Viruthagiri Thangavelu. 2008. Evaluation of 43: 237-240 medium components by placket-Burman statistical11. Eggert T. Van Pouderoyen G, Dijkstra BW, Jaegerk. design for lipase production by Candida rugosa I 2001. Lipolytic enzymes Lip A and Lip B from and kinetic modeling. Chinese Journal of Bacillus subtilis differ in regulation of gene Biotechnoogy. 24(3): 436-444. 4234 | Page