Yasmin C. Attar*, Sayali Randive, Sadiccha Patil and Mrudula M. Bendigeri

Department of Microbiology, Rajaram College, Vidyanagar, Kolhapur - 416 004, India

Abstract

The biosurfactant producing Acinetobacter sp. YMP which showed vegetable oil degradation potential on MSM was studied for lipase production. The production of lipase was confirmed on fat agar & lipase activity was measured by titrimetric method. The effect of environmental factors (agitation, time span , substrate concentration , pH & temperature ) on lipase production was studied by using Acinetobacter sp. YMP. The enzyme was further purified by Ammonium sulphate (60%) precipitation technique. The maximum lipase production by Acinetobacter sp. YMP was found to be within 120 hours at 450 rpm, 3.0 pH , 27° C temperature & at 2% substrate concentration. This species was also able to show upto 8 % oil degradation potential on MSM broth & agar.

Keywords

Biosurfactant; MSM (Mineral salt medium); Lipase

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Copy the following to cite this article: Attar Y. C, Randive S, Patil S, Bendigeri M. M. Study of Lipase Production by Acinetobacter sp. YMP. Biomed Pharmacol J 2012;5(2)

Copy the following to cite this URL: Attar Y. C, Randive S, Patil S, Bendigeri M. M. Study of Lipase Production by Acinetobacter sp. YMP. Biomed Pharmacol J 2012;5(2). Available from: http://biomedpharmajournal.org/?p=2568

Introduction

The  lipases  (triglycerolacylhydrolases E. C. 3.1.1.3)  are  one  of  the  most important  biocatalysts  catalysing  the  hydrolysis  of  triglycerides  to  glycerol  &  fatty   acids  &  can  carry  out  novel  reactions  in  both  aqueous  and  non  aqueous  media.¹ Among  lipases  of  plants,  animals  and  microorganisms²,  the  microbial  lipases  find immense  application,  this  is  because  microbes  can  be  easily  cultivated  and  their lipases  can  catalyse  a  wide  variety  of  hydrolytic  and  synthetic  reactions³ .Lipases are  useful  in  a  variety  of  biotechnological  fields  such  as  food  and  dairy  industry (cheese  ripening,  flavour  development),  paper  manufacturing , detergent formulation,  pharmaceutical  preparations  (naproxen,  ibuprofen),  agrochemicals industry  (insecticides,  pesticides)  and  oil  processing  chemical  industries  (fat  and  oil hydrolysis,  biosurfactant  synthesis)⁴  &  many  newer  areas.   Each  type  of  application requires  unique  properties  with  respect  to  specificity ,  stability,  temperature  & pH  dependance.  Many microorganisms like bacteria, fungi, yeast produce extracellular⁵ lipases.  Among  these ,  the  lipase  produced  by  biosurfactant  producing  organisms  shows  high  potential  to  degrade  the  hydrocarbons,  oils  and  fats  than   the  lipase produced  by  ordinary  microorganisms.  In  addition ,  they  show  stability  towards external  environmental  conditions  like  pH,  temperature,  substrate  concentration etc.  So  we  have  studied   production,  purification  & standardisation  of  lipase  by biosurfactant  producing  organism  Acinetobacter  sp. YMP.

Materials And  Methods

Isolation  of  organism                                                                                                        

Biosurfactant  producing  Acinetobacter  species  YMP  was  isolated  from oily  waste  collected  from   Farsan  industry  located  at  Unchgaon ,  Kolhapur . It’s cultural  characteristics  were  studied  &  the  species  was  confirmed  by  16 S  r RNA  sequencing.

Detection  and  confirmation  of  enzyme  production  of  Acinetobacter  sp. YMP .

A  loopful  of  suspension  of  Acinetobacter   species  YMP   was  aseptically  streaked     on  sterile  egg  yolk  agar  medium  by  four  quadrant  streaking  method.  The  plates were  incubated  at  room  temperature  for  24-48  hours.  For  confirmation  of  lipase activity, the  organism  was  streaked  on  fat  (butter)  agar  by  four  quadrant  streaking   method  &  the  plates  were  incubated  at  room  temperature  for  24-48  hours.

Study  of  enzyme  lipase  of  Acinetobacter  sp.  YMP 

The  selected  isolate  was   inoculated  in  100ml  Medium A⁶  with  1% groundnut  oil  concentration  [(gm/lit)  starch:  20;   peptone: 20;   NH₄CI : 3.8;     MgSO₄: 1;   K₂HPO4 : 5;   groundnut   Oil:1%  at  pH=7]  and  incubated  for  24-72  hours on  rotary  shaker  at  450  rpm  at  room  temperature.

Then  the  cell  free  broth  was  obtained  by  centrifugation  at  3500 rpm for  30 min  and  the  supernatant   was  used  as  the  crude  enzyme  for  assay  of  lipase.  The  lipase  activity  was  studied  by  titrimetric  method ⁷ described  by  Umamaheshwari  at regular  intervals  of  24,  48  and 72  hours.

Study  of  effect  of  environmental  factors  on  lipase  production by  Acinetobacter  sp.  YMP 

Various  environmental  factors   affecting   lipase  production. ( e. g. agitation ,  time span ,  substrate  concentration,   p^H  &  temperature.)   were  studied

Effect of   agitation   on   lipase   production  :

The  isolate  was  inoculated  in  medium  A  &  was  incubated  at  static condition &  on  rotary  shaker  at  450  rpm  at  room  temperature  for  144  hours.  The yield   of  lipase  was  determined  by  titrimetric  method  at  regular  intervals  of  24,  48 , 72,  96, 120  & 144  hours.

Optimization   of   substrate   concentration   for   lipase  production :

The  isolate  was  inoculated  in  medium  A  with  increasing  substrate concentrations  (groundnut  oil)  0.5%,  1%,  1.5%,  2%,   2.5%  on  rotary  shaker  at  450 rpm  for  120  hours.  The  optimum   substrate  concentration   was  determined  by titrimetric   method.

Effect   of   pH  on   the   lipase   production :

The  isolate  was  inoculated  in  medium  A  at  different  p^H  such  as  p^H =1, 2,  3,  5,  7,  9  & 11  &  incubated  on  rotary  shaker  at  4500  rpm  for  120  hours  & the  maximum  yield  was  determined  by  titrimetric  method.

Effect   of    temperature   on   the   lipase   production :

The  isolate  was  inoculated  in  medium  A  at  different  temperatures such  as  10°C,  27°C,  37°C,  55°C  &  incubated  on  rotary  shaker  for  120  hours  &  the maximum  yield  was  determined  by  titrimetric  method.

Purification  of  lipase  enzyme  produced  by  Acinetobacter  sp. YMP  by  precipitation  method.

The  organism  was  inoculated  in  medium  A  with 2%  oil  concentration at  pH  3.0  and  Temperature  27°C  on  rotary  shaker  at  450  rpm  for   120  hours. Then  equal  amount  of  broth  was  added  with  increasing  concentration  of ammonium  sulphate  (10%, 20%….70%)  and  enzyme activity  was  detected  by titrimetric  method.

Result and Discussion

The  enzyme  production of  the  isolate  was  detected  by  observing  the  zone  of  opalascence   around the  colonies  on  egg  yolk  agar  plate  &  further  confirmed  by  observing  transluscent  zone  around  the  colonies   on  fat (butter)  agar  plates.

We  studied  the  effect  of  various  environmental  factors  (time  span,  agitation, substrate  concentration ,  pH  &  temperature)  on  lipase  production  by  Acinetobacter sp.  YMP  & the  results   of  this  study  are  as  described  in  Table 1— Table 4.

Table 1: Agitation  (rpm)  Vs   Enzyme  units (mg/ml)

TIME  SPAN (HOURS)	ENZYME  UNITS  (mg/ml)
	Under static condition	Under  agitated  condition              (450 rpm)
24	0.1	0.13
48	0.14	0.25
72	0.2	0.28
96	0.22	0.3
120	0.2	0.32
144	0.18	0.28

 

Table 2 : Substrate  concentration  (%)  Vs.  Enzyme  units  (mg/ml)

Substrate  concentration (%)	Enzyme  units (mg/ml)
0.5	0.14
1	0.32
1.5	0.32
2	0.39
2.5	0.26

 Table 3 : pH  Vs Enzyme units (mg/ml)

pH	Enzyme units(mg/ml)
1	0.30
2	0.42
3	0.54
5	0.33
7	0.30
9	0.03
11	0.005

Table 4: Temperature ⁰C   Vs  Enzyme units (mg/ml)

Temperature 0 C	Enzyme units(mg/ml)
10	0.18
27	0.47
37	0.24
55	0.19

Thus,   the   maximum  yield   of   enzyme  was  obtained  within  120  hours  under agitated  condition  (450 rpm),  at  2 %  substrate  cocentration ,  at  3  pH &  at  27⁰ C.

Lipase  enzyme  was  purified  by  precipitation  with   ammonium  sulphate  in  increasing  concentration.  The  maximum  precipitation  and  enzyme activity   was  observed  at  60%   ammonium  sulphate  concentration .

Conclusion

By  using  biosurfactant  producing  Acinetobacter  sp. YMP ,  enzyme lipase  was  produced.  It’s  activity  was  determined  by  titrimetric  method.  Also , optimum  conditions  for  enzyme  production  were  determined.  The  enzyme  was further  purified  by  ammonium  sulphate   precipitation  method. Thus , lipase produced  by  biosurfactant  producing  Acinetobacter  sp.  YMP  can  be  exploited  in various  areas  of  Industrial  Microbiology  and  Biotechnology.

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