29-04-2014, 12:32 PM
Thermostable, alkaline tolerant lipase from Bacillus licheniformis using peanut oil cake as a substrate
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ABSTRACT
Thermostable alkaline tolerant lipase producing Bacillus licheniformis isolated from marine environment.
Peanut oil cake was used as substrate for enzyme production and purified to 3.6 fold with a specific
activity of 148.4 U/mg of protein with molecular weight of 35 kDa. The enzyme was 100% stable at 60°
C, it
retains more than 80% activity after 1 h incubation at 70°
C and pH 9-11.5. Metals like Ca2+, Ba2+ and Mg2+
2+
3+
enhanced whereas Ni and Fe inhibited the enzyme activity. No effect on enzyme activity found when
treated with Tween 80 and sodium deoxycholate, but completely inhibited with SDS.
INTRODUCTION
Lipases (EC 3.1.1.1) and esterases are act on the
hydroxyl ester bonds present in triglycerols, liberating
fatty acids and glycerols. Esterases act on water soluble
substrates with a preference for short fatty acid chains
but true lipases work at water-lipid interface and their
major substrates are long chain triglycerols (Jaeger et al.,
1994). Much attention is paid to lipases of microbial
origin, such as Candida sp., Pseudomonas sp., Bacillus
sp., and Rhizopus sp. for their unsurpassed roles in
biotechnology. They function as biocatalyst, capable of
catalyzing hydrolytic ester cleavage, transesterification,
alcoholysis, acidolysis, and aminolysis and are thus
widely used in the food, detergent, chemical and
biomedical industries (Pandey et al., 1999).
Lipase production using peanut oil cake
For the production of lipase, the bacterial cells were inoculated in
1000 ml erlenmeyer flasks containing 300 ml production media
containing (g/L) soybean 20, peanut oil cake 20, soluble starch 10,
K2HPO4 5, Na2 CO3 10, NaCl 5 and pH of the medium was adjusted
to 9.0 using 6 N NaOH (Sodium carbonate was sterilized separately
and added to the medium) were incubated at 55°C on shaker
incubator (125 rpm). The growth was measured at every 6 h
intervals in terms of optical density at 660 nm (Systronics, Visiscan
167) and cell free supernatant was used as crude enzyme.
Lipase assay
Estimation of lipase activity was carried out by titrating the fatty
acids liberated from olive oil with an alkali (Kunio et al., 1994). The
liberated fatty acids were titrated with 1 mol/L NaOH solution.
Simultaneously, a blank titration was also performed. One unit (U)
was defined as the amount of lipase capable of releasing 1 μmol of
titratable fatty acid per min under the assay conditions used.
Effect of Temperature, pH on enzyme activity and stability
The effect of temperature on enzyme activity was tested by
preincubating the reaction mixture (substrate + crude lipase solution
and 1 ml 100 mM Tris HCl buffer, pH 9.0) at temperatures ranging
from 30 to 90°
C. To check the thermostability, activity of the
enzyme incubated at different temperature for 1 h was checked at
different time intervals (0 to 60 min). The effect of pH on the
enzyme was determined by preincubating with four different buffers
(100 mM): citric acid-Na2 HPO4 (pH 4 to 6), phosphate buffer (pH 6
to 8), boric acid-NaOH buffer (pH 8 to 9.5) and phosphate-NaOH
buffer (pH 9.5 to 12), hydroxide chloride buffer (12 to 13) for 2 h
and the residual activity were determined by the standard assay.
Effect of EDTA detergent and various metals on lipase activity
Effect of detergents on lipase activity was analyzed by incubating
enzyme for 1 h at 45°C in 100 mM Tris HCl buffer (pH 9.0)
containing 0.1 or 1% (w/v) detergent. To determine the effects of
various metal ions on the lipase activity, enzyme assay was
performed for 1 h in the reaction mixture as described previously,
with EDTA, SDS and different metal ions such as MnSo4, FeSo4,
CoCl2, MgSO4, CuSO4, CoSO4, MgCl2, CaCl2, FeCl3, BaSO4, HgCl2
and NiSO4 at a final concentration of 1 mM.