1. Investigation of Pancreatic Lipase in Biodiesel Production
Cody J. Lloyd, Cody T. Lloyd, and Dr. Walter Patton
Lebanon Valley College, Program in Biochemistry and Molecular Biology, Annville, PA 17003-1400
Abstract
LVC BIOCHEMISTRY & MOLECULAR BIOLOGY B BMC
Chemistry
Biology
Introduction
Objective
Lipase Assay
Lipase Purification
Production of Biodiesel
Results
Conclusion
References
Major Concerns
Biodiesel is a non-petroleum based fuel source that has similar flow and
combustion properties as petroleum-based diesel. Therefore, biodiesel can be
blended with petroleum-based diesel, to make fuel blends, or alone as a substitute.1
Additionally, biodiesel is significantly less toxic and more environmentally friendly. The
acid catalyzed transesterification of triacylglycerols (TAGs), a.k.a. fats, with primary
alcohols is the most common way to synthesis biodiesel, but this method is not
completely environmentally friendly due to the use of liquid acids. Lipase, an enzyme
that hydrolyses TAGs into two free fatty acids (FFAs) and a monoacylglycerol, has the
ability to use primary alcohols as a nucleophile resulting in the transesterification of
TAGS. Lipase’s enzymatic mechanism is currently being studied as an alternative
method for biodiesel production.
Pancreatic Lipase was obtained from a digestive enzyme supplement, Ultra-
Zyme, and an ethanolic olive oil emulsion was used to track lipase activity. Partial
purification of pancreatic lipase was performed and lipase was then used to perform a
transesterification of olive oil TAGs. HNMR was used to determine the ratio of (FFAs)
to biodiesel. No biodiesel production was observed.
In this project, we aim to use pancreatic lipase as an alternative method for
the transesterification of olive oil TAGs with primary alcohols in the production of
biodiesel.
O CH
H2C
H2C
O
O
O
C
O
R3
R2
R1
O
H2O
H2O
Triacylglycerols
R2 O CH
H2C OH
H2C OH
O
HO
R1
O
HO
R3
O
1
2
Monoacylglycerol Free Fatty Acids
O CH
H2C
H2C
O
O
O
C
O
R3
R2
R1
O
MeOH
MeOH
Lipase
1
2
Lipase
H3C
O R1
O
H3C
O R3
O
R2 O CH
H2C OH
H2C OH
O
MonoacylglycerolTriacylglycerols Biodiesel
The Pancreatic enzyme lipase is essential for the absorption of fats. Lipase is
secreted, by the pancreas, into the lumen of the small intestine where it degrades
consumed fats. Lipase is characterized as an esterase, which means that it degrades
molecules by hydrolysis.
• Ultra-Zyme pills contained all pancreatic enzymes
• Methanol is an Organic Solvent
• Lipase activity is dependent on it coenzyme - Co-Lipase
• Detection of biodiesel for small yields
Free Fatty Acid H NMR4 Biodiesel H NMR4
0
0.1
0.2
0.3
0.4
0.5
0.6
0 50 100 150 200 250 300
Absorbance(400nm)
Time (s)
Effect of MeOH concentration of Lipase Activity
5% MeOH
10% MeOH
15% MeOH
20% MeOH
25% MeOH
30% MeOH
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 50 100 150 200 250 300
Absorbance(400nm)
Time (s)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 50 100 150 200 250 300
Absorbance(400nm)
Time (s)
Ethanolic Lipase Assay
1.35
1.4
1.45
1.5
1.55
1.6
0 50 100 150 200 250 300
Absorbance(400nm)
Time (s)
Methanolic Lipase Assay
Lipase activity was determined by spectrophotometry. An ethanolic olive oil
solution was emulsified, by sonication, into 25 mM Tris buffer containing 15 mM
sodium deoxycholate (pH 8.8).3 Lipase assay was performed at room temperature
tracking the hydrolysis of TAGs, which is characterized by emulsion disappearance.
Ultra-Zyme pills were crushed and dissolved into 3.5 ml of .1 M Tris buffer, per
gram of Ultra-Zyme pill, to make FSE. FSE was centrifuged at 10,000xg for 10
minutes. Acid precipitation (pH 5.0) was performed on supernatant followed by
centrifugation at 10,000xg for 10 min.4 The resulting supernatant was the purest form
of lipase obtained. Other methods performed used AmSO4 or NaCl and resulted in an
absence of lipase activity.
Determining Optimal Concentration of Methanol
Increased methanol concentration resulted in decreased lipase activity. The
highest concentration of methanol, with prominent lipase activity, was 15 percent
methanol.
Method 1:
H NMR indicated that there was no presence of free fatty acids or biodiesel.
Method 2:
H NMR indicated that there was no presence of free fatty acids or biodiesel due
to contamination of water.
c
Previous research has determined optimal pH, bile salt concentration,
temperature, and ratio of methanol:TAG for the transesterification of TAGs for
immobilized lipase.5,3,6
Reaction 1 (based off of previous literature): Olive oil was dissolved into
methanol in a 4:1 methanol:fatty acid ratio and heated to 50°C. Sodium deoxycholate
was added to purified lipase solution, until 15 mM sodium deoxycholate, and heated
to 50°C. Methanolic olive oil solution was added to activated lipase solution until final
solution contained 15 percent methanol. Reaction ran at 50°C for 60 min. Aliquots
were taken every 10 min.
Reaction 2 (based off of lipase assay): Methanolic olive oil emulsion, created
identically to ethanolic olive oil emulsion, and added to purified lipase solution in a 9:1
emulsion:lipase solution ratio. Reaction ran at room temperature for 1 hour.
1Lotero, Ind. Eng. Chem. Res., 2005, 44, 5353-5363
2Biodiesel, America’s Advanced Biofuel. Gorge Analytical. Retrieved April 10, 2015, fromhttp://www.biodiesel.org/what-is-biodiesel/biodiesel-basics
3Shihabi, Clinical Chemistry. 1971, 17, 1151-1153
4Ji, J. Basic Microbiol. 2015, 54, 1–11
5Borgstrom, Eur. J. Biochem, 1973, 37, 60-68
6 Leca, Romanian Biotechnological Letters, 2010, 15, 5618-5630
7 1H-NMR Spectroscopy of Fatty Acids and Their Derivatives. The AOCS Lipid Library. Retrieved April 10, 2015, http://lipidlibrary.aocs.org/nmr/1NMRsat/index.htm
Lipase activity was still present in methanolic lipase assay. Although, methanolic
lipase assay showed decreased lipase activity when compared to ethanolic lipase
assay.
Production of biodiesel was not obtained from the enzymatic mechanism of
pancreatic lipase in reactions performed.