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 ChemistryBiology

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

OO

C

O

R3

R2

R1

O

H2O

H2O

Triacylglycerols

R2 O CH

H2C OH

H2C OH

O

HO

R1

O

HO

R3

O1

2

Monoacylglycerol Free Fatty Acids

O CH

H2C

H2C

O

OO

C

O

R3

R2

R1

O

MeOH

MeOH

Lipase

1

2Lipase

H3C

O R1

O

H3C

O R3

OR2 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 50 100 150 200 250 3000

0.1

0.2

0.3

0.4

0.5

0.6

Effect of MeOH concentration of Lipase Activity

5% MeOH

10% MeOH

15% MeOH

20% MeOH

25% MeOH

30% MeOH

Time (s)

Abs

orba

nce

(400

nm)

0 50 100 150 200 250 3000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Time (s)

Abs

orba

nce

(400

nm)

0 50 100 150 200 250 3000

0.10.20.30.40.50.60.70.80.9

1

Ethanolic Lipase Assay

Time (s)

Abs

orba

nce

(400

nm)

0 50 100 150 200 250 3001.25

1.3

1.35

1.4

1.45

1.5

1.55

1.6

Methanolic Lipase Assay

Time (s)

Abs

orba

nce

(400

nm)

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-basics3Shihabi, Clinical Chemistry. 1971, 17, 1151-11534Ji, J. Basic Microbiol. 2015, 54, 1–11 5Borgstrom, Eur. J. Biochem, 1973, 37, 60-68 6 Leca, Romanian Biotechnological Letters, 2010, 15, 5618-56307 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.