ESTERIFICATION OF FATTY ACIDS USING PARTIALLY PURIFIED DVL-2 LIPASE

Prof. V.K. Gupta Department of Biochemistry Kurukshetra University, Kurukshetra (For Biosym 2013 Murthal)

Introduction
Enzymatic processes are generally preferred over Chemical methods in laboratories and industries mainly because these are: highly specific (no bye products formed) environment friendly mild reaction conditions India imports about 70% of the total enzyme consumption. Major industrial enzymes are protease, - amylase, xylanase, lipase, cellulase, penicillin acylase, laccase, glucose isomerase, tannase, pectinase, phytase, etc. The blooming industrial enzyme market is one of the major revenue generators in the life sciences industry sector

The world market for enzymes is estimated to grow 8% per year to eight billion dollar in 2013. According to Global Industry Analysts Inc., the global market for enzymes is expected to reach 4.3 US billion dollar by the year 2015

Lipases
Lipases (triacylglycerol acylhydrolase; EC 3.1.1.3) comprise a group of hydrolytic enzymes which catalyze reversibly the hydrolysis and synthesis of triacylglycerols at the oil-water interface

Lipases catalyze the hydrolysis of long chain acylglycerol at an oil- water interface are highly diversified in enzymatic properties and substrate specificity can also catalyze esterification, interesterification, and transesterification reactions in non-aqueous media to synthesize a growing range of products of potential industrial interest reactions usually proceed with high chemo-, regio- and/or enantioselectivity, making lipases an important group of biocatalysts

Microbial lipases are commercially significant because of: great variety of catalytic activities available high yields possible ease of genetic manipulation regular supply due to absence of seasonal fluctuations and rapid

growth of micro organisms or inexpensive media

more stable than the plant and animal enzymes their production is more convenient, safer and can be obtained in

bulk at low cost

Kitchen waste

Dairy waste

Potential Sample collection sites for isolation of lipolytc bacteria

Slaughter house waste

Fat and oil industry waste

Steps of Isolation & Screening

Collection of samples

Identification of culture
Plated on Nutrient agar medium
Selection of bacteria for producing largest zone of hydrolysis & highest Lipase activity

Qualitative assay of Lipase producing Bacteria (Tributyrin Agar )

Quantitative assay of Lipase producing micro organism

Sample from dairy industry waste

Bacterial colonies on NA plate

Electron microscopy
Zone of hydrolysis on TBA
Culture (DVL-2) was identified as Bacillus sp. DVL-2

16 S rDNA sequencing

Orange flourescence on Rhodamine olive oil plate

Identification of Culture (DVL-2) using 16 S rDNA sequencing

Consensus Sequence DVL-2 (1406 bp) GAGCAGGACAGAAGGGAGCTTGCTCCCGTGATGTTAGCGGCGGACGGGTGAGTAACACG TGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTT GAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCAT TAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATC GGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCA ATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCT GTTGTTAGGGAAGAACAAGTGCGAGAGTAACTGCTCGCACCTTGACGGTACCTAACCAGAAAGC CACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG GGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGG GTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAA ATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGA GGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAG TGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGG GAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATG TGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGA TAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAG ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCAC TCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTT ATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCAAGACCGCAAGGTTTAG CCAATCCCATAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAAT CGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT CACACCACGAGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCTATTATGGTAGC.

Lipase production in SmF

Production medium 1.75 % CSL, 1.25 % peptone, 0.75 % Tween 80 and 0.06 % MgSO4 (pH 8) SmF conditions used Inoculum size 3.0 %; Incubation temperature , 30 C, Incubation time 24 h; agitation rate 200 rpm Production medium (after 24 h) was centrifuged at 10,000 x g for 20 min at 4C The pellet (biomass) was collected and weighed.

The harvested cells were sonicated to release the intracellular lipase

Lipase assay
Titrimetric method using tributyrin as substrate
Spectrophotometric method using pNitrophenyl palmitate as substrate

Purification
Bacillus sp. DVL-2 cells were grown under
optimized SmF

Cell free extract (CFE) was obtained after sonication of the bacterial cells

The enzyme was purified in two steps : Ammonium sulfate fractionation HIC using Phenyl Sepharose CL-4B.

Most of activity was recovered in 30-70 % fraction and was loaded Phenyl Sepharose CL-4B column

Elution profile of Bacillus sp. DVL-2 lipase through Phenyl sepharose CL-4B column

Purification of Bacillus sp. DVL-2 lipase

Determination of MW by SDS-PAGE

Lane1: MW markers Lane2: Purified lipase

(a)
(b) (c) (d) (e)

Determination of MW by SDS-PAGE (a) = -galactosidase (116kDa)

(f)

(g)

(b) = BSA (66.2kDa)

(c) = ovalbumin (45.0kDa) (d) = lactate dehydrogenase (35.0kDa) (e) = REase Bsp98I (25.0kDa) (f) = -lactoglobulin (18.4kDa) (g) = lysozyme (14.4kDa) DVL-2 Lipase 39.8 kDa

Temperature optima

Temperature optima : 30 C

Thermostability of purified lipase

Enz was pre-incubating at different temperatures for 2 h and then lipase activity was determined DVL-2 lipase was most stable at 30-37 C retaining nearly 100 % of activity even after 2 h of incubation .

pH optima

Maximum lipase activity was observed over a broad pH range (6.0 to 10.0) with a slightly higher activity at pH 8.0. So its pH optimum was considered as 8.0.

To find out pH optima lipase assay was carried out at different assay pH

pH stability

The pH stability of the purified enzyme was investigated by pre-incubating it with buffers of varying pH (3.0-12.0) for different time intervals (30-240 min) Residual enzyme activity was calculated with respect to that of the control in which buffer was replaced by distilled water. The lipase purified from Bacillus sp. DVL-2 showed stability over a wide pH range (6.0-10.0).

Effect of organic solvents(25% v/v)

The DVL-2 lipase was found to be 100% stable in xylene, methanol, ethanol, toluene, hexane and DMSO after 12 h of incubation showing residual activity of 112, 104,101, 114, 99.2 and 101 % respectively as compared to control. In xylene and toluene, the enhanced lipase activity might be due to their activating effect. After 24 h of incubation, relative activity of the lipase in xylene, toluene, and DMSO was >90 %, in methanol (88.2 %), ethanol (82.1 %) and hexane (72.3 %). The DVL-2 lipase retained >70% of lipase activity in DMSO, toluene, and xylene even after 36 h of incubation. The DVL-2 lipase was found most stable in DMSO followed by toluene, xylene methanol and ethanol.

Effect of surfactants (0.1 %, w/v)

Anionic detergents such as deoxycholic acid, sodium deoxycholate, cholic acid and lithocholic acid were found to stimulate the lipase activity by 23%, 20%, 17% and 7% respectively whereas sodium taurocholate and SDS inhibited the lipase activity by 12% and 38% respectively. Among the non-ionic detergents, Brij 52 had a slight stimulatory effect (5%) whereas Brij 96 and Brij 58 inhibited the lipase activity by 12% and 17% respectively.

140 Residual activity (%) 120 100 80 60 40 20 0 100 88

120 107 98 99

123

117 99 105 83 89 88

62

Storage stability (4 C)of purified lipase

To determine the storage stability of the purified lipase, the enzyme was stored at 4 C and samples were collected at 15 days intervals for determination of lipase activity. Residual enzyme activity (%) was calculated by taking lipase activity of 0 time as 100 %.

120 100 100 Residual activity (%) 80 60 40 20 98 95 82 70

0
0 15 30 Days 45 60

Esterification of fatty acids using Bacillus sp. DVL-2 lipase

Lipase (25 IU) from Bacillus sp. DVL-2 was used for esterifcation of fatty acids (oleic, palmitic, stearic, and lauric acid) and ethanol in 1:1 ratio in hexane The reaction was carried out at 37 C with shaking at 100 rpm for 18 h with heat inactivated free enzyme as control. Ester formation was detected through analytical TLC (performed using pre-coated silica gel 60 F254 MERCK TLC plates) and 1H NMR. Spots were visualized by immersing TLC plates in 10% H 2SO4 in ethanol or 3-5% 2, 4dinitrophenylhydrazine (dissolved in conc. H2SO4, H2O and ethanol in the ratio 3: 4: 5) followed by heating on a hot plate. In 1H NMR, chemical data for protons are reported in parts per million (ppm) scale downfield from tetramethylsilane (TMS) and are referenced to the residual proton in the NMR solvent (CDCl3: 7.26).

TLC showing esterification of oleic acid and ethanol using DVL-2 lipase

Ethyl oleate

Oleic acid

S: substrate (oleic acid) D: Reaction with enzyme C: Cospot (S+D), E+D (Enzyme + reaction) E+S (Enzyme + Substrate)

Monday, November 18, 2013

28

1H

NMR (500 MHz, CDCl3)

5.38-5.30 (m, 2H), 4.11 (q, J=7.2 Hz, 2H), 2.28 (t, J=7.6 Hz, 2H), 2.05-2.00 (m, 3H), 1.62-1.56 (m, 6H), 1.30-1.24 (m, 20H), 0.89 (t, J=7.0 Hz, 3H).

Monday, November 18, 2013

29

Esterifcation of palmitic acid

Ethyl - Palmitate

Palmitic acid

R C

TLC showing esterification of Palmitic acid where S: substrate; R: Reaction with enzyme S: Substrate; R: Reaction mixture; C: Co-spot DVL-2; C: Cospot

Monday, November 18, 2013

30

 1H NMR (400 MHz, CDCl3) 4.12 (q, J=7.2 Hz, 2H), 2.28 (t, J=7.6 Hz, 2H), 1.67-1.58 (m, 2H), 1.30-1.24 (m, 27H), 0.88 (t, J=7.0 Hz, 3H). GC-MS

Monday, November 18, 2013

GC-MS calculated for C18H36O2 [M]+ 284, found 284

31

GC-MS of Ethyl-palmitate

Monday, November 18, 2013

32

Esterification of stearic acid and ethanol using DVL-2 lipase

Ethyl stearate

Substrate
S

S: substrate (oleic acid) R: Reaction with enzyme C: Cospot (S+D), E: Enzyme

Monday, November 18, 2013

33

Ethyl stearate

CDCl3): 4.12 (q, J=7.2 Hz, 2H), 2.28 (t, J=7.6 Hz, 2H), 1.62-1.58 (m, 2H), 1.28-1.24 (m, 31H), 0.89 (t, J1H-NMR of ethyl stearate 1H NMR (Fig. 4.5.9) confirmed the formation of ethyl-stearate is interpreted as: 1H NMR (500 MHz, =7.1 Hz, 3H).

Monday, November 18, 2013

34

TLC showing esterification of lauric acid and ethanol using DVL-2 lipase

1H-NMR

of ethyl laurate

1H
1H

NMR confirmed the formation of ethyl laurate is interpreted as:

NMR (500 MHz, CDCl3): 4.12 (q, J=7.2 Hz, 2H), 2.28 (t, J=7.6 Hz, 2H), 1.62-1.58 (m, 2H), 1.281.24 (m, 31H), 0.89 (t, J=7.1 Hz, 3H).

Monday, November 18, 2013

36

GC-MS confirming the formation of Ethyl laurate

GC-MS: calculated for C14H28O2 [M ]+ 228, found 228

Monday, November 18, 2013 37

Esterification using Immobilized DVL-2 lipase

Purified DVL-2 lipase was immobilized on glutaraldehyde activated aluminum oxide pellets Immobilization parameters statistically optimized using RSM number of pellets enzyme dose glutaraldehyde concentration and coupling time

Response surface plots showing cumulative effect of two variables on the immobilization of lipase while keeping other variables at 0 level Variables: A =number of beads; B =glutaraldehyde concentration; C =enzyme dose ; D = incubation period (a) interaction between factors A and B; (b) factors C and A; (c) factors C and B; (d) factors D and B ; (e) Perturbation graph showing the effect of variables on immobilization yield

Optimization of process parameters for Immobilization through RSM

All the RSM graphs were analyzed to determine the optimum value of each factor for maximum IY. The maximum IY of 78.20 % was obtained when 10 aluminum oxide pellets were treated with 4% glutaraldehyde and subsequent incubation with 0.04 mL of enzyme for 75 min.

Concentration of organic solvents: 25% (v/v) Incubation Period: 24 h

The immobilized lipase showed better stability than free enzyme in organic solvents

The immobilized lipase was found to be 100% stable in xylene, ethanol, methanol, toluene and hexane after 12 h of incubation. The stability in these solvents though declined after 24 h incubation as compared to 12 h yet the relative activity was greater than 90 %, except methanol (80 %)

Esterification of oleic acid using free and immobilized lipase

Immobilized lipase showed maximum 63% conversion of oleic acid into ethyl oleate in 16 h whereas free lipase showed maximum 60 % conversion of oleic acid into ethyl oleate in 24 h.

Esterification of oleic acid and ethanol in hexane to produce ethyl oleate by the free (solid circle) and immobilized lipase (hollow circle)