Suk Hoo Yoon Korea Food Research Institute 1/42
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1 Development of Phospholipases to Produce Structured Phospholipids Suk Hoo Yoon Korea Food Research Institute 1/42
2 Phospholipase D H H C O R R Z Fatty acyl chain -H Phosphatidic acid (PA) R O C H O - -CH 2 -CH 2 -NH 2 Phosphatidylethanolamine (PE) C H H O P O O Z -CH 2 -CH 2 -N(CH 3 ) 3 Phosphatidylcholine (PC) -CH 2 -CH(NH 3 ) - COO - Phosphatidylserine (PS) - Inositol Phosphatidylinositol (PI) 2/42
3 Phospholipase D - Hydrolysis(A), Transphosphatidylation(B) A CH 2 OCOR 1 R 2 COOCH O CH 2 O P OX O - phosphatidyl X PLD + H 2 O R 2 COOCH O + hydrolysis CH 2 OCOR 1 CH 2 O P OH O - phosphatidic acid X OH B CH 2 OCOR 1 R 2 COOCH O CH 2 O P OX O - + Y OH PLD transphosphatidylation CH 2 OCOR 1 R 2 COOCH O CH 2 O P O - OY X OH phosphatidyl X phosphatidyl Y 3/42
4 Phospholipase A 1 H H C O R R Z Fatty acyl chain -H Phosphatidic acid (PA) R O C H O - -CH 2 -CH 2 -NH 2 Phosphatidylethanolamine (PE) C H H O P O O Z -CH 2 -CH 2 -N(CH 3 ) 3 Phosphatidylcholine (PC) -CH 2 -CH(NH 3 ) - COO - Phosphatidylserine (PS) - Inositol Phosphatidylinositol (PI) 4/42
5 Lyso-Phospholipids Greater emulsion activity than PL, pharmaceutical agents, food preservatives - fat digestion in animal duodenum - cell autolysis by solubilizing bacterial membrane - natural surfactant with low toxicity - synergist with other surfactants - gentle solubilizing agent - inhibition of prostaglandin, etc. 5/42
6 Sources of PLD Carrot, cabbage Actinomycetes - Streptomyces, Actinomadura, Streptoverticillum etc Screening from soil & Cultivation, Enzyme Purification & Characterization 6/42
7 Screening of Actinomycetes 30g soil drying overnight dissolve in 6% yeat extract, 0.05% sodium dodecylsulfate heat at 40 C for 20min cultivate on HV agar plate containing nalidixic acid(20mg/l) at 30 C for 3 weeks Isolate colony and cultivate on Bennet agar plate 7/42
8 Cultivation of Actinomycetes Cultivate colony isolated in test tube containing peptone broth at 30 C for 3 days Fermentation using BioFlo IIC - working volume 1.8liter, 5% inoculation, 30 C, ph 7.2, 1 v/v/m, 600rpm, 3 days Centrifuge culture broth to obtain supernatent containing extracellular phospholipase D 8/42
9 Purification and Characterization of PLD Ultrafiltration Amicon PM10 Ion exchange chromatography CM Sephadex C-50 Gel permeation chromatography Sephadex G-100 FPLC Mono S (HR 5/5) SDS-PAGE, ph, temperature, metal ions 9/42
10 Hydrolytic activity of PLD 1 unit - 1μmole choline produced per min, phosphatidylcholine(pc) emulsion in Nacitrate buffer (ph 6), enzyme solution, 37 C, 10min reaction, boiling with EDTA Choline oxidase, peroxidase, Tris-Cl buffer (ph 8), 37 C, 20min reaction, OD at 500nm 10/42
11 Transphosphatidylation activity of PLD Egg yolk PC, glycerol, acetate buffer (ph 5.6), enzyme solution, 25 C, 30min reaction, reaction stop by citric acid Extract PL using diethyl ether-etoh(4:1), concentrate, develop TLC 1 st TLC CHCl 3 :MeOH:NH 4 OH(130:60:8) 2 nd TLC - CHCl 3 :MeOH:acetic acid:h 2 O(170:25:25:6) 11/42
12 Quantify PC, phosphatidylglycerol(pg), phosphtidic acid(pa) T activity (%) = 100 x PG / (PC+PG+PA) 12/42
13 Selection of Actinomycetes 1,100+ soil samples 131 HA>0.3u/ml 23 TA>20% Actinomycetes KF923, HA 12u/ml, TA 95% 13/42
14 14/42 Cultivation of Act. KF923
15 15/42 Ion exchange chromatography of PLD
16 16/42 Gel permeation chromatography of PLD
17 17/42 FPLC of PLD on Mono S
18 SDS-PAGE of PLD 55kD 18/42
19 19/42 Purification of PLD from Act. KF923
20 Effects of ph and temperature on TA of PLD 20/42
21 Effects of ph and temperature on stability of PLD 21/42
22 22/42 Effects of metal ions on TA of PLD
23 Sources of PLA 1 Intracellular PLA1 rat liver, brain & heart of human & bovine, E.coli, B. megaterium Extracellular PLA1 Serratia sp. Screening from soil & Cultivation, Enzyme Purification & Characterization Production of LPL & Optimization 23/42
24 Experimental methods Cultivation of Serratia sp. for PLA1 production - M9 minimal salt medium for seed culture - 3 liter fermentor, nutrition broth - aerobic, 14hrs at 30 C Enzyme preparation - crude extract, precipitation, dialysis - QAE-Sepharose ion exchange column - Sephacryl S-200 gel filtration column 24/42
25 Two-phase system - 10ml Phospholipon 90G in solvents, 10ml PLA1 (32units) and CaCl 2 in aqueous phase, ph 8, 40C Emulsion system - Phospholipon 90G, sodium deoxycholate in Tris-HCl buffer, add PLA1 and CaCl 2 solution 25/42
26 26/42 Analytical methods Activity determination of PLA1 - Hydrolysis, FFA by phstat (egg yolk lecithin) or TLC-FID - Activity staining, water-insoluble FA Substrate specificity of PLA1 - TLC-FID, 2 developments, monopalmitin Molecular mass by Matrix Assisted Laser Desorption Ionization (MALDI) mass spec
27 Ion exchange chromatography of PLA 1 27/42
28 28/42 Gel permeation chromatography of PLA 1
29 29/42
30 SDS-PAGE of PLA 1 30/42 Std 70%AS QAE S-200 Act. Stain on PL
31 31/42 MALDI-TOF Spectrum of PLA 1
32 32/42 Rat liver, brain
33 33/42 His, Cys
34 34/42
35 35/42 Effects of calcium conc n on PLA 1
36 Effects of ph on PLA 1 activity and stability S A 36/42
37 Effects of temp on PLA 1 activity and stability S A 37/42
38 Production of LPL in two-phase and emulsion systems T E 38/42
39 Effects of organic solvents in two-phase system 1. Ethyl acetate 2. Diethyl ether 3. Butyl acetate 4. Isopropyl ether 5. Benzene 6. Chloroform 7. Butyl ether 8. Cyclohexane 9. Hexane 10. Heptane 11. Isooctane 12. Decane 13. Dodecane 39/42
40 Effects of organic solvents on stability of PLA 1 Ethyl acetate Diethyl ether Butyl acetate Benzene Butyl ether Cyclohexane 40/42
41 Effects of PL conc n on LPL production in butyl acetate 2.5% 5% 10% 20% 41/42
42 Conclusion PLD - specific activity of 567 unit/mg, MW 55kD, optimum ph 6.0, temperature 60 C, no essential requirement for special metal ions PLA1 extracellular MW 43kDa (monomer) - Optimum ph 8.5, temperature at 50 C, stable up to 70 C - Enzyme activity inhibited by EDTA, and recovered by Ca ++ - High lipolytic activity for phosphatidylserine - Reaction yield of LPL in two-phase system higher than that in emulsion system - Highest catalytic activity and stability of the enzyme in butyl acetate 20% PL completely hydrolyzed into LPL in two-phase system 42/42
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