(Wff. uqun &Jhfl) - thaiscience.info for ThaiScience/Article/2/10005650.pdf · acid about 60% of...
Transcript of (Wff. uqun &Jhfl) - thaiscience.info for ThaiScience/Article/2/10005650.pdf · acid about 60% of...
(Wff. uqun &Jhfl)
-u-
-fl-
Thesis Title
Thesis Credits
Candidate
Supervisors
Degree of Study
Division
Academic Year
Synthesis of Stabilized Essential Fatty Acid by Yeasts and
Chemical Method
1 2
Mr. Paitoon Suksatavorapan
Assoc. Prof. Dr. Kanit Krisanangkura
Asst. Prof. Narumon Jeyashoke
Master of Science
Biotechnology
1 9 9 4
Abstract
The goal of this study was to synthesize relatively stable essential fatty acid of n-3
and n-6 series by chemical and biological method.
The first attempt was to elongate alpha-linolenic acid to eicosatrienoic acid or fatty
acids of longer chain length with Rhodotorula glutinis and yeast strain 572. The yeast grew
rapidly at 30°C. in medium with or without linseed soap which contained alpha-linolenic
acid about 60% of total fatty acid. Analysis of fatty acids from yeasts by gas
chromatography showed that the yeast could assimilate alpha-linolenic acid into the cell but
did not elongate. Assimilation of fatty acid as well as growth of yeast decreased as
temperature decreasing. It was concluded that the yeasts, R. gUnis and yeast strain 572,
could not convert linseed oil alpha-linolenic acid to any fatty acid of longer chain length.
Attempt biohydrogenate fish oil anaerobically with Sacc~omyces cerevisiae was
mlsuccessful. Lipid isolated from yeast treated with fish oil did not contain any
hydrogenated fatty acid of eicosapentaenoic acid or docosahexaenoic acid, suggesting that
the yeast was not able to biohydrogenate at this condition.
Hydrazine reduction of alpha-linolenic acid of linseed oil was very promising.
Analysis of reduction products by gas chromatography and mass spectrometry indicated that
the product contained one double bond at the n-6 (o&a&c-12-enoic acid) and n-3 (octadec-
1%enoic acid) position. The optimum molar ratio of fatty acid to hydrazine was 1 to 8 in
ethanol and amount of potassium ferricyanide was 0.2 gram per 5 millilitre. When water
was used as hydrogenation solvent, yields of octa&c-12-enoic acid and octadec-15-enoic
acid were increased from 14.1% and 6.4% to 15.4% and 8.7% respectively, but there was
no selectivity on the double bond position. At pH 9.9, rate of reaction was faster and easily
controlled. Reaction rates were highest at temperature 70 to 90°C but the maximum yields
were equal to that obtained at 30°C .
Keywords : Biohydrogenation / Essential fatty acid / Hydrazine reduction /
Linolenic acid / Linseed oil / Synthesis / Yeast
1.2.1.1 1.2.1.1 IUiuoil43laIUiuoil43la
1.2.1.2 1.2.1.2 %lllhlJmml%lllhlJmml
1.2.1.3 1.2.1.3 oy?bhR"ihoy?bhR"ih
U
9
Q
II
Q
Q
iu
1
1
2
2
3
3
3
4
4
4
4
5
7
7
11
11
15
16
2 0
2 0
2 0
-%-
1 . 3 . 3 . 1 1 . 3 . 3 . 1 er1nhJer1nhJ
1 . 3 . 3 . 2 1 . 3 . 3 . 2 Wf~n"hWf~n"h
1 . 3 . 3 . 3 1 . 3 . 3 . 3 ihhlt51ihhlt51
2 12 1
2 12 1
2 22 2
2 22 2
2 52 5
2 52 5
2 52 5
2 82 8
2 82 8
2 82 8
3 03 0
3 03 0
3 03 0
3 63 6
3 63 6
3 83 8
4 04 0
4 14 1
4 34 3
4 44 4
4 54 5
4 54 5
4 74 7
4 94 9
5 05 0
5 05 0
5 05 0
5 05 0
5 15 1
-All-
51
51
51
52
52
52
52
53
53
5 4
55
55
56
57
57
65
71
75
79
8 4
87
9 0
93
96
HiL
OllSld Hlb
1 . 1
1.2
1 . 3
1.4
1 . 5
1.6
1.7
1 . 8
1.9
3.1
3.2
3.3
3.4
3.5
3.6
6
26
26
27
29
37
37
39
40
61
66
66
7 4
99
1 . 1
1.2
1 . 3
1.4
1 . 5
1 . 6
1.7
1.8
1 . 9
1.10
1.11
3 . 13 . 1
3 . 23 . 2
3 . 33 . 3
3 . 43 . 4
3 . 63 . 6
3 . 73 . 7
3 . 83 . 8
99
1 01 0
1 21 2
1 31 3
1 41 4
1 51 5
1 61 6
1 81 8
1 91 9
1 91 9
2424
5 85 8
5 95 9
6 06 0
6 36 3
64
6 76 7
68
6 96 9
70
73
85
86
8 9
9 1
9 2
9 4
9 5
9 7
n. =
un. =
UQ. =
R. =
Oobl. =
AA =
ALA =
DHA =
DHGLA =
EPA =
G C =
GC-MS =
GM =
PUFA =
m/z, m/e =
nr'unr'u
ikl6nfuikl6nfu
4 4 *LL*LLaJQQR913aJQQR913
hshs
EMllWlCihJffEMllWlCihJff
Arachidonic Acid
alpha-Liuolenic Acid
Docosah~xaenoic Acid
di-homo-gamma-Linolenic Acid
Eicosapentaenoic Acid
Gas Chromatography
Gas Chromatography and Mass Spectrometry
gamma-Linolenic Acid
Polyunsaturated Fatty Acid
Mass/Charge Ratios
-2--2-
1.2 YE&
1.2.1 m (Lipids) [ 8, 9, 10 I
-3-
-4-
1.2.2 fQd!Uh (Fatty acids)
-5
a1.2.3 fl7rlgun(lrofIT&Wl* (Nomenclature)
0 - terminus CH,--CH,CH,-CH,--CH,--CHz--CH,COOH Ca&oxgl-
terminus
A numbering 10 9 8 7 6 5 4 3 2 1
normnumbering 1 2 3 4 5 6 7 8 9 10
Letter designation o o-1 s Y P cx
-6-
12:o
140
1 6 : 01 6 : 0
l&O
2o:o2o:o
24LO24LO
1611 A9;n-7
18:l A9;n-9
1812 A9,12;n-6
18:3 A9,12,15;n-3
20:420:4 fi,8,11,14;11-6fi,8,11,14;11-6
LNlliC
MyriStiC
Palmitic
stearic
AlWhidiC
Lignoceric
Palmitoleic
Oleic
Linoleic
Lholenic
AWAidOIliC
Dodecanoic 4 4 . 24 4 . 2
Tetradecanoic 5 3 . 95 3 . 9
Hexadecanoic 6 3 . 16 3 . 1
Octadecanoic 6 9 . 66 9 . 6
Eicosanoic 7 6 . 57 6 . 5
Tetracosanoic 8 6 . 08 6 . 0
Hexadecenoic -- 0 . 5 0 . 5
Octadecenoic 1 3 . 41 3 . 4
Ocbdecadienoic -- 5 . 0 5 . 0
Octadecatfienoic - 11.0
Eicosatetraenoic - 49.5
-7-
1.2.4 flWI!¶lfiU#Ub (Essential fatty acids)
ti1.2.5 fi.mT4lflllwHftl~luY lh (Fatty acid synthesis)
-8-
Acetyl CoA + HC%- + ATP + h&my1 CoA + A D P + P i
-9-
MITOCHONDRIA
CITRATE ______ . a - - - - - _ _ > CITRATE
CoASH 1
ACETYL CoA
OXALOACETATE
ADP Pi
ATP
PYRUVATE c--------.
CYTOPLASM
2 ACETYL CoA
OXALOACETATE
MALATE
NADP +
NADPH
y.Ji 1.1 nlsrfl0~~6~%~6BU~c11d60nlsrfl0~~6~%~6BU~c11d60 oofloln~aJl~nou~~4uoofloln~aJl~nou~~4u
1 : citrate synthetase, 2 : citrate lyase, 3 : malate dehydrogenase,
4 : malic 5 :enzyme, pyruvate carboxylase [ 9 ]
-lO-
ATP 1 A D P + P i
ACETYL CoA
3-KETOACYL - ACP
D-3-HYDROXY - ACP
H2O 48
A2,3-trans-ENOL - ACP
NADPH
NADP +-3
7
ACYL - ACP (04)
L- MALONYL CoA, 2 NADPH
ACYL - ACP (C6)
L- MALONYL CoA, 2 NADPH
ACYL - ACP (03)
L- MALONYL CoA, 2 NADPH
ACYL - ACP (ClO)+titi
PALMITATE (Cld)
Jdi 1.2 r-IlYfSjl~Yl,,*,Y,~~u'uUr-IlYfSjl~Yl,,*,Y,~~u'uU 1: acetyl CoA carboxylase, 2: acxztyl-
transferase, 3: malonyl transferase, 4: synthetase, 5: reductase,
6: hydratase!, 7: reductase [ 91
-ll-
-1%
00
II
-0OC - *Cl-$ - C - SCOA Malonyl CoA
9 - (CH,)1, - C -SCoA
Palmitoyl CoA
-L CO,, CoASH
0 0
I I
CH, - (Cl-I&,, - C - ‘CH2 - C - SCoA
NADPH
ILN A D P +
H 0
I I
CH3 - (CH,),, - C - ‘CHZ - C - SCoA
I
OH
L 40
0
I
3 - (Cl-& - Cl-I = ‘CH - C - SCoA
NADPH
I”
NADP +
0
I
a-~,,-(a-i,),,-CH/CH,-C-SCoA Steamy1 CoA
-13--13-
t
; CH,(CHJ,- Cl3 = CH - (CIiJ,- : - SCoA
Phitoboyl - CoA
-14-
.n-6 term
C l&O a-, C18:l A9 -b cm2 A9,12 __, c m 3 A9,12,15
StWliC linoleic 01 - linolenic
I I
C18:3 A 6,9,12 C20:3 A11,14,17
C20:2 A8,ll C20:3 A8,11,14 C20:4 A8,11,14,17
1 di-hano- y l/ok& (DHLA) 1
C20:3 A5,8,11 C20:4 A5,8,11,14 C205 L&8,11,14,17
eicosatrienoic arachidonic (AA) eicosapentaenoic (EPA)
C225 A7,10,13,16,19
docosapentaenoic
C22:6 A4,7,10,13,16,19
docaahexaenoic (DHA)
-15-
GLYCEROL - 3 - PHOSPHATE
2 FATTY ACYL CoA
2 CoASH
PHOSPHATIDATE CDP - DIACYLGLYCEROL
DIGLYCERIDE
FATTY ACYL CoA
CoASH
CAMP
TRIGLYCERIDE PHOSPHOGLYCERIDE
-16-
CYTOPLASM lbWlDC.HONDRIA
------- > ACETYL CoA
CoASH AMP + PPi I ~-OXIDATION
FATI-Y ACYL CoA FA’ITY ACYL CoA
.- CAR&It 8
b CoASH
----> FATTY ACYL CARNITiNE
Jdd 1.7 ~l~~l~~~~~tl’ul(i7~Ut~~~Ul~~~ 1: fatty acyl CoA synthetase
2,3: fat&y acyl CoA camitine trausferase [ 9 ]
-17-
-1%
FATTY ACYL
1A 2,3-tram-E!NOYL CoA
L3-HYDROXYACYL CoA
NAD+ 7
NADH 1 3
3-KETOACYL CoA
CoASH
ACETYL CoA 1 4
FATTY ACYL CoA (n-2)
1
Lc FADH 2 + NADH + ACJZTYL CoA
FATTY ACYL CoA (n-4)
FADH 2 + NADH + ACETYL CoA
FATTY ACYL CoA (n-6)
FADH 1 + NADH + ACE!TYL CoA
FATTY ACYL CoA (n-8)
4
4
FADH,+ NADH+ACETYL CoA
ACE!TYL CoA
Jdd 1.8 nYtu?UnlY~(sl-88n~l~~UnYtu?UnlY~(sl-88n~l~~U 1: fatty acyl CoA dehydrogenase, 2: enolhydratase
3: L-3-hydroxyl CoA &hydrogenase, 4: 3 - ketothiolase [ 9 ]
-19-
ODD-CHAIN FATTY ACID
~-o~A~ON
I%n ACETYL CoA
PROPIONYL CoA
ADP
METHYL MALONYL CoA
2 ISUCCWYL COA
&I-cis-EBJOYL CoA
I 1
A2,3-trans-EBJOYL CoA
Ifl - OXIDATION
A2.3-cis-ENOYL CoA
D3-HYDROXYACYL CoA
L-3-HYDROXYACYL CoA
Ifl - OXIDATION
-2o-
1.2.8.1 l&i%lh!Il&i%lh!I k&%K (Hydrolysis)
CH,OCOR CHZOH
ICHOCOR + H,O --heat+ CHOH + 3 RCOOH
I ICH,OCOR CH20H
Triglyceride Water Glycerol 3 Free fatty acids
r4 w1.2.8.2 m (E!shification)
- 2 1 -- 2 1 -
0 0
I I
R - C - O H + H O R , - R - C - O R , + Hz0
CI$OCOR
I
CH,OH
I
CH20COR
Alkaliue catalyst ICHOCOR + CHOH ______, CHOCOR
I ICH,OCOR C%OH CH20H
COCOR
I+ CHOH
dH20HdH20H
Triglyceride Glycerol Diglyceride Monoglyceride
1.2.8.4 -- ae (Hydrogenation)
-22-
CH3(CH2)4 CH = CHCH2CH = CH (CH,),COOH - CH3<CH2> ,,COOHPt or Ni
Liuoleic acid Steak acid
1.2.8.5 l&???Jl~V&I,,&l%~U (Polymerization)
R + R - (RI2
Polymerization Reaction
1.2.8.6 v (Oxidation) [ 15 ]
-23-
R’ + A r - O H _____, RH + Ar-0.
active radical hindered phenol inactive radical
I R=
neutral molecule
till?idlf Butylatecl Hydroxyanisole (BHA) Butylated Hydroxytoluene (BHT) Propyl
Gallate (PG) AAQZ Tert-Butyl Hydroquiuone (TBHQ) l!%J&J
-24-
CH,(CHJ), - CE&- CH = CH - CH,- (C~,COOCH,
101 9
1 - H
-‘CH-CH=CH-C%- -CT-+CH=CH-‘CH-
11 10 9. 8 11 10 9 8
Jf-C!H=CH-°CH-C~- -q-eCH--CHP-
11 10 9 8 11 10 9 8
-*CH -CH=CH-C3$- + 0, -
-CH,-CH=CH- +
newintactfatty acid mokcukI
1’
11
-CH -I
CH=CH-CCH,-
0 - 0.
-CH-CH=CH---
I0 - d
-H
-‘a-I -CH-CH- + -CH-CH=CH-CX&-I
new radical O - O H
Third:10 9
ketone t-40 - cIqq,cH = CH - CH - a-L&~:,
Ib--OH
*
CH,(CHJ,CH - CH - CHO + OW~~,C~,
aldehydes dCObOk
Y
1.3.1 &$UQlfd(ll&$UQlfd(ll (vegetable oil)
Y
1.3.2 ~UWfl~Vli (animal oil)
-26--26-
oilsoulw T fatty acid oompodion(%)
6zOo-10~0 129 149 16~0 18~0 18:l 1 8 2 18:3
I
coconut 11.0-19.0 44.0-52.0 13.0-19.0 8.0-I 1 .o
co rn - 10.9
cotton 808d - 22.7
olh 11.0
pslm’ 6.0-13.0 40.0-52.0 14.0-18.0 7.0-9.0
psglut 9 .5
safflower b * - 6 .2
safnowar” 4 .8
- - 8 .9
i3oybQan - 10.3
8unfIower 5 .9
Wllblllh - 7 .0
’ dqnwn MlKKae llwnol:: [ 19 ]
bhigh linobic aoid
= highobiuaoid
LL
1.0-3.0 5.0-8.0 2 .5
1.8 58.0
2 .3 51.5
2 .2
1 .o-3.0
2 .2
2 .2
1 .3
4 .8
3 .8
4 .5
2 .0
24.2
17.0
72.5
11.019.0
44.8
11.7
75.3
39.3
22.8
19.5
22.2
7 .9
0.5-2.0
32.0
74.1
14.2
41.3
51.0
65.7
52.9
1
0 .7
0 .2
0 .6
-
0 .4
-
0 .3
6 .8
10.4
OilllomcQ fatty acid uxnpodion(%)
4zOo-12fl 149 16~0 16:l 18~0 18:l 18:2 OthQlX
lad 0 1 21 3 12 4 6 16 1
Paltry 0 1 2 7 9 7 4 5 1 1 1
beef 0 3 2 6 9 8 4 5 2 0
lamb 0 3 21 4 2 0 41 5 6
dkcOW 10 12 2 6 3 1 1 2 9 2 4
milk-goat 21 1 1 2 7 3 10 2 6 2 0
Qgg Yolk 0 0 2 9 4 0 4 3 1 1 4
-27-
Fatty acid Fatty acid composition ( 96 w/w
140 1.4
16:o 19.6
16:l 3 . 53 . 5
18:O 3 . 83 . 8
18: 1 1 3 . 81 3 . 8
18:2 0 . 70 . 7
18:3 0.1
18:4 0 . 40 . 4
2&l 3 . 03 . 0
20:4 2 . 52 . 5
20:s20:s 17.0
221221 1.0
22:s22:s 1.3
2233 29.8
Haddock
1.5
2 0 . 02 0 . 0
4 . 04 . 0
6 . 16 . 1
1 4 . 21 4 . 2
2 . 22 . 2
0 . 40 . 4
O SO S
2 . 62 . 6
3 . 33 . 3
1 2 . 01 2 . 0
0.1
2 . 42 . 4
246
Mackerel
8 . 68 . 6
1 7 . 61 7 . 6
10.0
2 . 22 . 2
1 4 . 81 4 . 8
1 . 01 . 0
0 . 80 . 8
2 . 02 . 0
8 . 68 . 6
0.9
9.4
1 0 . 21 0 . 2
1 . 21 . 2
8 . 78 . 7
Sprat8
6 . 06 . 0
2 1 . 52 1 . 5
5 . 35 . 3
2 . 42 . 4
1 6 . 51 6 . 5
1 . 61 . 6
1 . 31 . 3
3 . 33 . 3
7 . 07 . 0
0 . 70 . 7
8.1
1 2 . 01 2 . 0
1
Rainbow trout
3 . 53 . 5
1 3 . 31 3 . 3
4 . 84 . 8
3 . 83 . 8
1 8 . 71 8 . 7
5.5
5 .9
2.1
-
7 . 27 . 2
solsol
6 . 26 . 2
2 1 . 02 1 . 0
-2s
*1.3.3 Jhu'ufll~Q~Jhu'ufll~Q~ (microbial oil)
1.3.3.1 nlMtiltrnlMtiltr ( a l g a e ) ( a l g a e )
1.3.3.2 ALIEd& (bacteria)
-29--29-
dative % (w/w) of fat&y apyl Jpupll in lipids
Microorganism 140 MD 169 161 189 18:l 18:2 183 18~3 18t4 20x1
(a) (9
Artbrobnctor AK 19 3 43 5 - 15 24 0.3 - - - -
cMo#lln~ti 2 26 8 9 2 2 34 20 - - -
Dun~primabota 5 11 10 21 tr 6 6 10 2 7 14
Nowhkuin olwabdn 2 15 4 3 11 36 7 - - - 5
sowodwmunncufun tr 15 1 25 1 8 20 30 tr - -
Chdidarp.l07(NCYC 911) 3 44 5 - 8 31 9 1 - - -
ZMomycopkvwdis -15- - 2 5 7 2 4 1 - - -
HnnmrllIlnnniumun 1 24 3 - 4 30 25 12 - - -
tipomyoeetipafer h 37 4 - 7 48 3 - - - -
L.starkqi 32 2 - 7 53 5 - - - -
Rtlod~tio~ 1 24 1 - 13 46 13 3 1 - -
R&odotonliaghrtiaiz? 1 13 - - 7 53 20 5 - - -
R.gWifiS 21 - - 13 52 11 3 - - -
rtl-ichonparonoutnwum tr 30 - - 13 46 11 - - - -
MOldi
Atzpgibniclulnnn tf 20 3 - 12 39 13 - - - -
A.kJlWS 2 23 tr - tr 14 40 21 - - -
hf- lfhaoa 2346-24494--ic
-w= 1 20 2 - - 8 48 11 - 6 -
Spllcclolbeoa~n 8 23 1 - 3 37 1 - - - 7
-3o-
1.3.3.3 &IjhEIl (yeast and mold)
d*
w1.4.1 sY
-31-
-32-
-33-
Radwan iin: soliman [ 3 7 ] Ylcw.l@hl orlndlmn!9lolniiuii Mllfi
Aspetgiuus SP., ~~nic~um q., opium sp., PaedomJxx?s sp., Tri&o&ma sp.,
-34-
-35
ikhMJ,,,D,,l~t~u"~ 12.3%, "VtAA0~r/l~tU~u'" 38.5% AA~~"~~AA"lJJJldtUdw'" 0.9%
-36
1.4.2.1 m (substrate)
-37--37-
carbon source biomass lipid lipid yield fatly acid composition
(30(30 g/l)g/l) wbwb (w/w) (g11OOg. substrate) (%I
1 6 : 01 6 : 0 18:0 l&l l&2
glucose 10.2 3 3 . 23 3 . 2 1 1 . 91 1 . 9 3 3 . 03 3 . 0 1 2 . 01 2 . 0 4 2 . 94 2 . 9 7 . 37 . 3
sucrose 11.2 3 7 . 43 7 . 4 1 4 . 81 4 . 8 3 2 . 43 2 . 4 1 1 . 31 1 . 3 4 2 . 04 2 . 0 6 . 76 . 7
l a c t o s el a c t o s e 1 2 . 51 2 . 5 3 9 . 23 9 . 2 1 6 . 51 6 . 5 3 2 . 53 2 . 5 1 1 . 01 1 . 0 4 9 . 04 9 . 0 6 . 06 . 0
xylose 9 . 99 . 9 4 3 . 64 3 . 6 1 7 . 41 7 . 4 4 1 . 24 1 . 2 1 4 . 01 4 . 0 4 3 . 04 3 . 0 3 . 53 . 5
ethanol 8.5 30.1 10.0 2 6 . 52 6 . 5 1 2 . 51 2 . 5 4 9 . 04 9 . 0 8 . 98 . 9
Growth Substrate Fatty Acids ( relative 9b, w/w )
l&O 16:l 18:o l&l l&2 18:3 2o:o2o:o
corncorn o i lo i l F e dF e d 1 1 . 61 1 . 6 1 1.7 24.7 61.5 0.5 -
Recovered 11.3 5 .8 2.1 3 6 . 03 6 . 0 4 4 . 84 4 . 8 -- --
Linseed oil Fed 7,37,3 -- 3 . 83 . 8 2 0 . 02 0 . 0 1 5 . 01 5 . 0 5 3 . 95 3 . 9 --
Recovered 6 .7 2 .8 6 .7 3 6 . 03 6 . 0 1 8 . 41 8 . 4 2 9 . 42 9 . 4 --
Palm oil Fed 2 9 . 72 9 . 7 7 . 17 . 1 2 . 02 . 0 4 5 . 44 5 . 4 1 0 . 71 0 . 7 5 . 15 . 1 --
Recovered 26.3 7.3 7.6 46.5 1 0 . 31 0 . 3 1 . 91 . 9 --
Olive oil Fed 12.5 1.8 2.5 69.3 10.7 1.3 1.8
Recovered 11.7 2.5 2.5 6 9 . 76 9 . 7 1 2 . 51 2 . 5 1.1 -
-3a-
1 . 4 . 2 . 2 &+A71~17AQ3tj.&& ( g r o w t h r a t e )
-39-
Dillltioll
rate(hr5
0.02
0.04
0.06
0.08
0.10
Biomass Lipid Fatty acid composition
Cl40 Cl&O C18:O C18:l C18:2 C18:3
6.7 57.2 9.1 24.0 14.4 26.6 22.1 3.8
3.9 37.0 9.8 20.6 13.4 22.2 29.5 4.5
2.6 16.9 6.5 18.5 11.7 24.7 34.2 4.4
1.6 16.2 12.6 14.9 8.9 20.4 37.7 5.5
1.4 13.6 10.2 14.3 8.6 20.2 39.6 7.1
a&mole
0.822
0.947
1.063
1.123
1.207
aA / mole designates the degree of unsahration [ 51 ]
-4o--4o-
Growth Lxssolved-oxygen Fatty acid composition
kmp- ( Oc ) tensim@= Of Hg.) 160 16~1 17:l 1 8 : 0 18:l 18:2 l&3 &nolea
b3 0 7 5 17.5 5.5 1.4 tr 41.8 34.4 tr 1.18
2 0 14.7 3.4 tr tr 33.8 48.2 tr 1.31
5 15.1 3.4 2.4 2.9 29.0 45.0 tr 1.25
2 0 7 5 16.9 7.0 3.2 tr 27.4 36.8 8.6 1.37
2 0 17.2 6.8 3.0 tr 27.6 37.9 7.6 1.36
5 19.1 6.6 2.7 tr 27.7 37.0 6.7 1.29
1 5 7 5 13.9 7.1 1.0 tr 23.9 32.6 19.2 1.55
2 0 14.8 6.9 1.1 tr 24.2 31.6 21.3 1.60
5 15.6 7.5 tr tr 23.3 34.0 20.0 1.59
adegee of unsaturation [ 511
btrace amount detected less than 0.1 %
41-
-42-
2 N,H,, + 3 / 2 0, + oleic acid + 2 N, + 3 H,O + stearic acid
v-4 + 0, - 2 N, + 2 H,O
43-
-44--44-
1.4.3.3 fi?rllnzn?ofi?rllnzn?o
1.4.3.4 ihhUKffl¶fltlfld~ihhUKffl¶fltlfld~ (Catalyst and Oxidizing agent)
2 KOOCN=NCOOK + 4H,O +H,N - NH, + 4KHC0, + N,
47-
48-
49-
2.1.1 d
&u”l$ubfi (purified linseed oil) lJOW%l Daler-Rowney n)5EAVW&“snQ%J
d4AflU commercial grade, lilu’wll*l8~A%J commercial grade, 1BFl519W18AFi59l (hydrazine
hydrate, H4N2.H20) FI~lUAu’olu’u 24% kJ~%.Al~5 AAK!til58Fl~ (pyrrolidine) A8U
analytical grade lJOW%l Fluka, f’l59Wfl.h~AWl~~Uo”tl (heptadecanoic acid, C16H33COOH)
&law technical grade, &n^AnRkWI58fl6~VlQ%J (butylaled hydroxytoluene:BHT) llOW%l
Sigma, ~FIAWlt~?IAIlEli ( diethyl ether, (QH5 )20 ) &A&J analylical grade UOW%l J.T.
Baker Chemical, I~AA~nA3uUA~o;~5r~al?ud; ( potassium ferricyani&, &Fe(cN)G ) ‘IJO9
ll%?Vl May & Baker Ltd. Dagenham ~5tAW’&flr\IA, AWl!li~Uflfl8%56 (methylene chloride,
CH2C12 ), A8fIAW.A (hexane), AUYIIUOQ (methanol), AElVllUOlt (ethanol), fI5F1~6l~FWlEl~~~
(hydrochloric acid), fW&$?rl (sulfinic a c i d ) , Id66m763uulatwm%~4 (potassium
hydroxide), nWlEWi%nAv’u~U (glacial acetic acid), kl~%AkJ¶JFll~YOAUS+l (disodium
carbonate), np%la @(+)-glucose), ~~AA~~A~~ol~~~a~~5A~U~~~A~~ (potassium dihydrogen
phosphate), rAY~A~uUls~~5AoUr~~A~~ (disodium hydrogen phosphate), AAWl~A&l&lAv(~
(magnesium sulfate), AAOlJ%JAilWJ&AArl9l (ammonium sulfate), AAfWAA3tJllflAlDl5a' (calcium
chloride)
2.1.2 DlH?sA&&lPs
~tT~AEln~AAVl5fl (yeast extract) lJEl4 Difco Laboratories Detroit, sabouraud
dextrose 4% , sabouraud dextrose agar
-51-
2.1.3 ti
pH meter: Fisher Accumet Model 325 expanded scale research, analytical balance:
Sartorius model A2OOS, Centrifuge : Centronic uow% P.Selecta l&l S - 577, shakiug
incubator : Lab term WN Adoff Kuhrur A.G. ilJ CH-4052 ~5~65lfEf%T6%8~66tlU~, water
bath, heater: Sybrou thermolyue type 1000, n5~~l~nsQ~luR~laalU6~5n (celhrlose nitrate)
UUlFt~nWN 1.2 !lJnSOU, HnOWlWlFNlJUl~ 2.5 x 12 L’W~IlJ995, 6fl~W666dfl5lJldPln5l~6fl~W666dfl5lJldPln5l~
(gas chromatography, G-C.) model GC 14 A UENlJ%l shimadzu kJ~5Wkt66IJlJ flame
ionization detector (PID) dE6t%J6f&~~5XJ?tWn model C-R4A, 6n~fI~66f%~n5IJlkln51~
66tl~66lJ~~6l.kl%Ps6lJVl~ (gas chromatography-mass spectrometry, GC-MS) model GC 8086 MS
d53.lXlWd’XJ~d566nToJ Mass Lab ~&lE&J~66Yll66dtltd~&lE&J~66Yll66dtltd SP-2330 6fi#tW&J 80%
biscyauopropyl, 20% cyauopropylphenyl-polysiloxaue KllU~l1KllU~l1 30 6UPlT llUlF66#U~l~U&6tll~
0.25 0.25 u"tln"6WI5u"tln"6WI5 66fltflfIn'oJu'66fltflfIn'oJu' Gmegawax 320 (o-wax) 6fln”DIJkXJ polyethylene glycol fl1lUQ111fl1lUQ111
30 6I.6995 UUl9l6t&w’l~U&W 0.32 u”nii6Wls UEIW%l Supelco
44 4 22 4'4 'YY2 . 3 2.3 fI156Bl5tJll'PI'1SLfiOJLLAwfJlMl56PU~fI156Bl5tJll'PI'1SLfiOJLLAwfJlMl56PU~
-52-
2.3.2.1 Sabouraud dextrose agar (SDA) 66BE sabouraud dextrose 4% $4
sabouraud dextrose 30 fI%l n=a?u9Urf;“~Ulln=11~~~~~~~5~U*5~ 1 &I5 66#&.h6&1
2.3.2.2 Production media piaUlls=nDulla~~lU~l5~~~ 3.1
2.3.3.2 3.5% methanolic H2SO4 (~llf~~~~WWl5): h.h5F&l~~~ 1.9
&Id%5 &MllU8tI 80 &&%l5 166+-dYhl.hFl56fhJ 100 ih%h5#Jti6WllUtM
2.4.1.1 “?56&JUH”16& (inoculmn) 6%~6&hh=lWU SDA-slant lhd
QQ6M$hhN 24 l+‘Jh ol”~Ui~a~~d6o~~66~~rls~u?ol 10 Qti (loop) iWhll?F@IfU~
(erylenmayer flask) Wlfl 250 u”&ol5 &J55~tllH15 sabouraud dextrose 25 h%hl5 \ql!d
lhJ$! 30°91. QfWXfll56lJth66YY reciprocal 120 5OlJ~Ol.61~ UIU 24 &JhlJ 66hI!l?~%‘#6fh
-53--53-
-54-54
4 wd WCI2.4.4 ~156Pl5~~~~~U~dlws~1RsPl~~~51~ (G.C.)
-55-
-56-
-5s
PKNO TIME4 4.375 4.978 7.339 8.33
1 0 10.271 1 13.3
AREA HEIGHT CONC NAME-7099 1293 17.34 ME PALMITATE2 5 6 1 8 0.63 ME PALMITOLEATE
1660 1 6 6 4.06 ME STEARATE17776 1585 43.43 ME OLEATE6631 4 6 2 16.2 ME LINOLEATE1457 7 9 3.56 ME ARACHIDATE
-59-
PKNO TIME AREA6 4.33 62977 4.87 a 2 0
1 0 7.27 31381 1 8.27 426161 2 10.17 14501 3 13.17 9 5 31 4 14.1 7 3 4
HEIGHT CONC NAME1107 9.41 ME PALMITATE
48 1.22 ME PALMITOLEATE3 1 4 4.69 ME STEARATE
3928 63.66 ME OLEATE7 9 2.17 ME LINOLEATE
39 1.42 ME ARACHIDATE4 2 1 . 1 ME LINOLENATE(w31
-0
- 5
- 1 0
- 1 5
- 2 0
.
- 2 5
,J 1
rr-14 Cl8:3,n-3
PKNO T I M E AREA2 2.98 923 3. 33 144 3:97 73.5 4.88 37196 5.4 125‘7 6.49 55118 7.23 2179 8 . 8 2172
10 9.33 108741 1 10.38 40112 10.74 1184413 11’. 97 49714 13.19 4860315 14.66 1899
HE I GHT353
15 ’909
301029
36286
133346
129045
4229145
COW0.11 140.02 14.50.09 15.234.62 16.02
,o. 15 16.380 17.020.27 17.382.7 18.02
13.5 18.20. 5 18.54
14. -il 18.640.62 18.9-i
60.36 19.272. 36 19.59
CiRBOh; $
fll5El1HlTfll5El1HlT ( g / l ) ( g / l )
yeast extract
glucose
w2p04
Na2HPO4
MgS04.7H20
(NHq)2s04
CaC12.2H20
FeS04.7H20
CaC12.2H20
%lso,$7H’~o
CUSO4SH2O
CoCl2.6H2O
MIlSO4.H2O
(NHd2M0207
0 . 50 . 5
20.0
7.0
2.0
0.73
1.0
0.134
-
-
-
m?lJl~UOL%Okyl5il2
1.0
20.0
2.4
2.3
1.0
4.0
0.024
0.036
0.008
0.001
0.001
0.005
0.001
-62-
-63-
25 r
20t
tB medium 1
43 medium ltsoap
f medium 2
* medium 2tsoap
+ medium 9
* medium Stsoap
.0’ I I I I I I I
0 24 48 72 ee 120 144 166
Hours
4%
25c
20-
ClS-m
%$
ElO-
@ medium 1
+S medium l+soap
* medium 2
+ medium 2+soap
+ medium 3
* medium S+soap
-65-
-66-
01w1qmil 1 %I Lllo~~rU~nY~~uu’ulUlc~~U~~~~~
C16:O C16:l Cl&O C18:l C18:2 Cl&3 C22:O
1 36.7 1.8 24.7 28.3 4.3 4.0 0.4
lliihff$uaR 2 23.3 1.5 5.5 48.5 17.7 3.1 0.4
3 21.1 1.3 3.3 38.2 26.3 9.2 0.6
5 20.6 1.7 1.6 40.0 25.7 10.2 0.3
1 16.7 2.2 2.7 39.7 25.9 10.7 2.1
l~Ufl~~Ui)R 2 8.8 0.4 6.0 25.6 16.4 42.2 0.7
3 12.0 0.0 4.7 30.9 20.2 32.2 0.0
5 13.6 0.5 11.4 44.6 13.9 15.0 1.1
Dlwlq91’1il 1 Td 6llo~l~~~~luu’u~ul~nn;o~u”ao;
C16:O C16:l C18:O C18:l C18:2 C18:3 C22:O
1 20.1 0.9 3.0 41.4 27.0 7.5 0.0
lti13U~Yj~U~R 2 14.0 1.8 6.8 53.6 19.8 4.2 0.0
3 17.8 0.6 3.6 49.1 24.0 4.9 0.0
5 16.8 1.2 2.6 49.1 25.6 4.7 0.1
1 12.2 0.2 9.3 30.0 15.7 32.5 0.1
&Jfl$hJZJR 2 14.1 0.3 7.8 39.9 15.0 22.0 0.8
3 13.9 0.5 8.3 45.3 13.9 17.3 0.9
5 12.6 0.7 6.2 32.5 22.7 24.5 0.9
-67-
+=--l-! Cl8:O
f 15 Cl8:l,rv9- 1 0 f- - 1 i Cl8:2,n4
19 C18:3,n-3
- 1 5
PKNO6
A9
10
:s.1;141, ;16l/
- l-819302 1
-20
T I SlE4. ai4.514.993. 666.196.547.157. 3;- 71
*;:j9. 559.89
1-l-L 3 9-14.9413.6318.38
AREA2;
3383322
284581720
20124184167
44i351418
6235444
61512335
430- - -I/3
HE I GHT COW CXRBOh: ;12 0.02 12.63
699 2.42 13.951 0.23 14.89
4237 20.35 15.92216 1.23 16.56
2311 0 16.9322 0.13 1-i. 4930 0.12 1;. 6’;
451 3.3 1;. 914805 36.78 18.48
13 0.04 19.13000 25.35 19.37
31 0.34 19.97878 8.82 20.19
36 0.31 20.4530 0. 55 22.06
>
-69-
--00
lK(NO?
256
8899
10101 11 1
TIYE ARE-44. jr 883j. 73 i9966. 26 2906.61 48377.45 106i. 8 16108. 5; 320999.97 lOi90
12:05 2209
.
HE I GHT188
140843
-iQ919
20224201031
179
CONC1.92
17.390.6300.23? -
4;: ;623.474 . 8
CARBON q14. OS1616.64171;. 741818.5319.3120.23
-7o-
- 0
- -3
-10
- 1 5
- 2 0
-25
PKNO4567‘St9
101 1121314151617181920
T I ?ilEil.335.03- -
;: ;66. 397. 217.137.88.629.389.629.97
Il.5313.0714.9818.1233.85 -
-iREX1670369
387341474
2346928747
33077126787
287733&Ml
-- 38860869
48252341
2548339
HE I GHT CONC CiRBON 7358 0.59 14. 16
32 0.09 15.133945 13.64 16. 11
109 0. 52 16.653069 0 17.08
28 0-l li-. 6317 0.02 17.82
3409 8. 12 18.0712872 44.63 18.63
25 0. 1 19.0533 0. 08 19.17
3633 13.68 19.3560 0.31 20.04
3741 16.99 30.2521 0. 12 21.2
102 0.9 22.0611 0.12 23.w
-71-
-72-
-73-
e R~taDap+ 572
+ 5l2tmp
24 4 8 7 2 95
Hours
120 144 155
-74-
ti
2
3
4
5
2
3
4
5
C16:O C16:l C18:O C18:l C18:2 C18:3 C22:O I
38.4 2.1 7.0 50.1 1.8 0.4 0.4
32.6 1.7 4.7 51.4 8.4 1.2 0.0
39.5 2.9 4.3 51.9 0.6 0.5 0.3
31.1 3.7 2.8 56.0 4.9 0.4 1.1
8.3 0.3 6.4 29.5 18.4 36.1 0.9
22.3 0.4 12.6 28.0 15.4 19.2 2.2
30.4 0.0 11.5 27.2 14.2 16.7 0.0
30.2 0.6 10.4 30.3 15.2 12.4 0.9
2 30.9 0.8 8.8 58.9 0.4 0.3 0.0
3 24.9 0.3 6.4 66.0 2.5 0.0 0.0
4 15.8 0.3 3.3 50.0 24.5 6.2 0.0
5 13.6 0.9 1.9 51.4 25.9 5.9 0.3
2 24.4 0.5 17.7 56.4 0.7 0.4 0.0
3 38.9 0.0 20.3 34.8 4.3 1.8 0.0
4 33.7 0.4 15.8 42.2 5.7 2.2 0.0
5 32.0 0.0 14.2 29.9 11.3 11.1 1.4
-75--75-
-76-
-0
- 5
- 1 0- 1 0
- 1 5- 1 5
- 2 0- 2 0
- 2 5- 2 5
-- 3 0 3 0
PWO IlYEIlYEi JJ8 - 1 .- 1 . 56560 4.974.97
IO j . . 3j . . 3I I 3 . 7 23 . 7 2I:! 6 .6 . I I I II3 6 . 5 96 . 5 9II 7.077.071 51 5 7. 3 61 61 6 7 . 77I iI i R. 051 81 8 8 . 4 08 . 4 0I OI O 9 . 1 99 . 1 92 02 0 I O . I O . 552 12 1 1 11 12 22 2 1 2 . 0 61 2 . 0 62 32 3 I ? .I ? . 4 3 4 32 42 4 1 3 . 0 91 3 . 0 92 52 5 13.4713.472 62 6 1 3 . 8 31 3 . 8 32 i2 i I - 1 . 6 9I - 1 . 6 92 s2 s 16.2716.272 92 9 1 6 . 7 31 6 . 7 33 03 0 I i . 1 5I i . 1 53 13 1 1 8 . 1 91 8 . 1 93 '3 ' 1 9 . 6 21 9 . 6 23 33 3 1 9 . 9 91 9 . 9 93 43 4 29.2929.293 53 5 2 3 . 2 3 . SFSF16 2 1 . 7 42 1 . 7 4
1 dd Cl&OCl&Ozrzr Cl&l,n-0Cl&l,n-0
I-lo.f---- 4 14 1 tXM,n-3tXM,n-3
AREA H E I G H TH E I G H T3 1 03 1 0 9 59 5
3 8 1 93 8 1 9 9 ' ) '9 ' ) '715 I J OI J O318318 3 93 9
i 6i 6 I OI OIOi520IOi520 185381853816655'16655'
141;141;2790927909
1 8 01 8 04 1 34 1 3 4 84 8
2 0 1 62 0 1 6 2 0 02 0 048524852 i40i405 6 3 35 6 3 3 7187184 2 84 2 8 j lj l
3 7 0 3 3 ‘ I 0 9 1‘ I 0 9 11 8 7 5 5 6 I85i 1
3 2 6 13 2 6 1 36X36X12311231 2 82 83 5 53 5 5 I ?I ?6 1 36 1 3 S TS T
12211221 I I OI I O1 7 0 51 7 0 5 I70I70
5 . 1 65 . 1 6 3 33 3I304I304 9 59 55 1 65 1 6 3 83 8337337 2 82 8
2 5 9 72 5 9 7 2542547 37 3 ii
3 0 03 0 0 2 42 41 0 1 01 0 1 0 1 31 37 8 6 17 8 6 1 i l li l l
C O N C0 . 0 50 . 0 50 . 6 30 . 6 30.120.120 . 0 50 . 0 50 . 0 . 0 10 1
I i . 7 9I i . 7 92 9 . 62 9 . 60 . 2 30 . 2 30 . 0 70 . 0 70 . 3 30 . 3 3000 . 9 30 . 9 30 . 0 76 . 1 36 . 1 3
3 1 . 0 43 1 . 0 40 . 0 . 5 . 15 . 10 . 20 . 20 . 0 60 . 0 60 . 0 . II0 . 20 . 20 . 2 80 . 2 80 . 0 90 . 0 90 . 2 20 . 2 20 . 0 90 . 0 90 . 0 60 . 0 60 . 1 30 . 1 30 . 0 10 . 0 10 . 0 . OSOS0 . 0 . I iI i1 . 31 . 3
15.13IS.41IS.41Ii.72Ii.72l bl b16.2816.2816.5516.55
22Ii.03 ,I i . 2 3I i . 2 31 7 . 5 21 7 . 5 2I 8I 818.1;18.1;I S . I S . 4949I S . 6 1I S . 6 1I C 3 . i ;I C 3 . i ;18.8718.87I S 9 6I S 9 619.1719.1719.5219.521 9 . 6 21 9 . 6 21 9 . 71 9 . 71 9 . 8 91 9 . 8 92 0 . 1 52 0 . 1 52 0 . 2 12 0 . 2 12 0 . 2 62 0 . 2 62 0 . 3 42 0 . 3 42 0 . . 1 92 0 . . 1 9
-77-
- 0
-- 5 5
--1 0
-1 5
- 2 0
- 25
---- A I CZ6.n3- 30
Pi\0 T I NT I N AFiEA8 4 . 1 94 . 1 9 16759 5. ?I lil
IO 5.09 3042111 6.41 8 1 9 911 6.95 3 6 013 i. 26 132I II I i. 5 3 2 3
15 7 . 6 7 18421 6 i. 93 2 2 5 31; 5.3; 122-1IS 9.07 4 3 719 10.34 8656‘0 10.76 2 0 9 3 3‘1 11.31 2 1 412 11.93 1 8 6 82 3 12.71 135521 1 3 . 1 453IS 13.713.7 10562 6 14.56 I18427 16.15 6 3 02s 16,61 20X32 9 16.92 2 3 03 0 I i . 6i 3 3 831 18.04 3213 2 15.81 22s3 3 19.47 2 7 3 03 1 20.9 83;3 5 21.5 i S 9 7
3 6 22. T3 5 7 0;i 23. o-l 10973 8 2 6 . 3 6 1 23 9 2;. 21 2 4 5 240 28.46 2 3 6 411 29.4 3 3 8 6 2
I I E I C I I TI I E I C I I T321
2 544321092
3i1.l4 9
2 3 02 3 2II
.lO863Ii32Ii32
2 62 6IS1119
488 2
1 2 16 2
1 5 71 5 7
::3 32 0
264G O
7SO33i ii i2 9
?OiIi3
3231
-12 30 OR 2514 116
I--- I O C16:O
2 52 6
35 C205,n-3
co\c cm:o\ =I. 2 I-l.820.12 15.-I-i
31.T 1 65.8; 16. ?b0 . 26 16 . 560.09 16.730.23 16.530 16.931.61 IT.060 . 8.5 Ii. 250.31 17.516.21 IS
14.9s IS. 140.15 1s. 311 . 3 - i 1% 50.9i 18.i2 ,0.35 IS . 810.5; I S . 97O.SS 19. Ii0.45 19.521.49 19.610 . 16 19.670.21 19.820.23 19.ss0.16 20.03I. 9s 20.130.6 20.373. 63 20.4i0.41 20. GJ0. is 20.650.34 21.11I.75 21.221.69 21. 36
2-1 .23 2 1 . 4 7
1.8 21.51
-0
-3
-10
89 , ~ C16:Oll
li 15Cl6:l,n-7
Cl&O _- iF3 Clt%l,n4
PKSO T IYE6 3.19
9 1.971 0 6 . 11 1 6.5612 7. o.!
l-1 s. 03
1 6 8.7li IO. 3i1 8 10.961 9 13.032 0 12.4-L2 1 13.2122 !6.2623 16.762-I 17.1415 22.862 6 7- -/.31
1 9 9 53i96
15526521
1 4 2 6 7 83323 0 61 2 24 8 14 1 13573 8 4235
HEIGHT467
822 6 41 0 9
1080521315
IS10
q-.7>>I4 2 3
2 33250
146113 61 41 15 - l2923331 5
:o.\c0.330.090. 380.15
L5.536.13
0.0-l0.0200.760.047.19
38.70. 10.080.03
-0.130.110. 10 . 10. 06
C.?IRBO\ =1 3 . 5 914.0-L14.815. i71 616.2816.5316.521;. 0-Ili. 2-l1,. 3-l1 818.1618.4918.618.82
- 19.5219.6219.6920.6321.21
-79-
-8O--8O-
1010 Cl6:OCl6:O
-15
-20- 25 11 Cl@0Cl@01212 Cl6:lA9;n-9Cl6:lA9;n-9
13 Cl8:lAl2;n-6
- 301515 Cl6:2A9,12;n-6Cl6:2A9,12;n-6
1616 Cl6:2A9,15;n-3Cl6:2A9,15;n-3
1s Cl8:3A9,12,15;n3
iREi HE I GHT COSC CARBOX =610: 7 3 0 5.51 16
-71531 137S 20.5 1S2 2 6 0 6 1 x4 21.53 1s. 161 3 3 6 0 ;-‘2 12.72 1 s . 2 9
6717 306 6.42 1s. 5 213631 69s 1’. 9s 1s. 58
7829 40s 7.46 1s. 716 0 7 0 153 5.78 1 s . 97 1 3 5 310 6.79 15. !P
-81-
-82-
J
-84--84-
o~l~6anlnl5r~uo~"5a%uu"u~~ 2 n'urtcjulu"iln5du~u~~n'urtcjulu"iln5du~u~~ 1 1 w5ua:Fjw5ua:Fj hJ6hmlhJ6hml
dlhJnl5?f~n"E1dlhJnl5?f~n"E1 C18:ti9,12,C18:ti9,12, C18:2C18:2 A9,1S 66flf66flf C18:2A12,15C18:2A12,15 09lUdlkl09lUdlkl
20r
16
15 16 21 24 27
mole ratio* 4
+ 8
f 12
+ 16
+20
- 8 6 -- 8 6 -
oo 33 66 88 1 21 2 1 51 5 1 81 8 2 12 1 2 42 4 2 72 7 3030
Reactbnthne(hours)
mole ratio-- 4 4
++ 88
ff 12 12
* 16
++ 20 20
-87-
-88-
18-
18 -
zp14 -
. r
&2-
.-ilo-& _
h 8-
8 -
4 -
-m- 0.1 g.
+ 0 . 2 g .
t- 0 . 5 g .- l.Og.
+ 1.59.
0 8 8 0 12 15 18 21 24 27 80
-89--89-
l*r
88
Q 0.1 g.
-e 0 . 2 9 .0 . 2 9 .
f 0 . 5 g .
- 1 . 0 g .+ 1.6g.
i---ri---r 11 11 11II II II II iibb ~-m~-m
II 99 66 99 1 2 12 15 1 8 21 2 4 2 7 3 0
Reaction time ( hours )
-9O-
-91-
2 0 -
1 0 -1 0 -
1 6 1 6 --
14 -
ap
?2-?2--9
,o,o0
vv10 -
N-
i -
8 -
06-
r
8 73 %+ 4 6 %f 31 %+ 16 %+ 0 %
0 3 6 9 12 15 18 2 1 24 27 30
R-ume(hows)
-92-
IO-
Q -
. -
7-ap
I
2J a -
u
K 5-
9
e
! 4- -
3-
-a-a 7 3 %%44 4 6 %4 6 %
ff 3 1 %%++ 16 %%++ 0 %0 %
-93--93-
-94--94-
0 3 6 9 12 1 5 18 21 2 4 2 7 3 0
Reaction times(hours)
* pH 0.0
* pH 0.0
* pH 11.0
4 pH 12.0
+ pH 13.0
-95-95
10-
Q-
u-
7-ap-
u3 o-
8S-
g _
3'liil 4-
8a-
2-
l -
* pti 0.0
* pH 9 . 8f pH 11.0
* pH 12.0+ pH 19.0
0 3 Q 1 2 15 lb 2 1 24 27 30
Rsadion timens(houm)
-96-
Octadec-124noic add, 96
-9%
0 S 8 0 12 1S 18 21 24 2 72 7 soso
Fbwtion timcw, ( hours )
* 3o”c
+- 50°C* 7o”c* 90°C
-99--99-
~~olnnolmsvo~mltn"ali~~~~~6flij~~olnnolmsvo~mltn"ali~~~~~6flij [[ 8 5 ] 8 5 ] 66ftE66ftE [86][86] fJd#fJd#
-d[A] / d t = k [ A ]
i%Jn"6~~~~ll~l~~Xi%Jn"6~~~~ll~l~~X In Ml, / [Alo= -kt
6dO6dO [Al : concentration of substrate
(1)
(2)
WI,: concentration of substrate at any times
Ml0 : concentration of substrate at time zero
t : reaction times
k : rate constant
66tl~6lhUD~~U~d66tl~6lhUD~~U~d exponential hW~l~6?hJhW~l~6?hJ
- k tCAlt/[Al, = e (3)(3)
dD- k t
C A It = [Q-p (4)(4)
ki logarithms 1#6?hJ h[AJ - ln[A]* = -kt (5)
H% lxl[A]tlxl[A]t = = -kt + ln[A], (6)
~~6~uum'ln"ynu"lr6~u~1J~~6~uum'ln"ynu"lr6~u~1J , , y=mx+by=mx+b
olnnlrn~no~63~Unslr~~~~l~olnnlrn~no~63~Unslr~~~~l~ l~[C18:3]~f%Jl~[C18:3]~f%J tt ndqtUM$ndqtUM$ 30,50,7030,50,70 66QZ66QZ
9O”V. +h6lZldlQdi 3.28 - 3.31 o~:r~~11lU~Ul~Ut~U~~~6~1~~ -k 66R=ihJd$%Jl~U~~
db%ildb%il k 1Tilfh1Tilfh 3.077~10-~,3.077~10-~, 8.025~10-~,8.025~10-~, 15.55oX1O-315.55oX1O-3 66Qt66Qt 20.740~16~20.740~16~ 9WdlkJ9WdlkJ $0$0
6ihJUflW'htiVf~l~6ihJUflW'htiVf~l~ InkInk fhfh l/rl/r (OK.(OK. ) ) f1lfWlYl4~f1lfWlYl4~ 3 . 6 3 . 6 66~~nlP1?lu~uuo~6~unslrl~~~~66~~nlP1?lu~uuo~6~unslrl~~~~
I&I& 3.32 3.32
k(mi.t?)
-33 . 0 7 73 . 0 7 7 xx 10
-38 . 0 2 58 . 0 2 5 x 10
lnk
-- 5 . 7 8 45 . 7 8 4
-- 4 . 8 2 54 . 8 2 5
T ( OK. ) l,T(IP)
-33 0 33 0 3 3.30x3.30x 10
-33 2 33 2 3 3 . 1 0 3 . 1 0 x 10
-3 -31 5 . 5 5 51 5 . 5 5 5 x 10 - 4.163 3 4 33 4 3 2 . 9 2 2 . 9 2 x 10
-3 -32 0 . 7 4 02 0 . 7 4 0 x 10 -- 3 . 8 7 63 . 8 7 6 3 6 33 6 3 2 . 7 5 2 . 7 5 x 10
-lOO-
-2 , I I I
-3 -3.077x10 -3 mill-1
slope =
lIl[C18:3] A
-5
~~
ln[C18:3]ln[C18:3] 44 _ __ _
-8 - -
-lOl-
- 3-3
-1. dOp= -15.550x10 min
lIl[C18:3] 4
4
-5
1~
4
-2 I I I I
-3 - --1
lIl[C18:3] 4 - -
-5 - -
00 30 60 90
time (minute)
120 150
-102-
-3
-4
I n kI n k
l/Tl/T xl&K-'1xl&K-'1
-103--103-
-Ea/RTk = A e
A : frequency factor
R : gas constant , 8.314 j/mol.‘K
T : Kelvin temperature
( 7 )( 7 )
Ea : activation energy
Iti logarithm t%wwlnun1s~Xt%wwlnun1s~X
- Ea/RTInk = h eh e +lnA (8)
N%l Ink = -Ea/R( l/r)+lnA ( 9 )( 9 )
PllU?arRn~~Uuo~~~~"57dQl"~11~PllU?arRn~~Uuo~~~~"57dQl"~11~ 3 . 3 2 3 . 3 2 1414 mm = - = - 4.258~10~4.258~10~ K lnlWi1lnlWi1
*??u~~lwnunl~~(s)ua=*??u~~lwnunl~~(s)ua= mm = = -- Ea/ R !#
-Es/R = - Ea / 8.314 J/mol. K
= - 4.258~10’ K
-105-105
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8 .8 .
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