4 !!y for thaiscience/article/61/10005800.pdfsulfuric acid. experiment on protein adsorption with...
TRANSCRIPT
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Thesis Title
Thesis Credits
Candidate
Supervisors
Degree of Study
Department
Academic Year
Immobilization of Candida cylindracea lipase on rice hull ash
12
Miss Chitawan Tantrakoonsiri
Assoc. Prof. Dr. Kanit Krisnangkura
Asst. Prof. Narumon Jeyashoke
Master of Science
Biotechnology
1996
Abstract
Rice huh is very cheap agricultural waste and can be easily found in the country.
The ash mainly composes of silicon dioxide (SiO,) which is suitable for enzyme
immobilization. The binding capacity of rice hull ash can be increased by activating with
sulfuric acid. Experiment on protein adsorption with bovine serum albumin showed that rice
hull ash treated with 40% (volume/volume) sulfuric acid was the most effective adsorbent.
The activated ash could adsorb up to 80% of protein from the solution of 1 mg protein/ml,
but protein desorption in phosphate buffer was very high. In 0.01-0.1 M phosphate pH 6-8,
protein desorptions were 26-57%. Therefore, the immobilized enzyme was not suitable for
catalysis in aqueous system.
Immobilization of enzyme in organic solvent showed that 10% sulfuric acid
activated ash was suitable for immobilized of Candida cylindracea lipase. The optimum
amount of enzyme per gram of activated ash was 10 mg solid. Fifty microliters of water
were required to maintain the optimum activity of the enzyme. The immobilized enzyme
had optimum temperature and pH for hydrolysis of olive oil were 3O’C and 7.2, respectively.
The apparent K, and V,,, were 41 mM and 25 mole/hr/mg solid. Thermal stabilities
(half life) of the immobilized enzyme were increased to 42, 17 and 4 min at 50, 60 and 70°C
respectively but those of free enzyme were 16 and 3 min at 50 and 60 ‘C respectively.
Hydrolytic activity of immobilized enzyme in hexane at 30°C was 20.4 unit/mg solid which
was about l/3 of the free enzyme activity.
Keywords : Candida cylindracea / enzyme immobilization / enzyme in organic solvent /
lipase / rice hull ash / thermal stability
w
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
v
9
Q
Tf
%
QaI
1
1
3
6
7
9
14
16
19
20
21
28
28
34
35
37
41
J
2.3.5.1
2.3.5.2
2.3.6.1
2.3.6.2
2.3.6.3
2 3 6 . 4
2.3.6.5
2.3.6.6
I&
41
43
44
45
45
46
47
47
48
49
49
49
49
50
50
50
51
51
51
51
52
52
52
53
3.1
3.2
3.3
3.1.1
3.1.2
3.1.3
3.3.1
3.3.2
3.3.3
3.3.4
3.3.5
3.3.6
3.3.7
WWlWi%l
nod;53
53
53
54
55
55
55
59
59
61
64
64
64
67
67
67
74
78
81
81
86
88
99
1.1
1.2
1.3
1.4
3.1
3.2
“u.2
“u.4
“u-5
“u.6
“u.7
“u.8
5
8
17
25
56
109
109
110
110
“u.9
“u.10
“u-11
“u.12
“u.13
“u.14
HI&111
111
112
112
113
114
q¶lii mY1
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
4
5
6
9
10
11
14
15
16
57
65
66
68
69
70
71
72
73
75
76
77
79
8 0
105
106
4
IHO-C-H
IHO2 -H1.
c! 3H20H AH2OH
Glycerol 1 -Monoglyceride
0 0
R2
1,2-Diglyceride Triglyceride
PH2C -OH
HO -C - R1
H - C -OH r+ HOC-R2
H2C -OH
H2C -COO-R1
> H -
7
-COO-R;! + 3H20
H2C -COO-R3
Triglyceride
ST%*nxnavGu5u~3
12:o
140
16:0
l&O
20:o
2401 1.
ns~%%~u~au~aJ~3
16:1, A9 ; n-7
18:1, A9 ; n-9
1~2, A9, 12 ; n-6
1~3, A9, 12, 15 ; n-3
20:4, A5, 8, 11, 14 ; n-6
h=fl~~ 3k39WXIHJ gelwaouawaa,"%
Law-k Dodecanoic 44.2
Myristic Tetradecanoic 53.9
Palmitic Hexadecanoic 63.1
Steak Octadecanoic 69.6
Arachidic Eicosanoic 76.5
Lignoceric Tetracosanoic 86.0
Palmitoleic Hexadecenoic -0.5
Oleic Octadecenoic 13.4
Linoleic Octadecadienoic -5
Linolenic Octadecatrienoic -11
Arachidonic Eicosatetraenoic -49.5
IfIJr
WI5196 1.2 ~~o;l9a~M~~~~dlPJ19OH~WL~UrCdOJdrBb~aaa~~~~PJgil~~7wfdler [lo]
@Wl~u~C.WAri, v I
Ir3MIlM01~~10
Alcaligenes sp. Amano
Achromobacter sp. Meito Sangyo
Aspergillus niger Amano, Fluka
Bacillus subtitis Towa Koso
Candida cylindracea Sigma, Amano, Meito Sangyo,
Boehringer-Mannheim,
Fltia, Aldrich
Zandida lipolytica Amano, F?luka
Chromobacterium viscosum Sigma, Toyo Jozo
Geotrichum candidum Sigma, Amano
Humicola lanuginosa Amano
Mucor miehei Amano, Novo
PeniciUium camemberti Rhone-Poulenc
Penicillium roqueforti Fluka
Phycomyces nitens Takeda Yakuhin
Porcine pancreas Sigma, Amano, Boehringer-
Mannheim, Ruka, Aldrich
Pseudomonas aeruginosa AIllUlO
Pseudomonas fluorescens Amano, Fluka
Pseudomonas sp. Sigma, Boehringer-Mannheim
Rhizopus arrhizus Sigma, Boehringer-Mannheim,
Huka
Rhizopus delemar Sigma, Amano, Fluka
Rhizopus japonicus Amano, Nagase, Sangyo
Rhizopus oryzae Osaka Saiken Lab.
Rhizopus sp. Amano, Serva
Wheat germ SigmaI
L%O13*’ (non-specific lipase)
0
H2OH
Triglyceride
3 R C O O H + H H
CH20H
Fatty acid Glycerol
Triglyceride 1,2(2,3)-Diglyceride
0“20”
\c“20”
+ RCOOH
11
+ 2RCOOH
11
1.6
rp rp rstt-
o + St
P
+ TV + IIt + 1; + P + St
1.3.2.2 fl'WJ~l~Wl~~Bfl9~~V~U (Fatty acid specificity)
12
miehei
13
1.3.2.3 Enantioselectivity
14
Lipase+ H20 ,) + + RlCO2H + R3CO2H
0Triglyceride
1,2_Diglyceride
2,3-DiglycerideI
0
II J
R2CO + RlCO2H + R3CO2H
2-Monoglyceride
+ HO[fR2 + HO
1 -Monoglyceride
I
03-Monoglyceride
H20 Lipase
-OH
HO + R2CO2H
-OH
Glycerol Fatty acid
15
I OWnCH3
rOH
HO
- O H
Glycerol
16
I- TR3
0
Rl Mono-Substituted Triglyceride
+ CH3(CH2),&OOH - +
0Triglyceride Fatty acid
- l(CH2),CH3
I- TWH2)nCH3
0Di-Substituted Triglyceride
Composition I %
Silicon dioxide
Ahmkium oxide
Iron oxide
Calcium oxide
Magnesium oxide
Sodium oxide
Potassium oxide
Loss
(SiO,)
W,O,)
(Fe,O,)
00)
OW)
(Na,O)
6,O)
93.15
0.41
0.20
0.41
0.45
0.08
2.31
2.77
L Total I 99.78
18
19
Jod& (non polar)
20
22
23
24
25
Solvents log P Solvents log P Solvents log P
1. dimethylsulfoxide -1.3
2. dioxane -1.1
3. N,N-dimethylformamide -1.0
4. methanol -0.76
5. acetonitrile -0.33
6. ethanol -0.24
7. acetone -0.23
8. acetic acid -0.23
9. ethoxyethanol -0.22
10. methylacetate 0.16
11. propanol 0.28
12. propionic acid 0.29
13. butanone 0.29
14. hydroxybenzylethanol 0.40
15. tctrahydrofuran 0.49
16. diethylamine 0.64
17. ethylacetate 0.68
18. pyridine 0.71
19. butanol 0.80
20. pentanone 0.80
21. butyric acid 0.81
22. diethylether 0.85
23. benzylethanol 0.90
24. cyclohexanone O.%
25. methylpropionatc 0.97
26. dihydroxybenzene 1.0
27. methylbutylamine 1.2
28. propylacetate 1.2
29. ethylchloride 1.3
30. pentanol 1.3
3 1. hexanone 1.3
32. benzylformate 1.3
33. phenylethanol 1.4
34. cyclohexanol 1.5
3.5. methylcyclohexanone 1.5
36. phenol 1.5
37. m-phthalic acid 1.5
38. triethylamine 1.6
39. benzylacetate 1.6
40. butylacetate 1.7
41. chloropropane 1.8
42. acetophenone 1.8
43. hexanol 1.8
44. nitrobenzene 1.8
45. heptanone 1.8
46. benzoic acid 1.9
47. dipropylether 1.9
48. hexanoic acid 1.9
49. chloroform 2.0
50. benzene 2.0
5 1. methylcyclohexanol 2.0
52. methoxybcnzene 2.1
53. methylbenzoatc 2.2
54. propylbutylamine 2.2
55. pentylacetate 2.2
56. dimethylphthalate 2.3
57. octanone 2.4
58. heptanol 2.4
59. toluene 2.5
60. ethylbenzoate 2.6
61. ethoxybenzene 2.6
62. dibutylamme 2.7
63. pentylpropionate 2.7
64. chlorobenzene 2.8
65. octanol 2.9
66. nonanone 2.9
67. dibutylether 2.9
68. styrene 3.0
69. tetrachloromethane 3.0
70. pentane 3.0
7 1. ethylbenzene 3.1
72. xylene 3.1
73. cyclohexane 3.2
74. bcnzophenone 3.2
75. propoxybenzene 3.2
76. diethylphthalatc 3.3
77. nonanol 3.4
78. decanone 3.4
79. hexane 3.5
80. propylbenzene 3.6
8 1. butylbenzoate 3.7
82. methylcyclohexane 3.7
83. ethyloctanoate 3.8
84. dipentylether 3.9
85. benzylbcnzoatc 3.9
86. decanol 4.0
87. heptane 4.0
88. cymene 4.1
89. pcntylbenzoate 4.2
90. diphenylether 4.3
91. octane 4.5
92. undecanol 4.5
93. ethyldecanoate 4.9
94. dodecanol 5.0
95. nonane 5.1
96. dibutylphthalate 5.4
97. decane 5.6
98. undecane 6.1
99. dipentylphthalate 6.5
100. dodecane 6.6
101. dihexylphthalate 7.5
102. tetradecane 7.6
103. hcxadecane 8.8
104. dioctylphthalatc 9.6
105. butyloleate 9.8
106. didecylphthalate 11.7
107. dilaurylphthalate 13.7
26
Aq = d%lm water fled supportIEUXM water %U organic solvent
27
l-5.2.4
6Tfpd celite, duolite, cellulose, ethyl cellulose, silica gel, kieselguhr, clay, kaolin, alumina,
titania, nylon, sepharose, sephadex, activated carbon, avicel 66% porous glass L&6&4 0%
29
3 0
31
32
33
34
35
36
37
38
39
40
41
42
43
44
1.
2.
3.
1.
2.
3.
46
- Copper (II) sulfate - 5 hydrate (CuSO, _ 5H,O), Folin - Ciocalteu ‘s phenol
reagent, Di sodium tartrate (C,H,Na,O, . 2 H,O), Cupric acetate (Cu (CH, COO),) 66tX
Pyridine (C,H,N) a&-! analytical grade “uWl¶J~~‘ll E-Merck ~3X’lWJt33~¶.6?!
- Sodium hydroxide (NaOH), Sodium carbonate (Na,CO,) 66KZ Isooctane (CsH,&
6%.6 analytical grade 6u@W?~W Farmitalia Carlo Erba ~~X6’W~~l”a
- Potassium sodium tartrate (COOK(CHOH),COONa_4H,0) 6dod analytical grade
WWl?~‘W’l May and Baker LTD. ~‘X:aWW&l,“snt+J
- Hydrochloric acid (HCl), Sulfuric acid (H,SO,) 66tX Hexane (C,H,,)
6&6analytical grade ?JWl¶J~~~ J.T Baker Chemical ~X~6~ff~~~~~63.l?f61
- Palmitic acid (CiSH,,COOH) 6fll.6 technical grade ‘WISXI?PJ”W BDH Chemical
LTD. Poole ~3Z:aW&fltjpr
- ~lol’odolZ”Bn ( O l i v e O i l ) “UtXlpI?~~ Sigma Chemical CO., St. Louis,
MO, USA.
- Bovine serum albumin initial fractionation by cold alcohol precipitation fraction
V 9699% Albumin “uWlpI?~W Sigma Chemical CO., St. Louis, MO, USA.
- ao&w.~aanlnol” Candida cylindracea TKk%l’l Sigma Chemical CO.,
zJ66wl~~~ 905 gG36/oln.ros66&, 4570 i$~/ah&Js~Pd
48
- 6Wl6Wl (Muffle furnance) “UWI Lenton thermal designs $pd UAF 6/15
- ~‘ttSWW1 (Motar)
- PE66fI395tlU%Odl~ 63 t&X 106 ~Wl3t?II-!%t%l Endecotts LTD, London, England. .
- ~fl~tN38UWH Retsch ?I.! US 1000
- 6fl~CN6WJlWJIJ~IlgtI6T4~~ (Shaking incubator) 1I.I Model G 25 WI9 New
Brunswick Scientific CO., NJ, USA.
- ~tlWlJ89 Eyela Tokyo Rikakikai CO., LTD. lpd ND0600 ND* 1
- k&Pit4 2 d~KWJW3~~~ Sartorius 1” 1409 BMP 7-21 .
- M6h~J 4 <t~odWt3~&&l Oertling Jpd Model NA 264
- 6fl~i3?hfllTi~FlfIdPd66~~ (Spectrophotometer); spectronic 21 Bausch & Lomb1 *
- Sfl~i+dh’MU (Vortex mixer); Scientific Industries ~M~~WJ?fIl
- 6fl~~~~~~~l%J6%.6fl3~~1~ (pH meter) ipl’t3 MettJer 1” Delta 340* . I
- 6~~~9~~6w3us~alu6~~~~ (High speed micro refrigerated centrifuge MTX-150)
49
23.1 "1~6ahJ~~l66"au
5 0
23.4
51
52
V
Slope
54
10
20
30
40
SO
_ _
5 a.m./ 10 ML/ 30 ml_/
10 am. 10 l.m. 10 BJa.
2 5.8 7.9
2 6 9.1
2 8 9.1
2 8 11.7
1.6 4.4 6.8
so WLJ
10 ala.
7.9 9.2 8.9
7.9 11.9 11.7
14.8 16 16.9
15.6 19.2 19.7
8.3 9.8 14.1
70 BJfl./
10 m.
90 ML/
10 BJA.
@IO) 5 ufl./ 10 ML/ 30 m-L/
10 aJa. 10 m=I. 10 BJa.
10 2 5.8 8.4
20 2 6.3 10.3
30 2 8 11.7
40 2 8 13.4
SO 1.6 5.8 8.1
10 m.
7.9
9.2
14.8 16
15.6 19.2
8.9
10 81a.
9.2
11.9
9.8
90 WI./
10 aJa.
8.9
11.7
16.9
19.6
14.6
1
80
20
10
0 I I I I I I I I I ---I
+ 10% nset + 20% fncl * 30% frim
-c- 40% fricl + 50% fn@l
59
60
0 10 20 30 40 50 60 70
61
62
5.5 6 6.5 7 7.5 8 8.5
+O.l M --g 0.05 M + 0.02 M -X- 0.01 M
63
6 0
SO
4.0
3 0
20 I I I I I I
0 10 2 0 3 0 4.0 SO 60 70
+pH6 -E- pH 6.5 *pH7 -X- pH 7.5 -XC- pH 8
64
66
20
19
I8
17
16
15
I I I I I I i
0 25 50 75 100 12s 150 17s
IJ%mM~I (w-&w)
67
68
19
17
11
10
9
8 i
5.5 6 6.5 7 7.5 8 8.5
ihtf
69
90
70
60
6 6.5 7 7.5 8 8.5
70
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
I I I I I
71
so t
30 4
72
300
250
200
150
100
50
0 4
0 5 10 15 20 2s 30
Ihedaouacak (am)
73
20
19
17
16
15
14
13
12
11
10
9
0 5 10 15 20 2s 30
d%JIaaouY%k (am.)
74
75
0 0.2 0.4 0.6 0.8 1 1.2 1.4
d?aJI~~IGUaJ~nen (ml.)
76
-0 I__lY!rz- 18 I s 9 10 1-I
56
PKNO TIME
33 4.75
34 5.10
48 9.08
49 9.64
SO 11.03
AREA HEIGHT CONC NAME
17350 2746 8.98 ME PALMITATE
1584 191 0.82 ME PALMITOLEATE
5675 4S2 2.94 ME STEARATE
15236 10991 78.94 ME OLEATE
7990 575 4.14 ME LINOLEATE
77
I I I I I I I I I
-30 -20 -10 0 10 20 0 4.0 50 60 70 80 90 loo
(mllmmol)
78
79
0 20 40 60
l%ll (Insi)
8 0 100 120
I I I I IT “I I II
II I
82
84
86
87
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Brink, L.S., Tramper, J., Luyben, KM. and Riet, K-V., 1988, “Biocatalysis in
Organic Media,” Enzyme Microbial Technology, Vol. 10, pp. 736-743.
Khmehritsky, Y-L., Levashov, A-V., Klyachko, N-L. and Moutinek, K., 1988,
“Engineering Biocatalytic Systems in Organic Media with Low Water Content,”
Enzyme Microbial Technology, Vol. 70, pp. 710-724.
Bosley, J.A. and Clayton, J-C., 1994, “Blueprint for a Lipase Support : Use of
Hydrophobic Controlled-Pore Glass as Model Systems,” Biotechnology and
Santaniello, E., Ferraboschi, P. and Grisenti, P., 1993, “Lipase-Catalyzed
Transesterification in Organic Solvents : Applications to the Preparation of
Enantiomerically Pure Compounds,” Enzyme Microbial Technology, Vol. 15,
vv. 367-382.
89
11.
12.
13.
14.
1s.
16.
17.
18.
19.
20.
Malcata, F-X., Reyes, H-R., Garcia, H-S., Hill, C.G. and Amundson, C-H., 1992,
“Kinetics and Mechanisms of Reactions Catalysed by Immobilized Lipases,”
Enzyme Microbial Technology, Vol. 14, pp. 426-446.
33;“: tf&~33TPd, 2537, “%an33a.k&=t,” “74Jo=~Plnl~~~“?s~~~~~~~~~~~~.
hfi &; 19 t%13 ?i% : ?k~&w~\%Pd~~“%-U~l~, a”pa; 2-4 wq~fllt3%.l 2537,
a~~~~aw~9pd%~~~~~~~~~“~~~~~~, flolal., plea; 85-87.
Rua, M-L., Maurino, T-D., Fcmendez, V.M., Otero, C. and Ballesteros, A., 1993,
“Purification and Characterization of Two Distinct Lipases from Candida
cylindracea, ” Biochemica et Biophysics Acta, Vol. 1156, pp. 181-189.
Sonnet, P-E., Foglia, T.A. and Baillargeon, M-W., 1993, “Fatty Acid Selectivity :
The Selectivity of Lipases of Geotrichum candidurn,” Journal of the American
Oil Chemists’ Society, Vol. 70, No. 10, pp_ 1043-1045.
Hoshino, T., Yamane, T. and Shimizu, S., 1990, “Selective Hydrolysis of Fish Oil
by Lipase to Concentrate n-3 Polyunsaturated Fatty Acids,” Agricultural Biological
Chemistry, Vol. 54, No. 6, pp. 14S9-1467.
a%JV’U3sr” t?f’%tWS;, 2536, ‘&a%-&~~ &%k~Wkikt, dlpd”nw”U&%t~33&,
Wn;? 14-59.
Tanaka, Y., Hirano, J. and Funada, T., 1992, “Concentration of Docosahexaenoic
Acid in Glyceride by Hydrolysis of Fish oil with Cmdida cyltidracea Lipase,”
Journal of the American Oil Chemists’ Society, Vol. 69, No. 12, pp_ 1210-1214.
Yadwad, V-B., Ward, 0-P. and Noronha, L.C., 1991, “Application of Lipase to
Concentrate the Docosahexaenoic Acid (DHA) Fraction of Fish Oil,” Biotechnology
and Bioengineering, Vol. 38, No. 8, pp_ 956-959.
Hills, M-J., Kiewitt, I. and Muherjee, K-D., 1990, “Enzymatic Fractionation of Fatty
Acids : Enrichment of y-Linolenic Acid and Docosahexaenoic Acid by Selective
Esterification Catalyzed by Lipase,” Journal of the American Oil Chemists’
Society, Vol. 67, No. 9, pp_ 561-564.
Haraldsson, G.G. and Hoskuldsson, P-A., 1989, “The Preparation of Triglycerides
Highly Enriched with w-3 Polyunsaturated Fatty Acids via Lipase Catalyzed
Interesterification,” Tetrahedron Letters, Vol. 30, No. 13, pp_ 167 1 - 1674.
90
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Muherjee, K.D. and Kiewitt, I., 1991, “Enrichment of y-Linolenic Acid from Fungal
Oil by Lipase-Catalysed Reactions,” Applied Microbiology Biotechnology, Vol. 35,
pp_ 579-584.
Hills, M-J., Kiewitt, I. and Muherjee, K-D., 1990, “Lipase from Brassica napus L
Discriminates against Cis-4 and Cis-6 Unsaturated Fatty Acids and Secondary and
Tertiary Alcohols,” Biochemica et Biophysics Acta, Vol. 1042, pp. 237-240.
Shimada, Y., Sugihara, A., Maruyama, K., Nagao, T., Nakayama, S., Nakano, H.
and Tominaga, Y., 1995, “Enrichment of Arachidonic Acid : Selective Hydrolysis of
a Single-Cell Oil from Mortierefla with Candida cylindracea Lipase,” Journal of the
American Oil Chemists’ Society, Vol. 72, No. Ii, pp_ 1323-1327.
McNeill, G.P. and Sonnet, P-E., 1995, “Isolation of Erucic Acid from Rapeseed Oil
by Lipase-Catalyzed Hydrolysis,” Journal of the American Oil Chemists’ Society,
Vol. 72, No. 2, pp. 213-218.
Mbayhoudel, K. and Comeau, L.C., 1989, “Obtention selective del’acide
petroselinique a partir de fhuile de fenouil par hydrolyse enzymatique,”
Rev. Fr. Corps. Gras., Vol. 36, pp_ 427-431.
Hayes, D-G. and Kleiman, R., 1992, “Recovery of Hydroxy Fatty Acids from
Lesquerella Oil with Lipase,” Journal of the American Oil Chemists’ Society,
Vol. 69, No. 10, pp. 982-985.
Wade, J-R., 1991, Organic Chemistry, 2nd ed., New York, Prentice-Hall, pp. 223-276.
Mustranta, A., 1992, “Use of Lipases in the Resolution of Racemic Ibuprofen,”
Applied Microbiology Biotechnology, Vol. 38, pp. 61-66.
Tsai, SW. and Wei, H-J., 1994, “Enantioselective Esterification of Racemic
Naproxen by Lipases in Organic Solvent,” Enzyme Microbial Technology, Vol. 16,
pp_ 328-333.
Hedstrom, G., Backhmd, M. and Slotte, J-P., 1993, “Enantioselective Synthesis of
Ibuprofen Esters in AOT/Isooctane Microemulsions by Candida cyhdracea Lipase,”
Biotechnology and Bioengineering, Vol. 42, No. 5, pp. 618-624.
91
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
Malcata, F-X., Reyes, H.R., Garcia, H-S., Hill, C.G. and Amundson, C-H., 1990,
“Immobiliid Lipase Reactor for Modification of Fats and Oils-A Review,”
Journal of the American Oil Chemist; Society, Vol. 67, No. 12, pp_ 890-910.
Winkehnann, G., 1992, Microbial Degradation of Natural Products, Germany,
Weinheim, pp. 244-245.
~Us~JaWm~~~~~~l~sl~ls66~~~~~l~, 2536,“t6fla¶J : WsaHa”o~~~~~Q6fll~L~~1 .
llHa9al5~agnl~Pdln~~Q,” a&&k+: kd 15-16 wq%f~lnIJ, I& 10.
Proctor, A., 1990, “X-Ray Diffraction and Scanning Electron Microscope Studies of
Processed Rice Hull Silica,” Journal of the American Oil Chemists’ Society, Vol. 67,
No. 9, pp_ 576-584.
Damel, S.A., 1976, Rice Hull Ash as a Piazzolanic Material, Thesis, Master of
Engineering, Structural Material Program, Asian Institute of Technology, pp_ 26.
Proctor, A., Adhikari, C. and Blyholder, G-D., 1995, “Mode of Oleic Acid
Adsorption on Rice Hull Ash Cristobalite,” Journal of the American Oil Chemists’
Society, Vol. 72, No. 3, pp. 331-335.
Proctor, A. and Palaniappan, S., 1989, “Soy oil Lutein Adsorption by Rice Hull
Ash,” Journal of the American Oil Chemists’ Society, Vol. 66, No. 11,
pp_ 1618-1621.
Proctor, A. and Palaniappan, S., 1989, “Adsorption of Soy Oil Free Fatty Acids by
Rice Hull Ash_” Journal of the American Oil Chemists’ Society, Vol. 67, No. 1,
pp_ 15-17.
Proctor, A., Tan, L.C. and Palaniappan, S., 1992, “Phospholipid Adsorption onto
Rice Hull Ash from Soy Oil Miscellas,” Journal of the American Oil Chemists’
Society, Vol. 69, No. 10, pp. 1049-1050.
Liew, K-Y., Yee, A.H. and Nordin, M-R. 1993, “Adsorption of Carotene from Pahn
Oil by Acid-Treated Rice Hull Ask”Journal of the American Oil Chemists’ Society,
Vol. 70, No. 5, pp_ 539-541.
Laane, C. and Tramper, J., 1990, “Tailoring the Medium and Reactor for
Biocatalysis,” Chemtech, Vol. 20, No. 8, pp_ 502-506.
92
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
Gorman, L.S. and Dordick, J.S., 1992, “Organic Solvents Strip Water Off Enzymes,”
Biotechnology and Bioengineering, Vol. 39, No. 4, pp_ 392-397.
Klibanov, A.M., 1986, “Enzymes that Work in Organic Solvents,” Chemtech,
Vol. 16, No. 6, pp_ 354-359.
Dordick, J.S., 1989, “Enzymatic Catalysis in Monophasic Organic Solvents,”
Enzyme Microbial Technology, Vol. 11, pp_ 194-211.
Dordick, J-S., 1992, “Designing Enzymes for Use in Organic Solvents,”
Biotechnology progress, Vol. 8, No. 4, pp. 259-267.
Aldercreutz, P. and Matiasson, B., 1987, “Aspects of Biocatalyst Stability in Organic
Solvents,” BiocataJysis, Vol. 1, pp. 99-108.
Goderis, H-L., Ampe, G., Feyten, M-P., Fouwe, B-L., Guffens, W.M., Cauwenberg,
S.V. and Tobback, P.P., 1987, “Lipase-Catalyzed Ester Exchange Reactions in
Organic Media with Controlled Humidity,” Biotechnology and Bioengineering,
Vol. 30, No. 2, pp. 258-266.
Zaks, A. and Khbanov, A.M., 1984, “Enzymatic Catalysis in Organic media at
lOO”C,” Science, Vol. 224, pp_ 1249- 1251.
Zaks, A. and Klibanov, A.M., 1988, “Enzymatic Catalysis in Nonaqeous Solvents,”
The Journal of Biological Chemistry, Vol. 263, No. 7, pp_ 3194-3201.
Elliott, J-M. and Parkin, K-L., 1991, “Lipase-Mediated Acyl-Exchange Reactions
with Butteroil in Anhydrous Media,” Journal of the American Oil Chemists’ Society,
Vol. 68, No. 3, pp_ 171-175.
Brink, L.S. and Tramper, J., 1985, “Optimization of Organic Solvent in Multiphase
Biocatalysis,” Biotechnology and Bioengineering, Vol. 27, No. 8, pp. 1258-1269.
Laane, C., Boer-en, S. and Vos, K., 1985, “On Optimizing Organic Solvents in
Multi-Liquid-Phase Biocatalysis,” Trends Biotechnology, Vol. 3, pp. 251-252.
93
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
Rekker, R-F., 1977, The Hydrophobic Fragmental Constant, Amsterdam, Elsevier.
Laane, C., Boeren, S., Vos, K. and Veeger, C., 1987, “Rules for Optimization of
Biocatalysis in organic Solvents,” Biotechnology and Bioengineering, Vol. 30,
No. 1, pp_ 81-87.
Reslow, M., Adlercreutz, P. and Mattiasson, B., 1987, “Organic Solvents for
Bioorganic Synthesis l.Optimization of Parameters for a Chymotrypsin Catalyzed
Process,” Applied Microbiology Biotechnology, Vol. 26, pp_ l-8.
Zaks, A. and Klibanov, A.M., 1988, “The Effect of Water on Enzyme Action in
Organic Media,” The Journal of Biological Chemistry, Vol. 263, No. 17,
pp_ 8017-8021.
Reslow, M., Adlercreutz, P. and Mattiasson, B., 1988, “On the Importance of the
Support Material for Bioorganic Synthesis : Influence of Water Partition between
Solvent, Enzyme and Solid Support in Water-Poor Reaction Media,” Journal of
Biochemistry, Vol. 172, pp_ 573-578.
Zaks, A. and Klibanov, A.M., 1985, “Enzyme-Catalyzed Processes in Organic
Solvents,” Proceedings of The National Academy of Sciences of the United States
of America, Vol. 82, pp. 3192-3196.
Wehtje, E., Adlecreutz, P. and Mattiasson, B., 1993, “Improved Activity Retention of
Enzymes Deposited on Solid Supports,” Biotechnology and Bioengineering, Vol. 41,
No. 2, pp. 171-178.
Yamane, T., Ichiryu, T., Nagata, M., Ueno, A. and Shimizu, S., 1990,
“Intramolecular Esterification by Lipase Powder in Microaqueous Benzene:
Factors Affecting Activity of Pure Enzyme,” Biotechnology and Bioengineering,
Vol. 36, No. 10, pp_ 1063-1069.
Yang, D. and Rhee, J.S., 1992, “Continuous Hydrolysis of Olive Oil by Immobilized
Lipase in Organic Solvent,” Biotechnology and Bioengineering, Vol. 40, No. 6,
pp_ 748-752.
Brady, C., Metcalfe, L., Slaboszewski, D. and Frank, D., 1988, “Lipase Immobilized
on a Hydrophobic, Microporous Support for the Hydrolysis of Fats,” Journal of the
American Oil Chemist: Society, Vol. 65, No. 6, pp. 917-921.
94
64.
65
66.
67.
68.
69.
70.
71.
72.
73.
Montero, S., Blanco, A., Virto, M.D., Landeta, L.C., Agud, I., Solozabal, R.,
Lascaray, J-M., Renobales, M.D., Llama, M.J. and Serra, J.L., 1993, “Immobilization
of Candida rugosa Lipase and Some Properties of the Immobilized Enzyme,”
Enzyme Microbial Technology, Vol. 15, pp_ 239-247.
Lavayre, J. and Baratti, J., 1982, “Preparation and Properties of Immobilized
Lipases,” Biotechnology and Bioengineering, Vol. 24, No. 4, pp. 1007-1013.
Kimura, Y., Tanaka, A., Sonomoto, K., Nihira, T. and Fukui, S., 1983, “Application
of Immobilized Lipase to Hydrolysis of Triacylglyceride,” Europe Journal of Applied
Microbiology Biotechnology, Vol. 17, pp. 107-112.
Negishi, S., Sato, S., Mukataka, S. and Takahashi, J., 1989, “Utilization of Powdered
Pig Bone as a Support for Immobilization of Lipase,” Journal of Fermentation and
Bioengineering, Vol. 67, No. 5, pp_ 350-355.
Battistel, E., Bianchi, D., Cesti, P. and Pina, C., 1991, “Enzymatic Resolution of
(S)-(+)-Naproxen in a Continuous Reactor,” Biotechnology and Bioengineering,
Vol. 38, No. 6, pp_ 659-664.
Hoq, M-M., Yamane, T. and Shimizu., S., 1985, “Continuous Hydrolysis of Olive
Gil by Lipase in Microporous Hydrophobic Membrane Bioreactor,” Journal of the
American Gil Chemist: Society, Vol. 62, No. 6, pp. 1016-1021.
Garcia, H-S., Malcata, F-X., Hill, C.G. and Amundson, C-H., 1992, “Use of Candida
nzgosa Lipase Immobilized in a Spiral Wound Membrane Reactor for the Hydrolysis
of Milkfat,” Enzyme Microbial Technology, Vol. 14, pp. 535-545.
Ruckenstein, E. and Wang, X., 1993, “Lipase Immobilized on Hydrophobic Porous
Polymer Supports Prepared by Concentrated Emulsion Polymerization and their
Activity in the Hydrolysis of Triacylglycerides,” Biotechnology and Bioengineering,
Vol. 42, No. 7, pp_ 821-828.
Pronk, W., Kerkhof, P.M., Helden, C.V. and Riet, K-V., 1988, “The Hydrolysis of
Triglycerides by Immobilized Lipase in a Hydrophilic Membrane Reactor,”
Biotechnology and Bioengineering, Vol. 32, No. 4, pp_ 5 12-518.
Pronk, W., Boswinkel, G. and Riet, K-V-,1992, “Parameters Influencing Hydrolysis
Kinetics of Lipases in a Hydrophilic Membrane Bioreactor,” Enzyme Microbial
Technology, Vol. 14, pp_ 214-220.
74.
75.
76.
77.
78.
79.
80.
81.
82.
Guit, R-M., Kloosterman, M., Meindersma, G-W., Mayer, M. and Meijer, E-M.,
1991, “Lipase Kinetics : Hydrolysis of Triacetin by Lipase from Can&da cylindracea
in a Hollow-Fiber Membrane Reactor,” Biotechnology and Bioengineering, Vol. 38,
No. 7, pp_ 727-732.
Tanigaki, M., Sakata, M. and Wada, H., 1993, “Hydrolysis of Soybean Oil by Lipase
with a Bioreactor Having Two Different Membranes,” Journal of Fermentation and
Bioengineering, Vol. 75, No. 1, pp. 53-57.
Rucka, M., Turkiewicz, B. and Zuk, J.S., 1990, “Polymeric Membranes for Lipase
Immobilization,” Journal of the American Oil Chemists’ Society, Vol. 67, No. 12,
pp. 887-889.
Gelux, M.A., Norde, W., Kalsbeek, H.V. and Riet, K-V., 1992, “Adsorption of
Lipase form Candida rugosa on Cellulose and Its Influence on Lipolytic Activity,”
Enzyme Microbial Technology, Vol. 14, pp. 748-754.
Shaw, J-F., Chang, R-C., Wang, F.F. and Wang, Y-J., 1990, “Lipolytic Activities
of a Lipase Immobilized on Six Selected Supporting Materials,” Biotechnology and
Bioengineering, Vol. 35, No. 2, pp_ 132-137.
Mosmuller, E.W.J., Franssen, M.R. and Engbersen, J-J., 1993, “Lipase Activity in
Vesicular Systems : Characterization of Candida cyhdracea Lipase and Its Activity
in Polymerizable Dialkylammonium Surfactant Vesicles,” Biotechnology and
Bioengineering, Vol. 42, No. 2, pp. 196-204.
Bilyx, A. Bistline, R-G., Haas, M. and Feairheller, S-H., 1991, “Lipase-Catalyzed
Triglyceride Hydrolysis in Organic Solvent,” Journal of the American Oil Chemists’
Society, Vol. 68, No. 5, pp. 320-323.
Virto, M.D., Lascaray, J-M., Solozabal, R. and Renobales, M.D., 1991, “Enzymic
Hydrolysis of Animal Fats in Organic Solvents at Temperatures Below Their Melting
Points,” Journal of the American Oil Chemists’ Society, Vol. 68, No. 5, pp. 324-327.
Virto, M.D., Agud, I., Montero, S., Blanco, A., Solozabal, R., Lascaray, J.M., Llama,
M-J., Serra, J-L., Landeta, L.C. and Renobales, M.D., 1994, “Hydrolysis of Animal
Fats by Immobilized Can&da nzgosa Lipase,” Enzyme Microbial Technology,
Vol. 16, pp. 61-65.
96
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
Haas, M-J., Cichowicz, D.J., Phillips, J. and Mareau, R., 1993, “The Hydrolysis of
Phosphatidylcholine by an Immobilized Lipase : Optimization of Hydrolysis in
Organic Solvents,” Journal of the American Oil Chemists’ Society, Vol. 70, No. 2,
pp_ 111-117.
Haas, M-J., Scott, K., Jun, W. and Janssen, G., 1994, “Enzymatic
Phosphatidylcholine Hydrolysis in Organic Solvents : An Examination of Selected
Commercially Available Lipases,” Journal of the American Gil Chemists’ Society,
Vol. 71, No. 5, pp_ 483-490.
Han, D. and Rhee, J-S., 1986, “Characteristics of Lipase - Catalyzed Hydrolysis of
Olive Gil of High Concentration in Reversed Phase System,” Biotechnology and
Bioengineering, Vol. 28, No. 8, pp. 1250-1255.
Kang, ST. and Rhee, J.S., 1909, “Characteristics of Immobilized Lipase-Catalyzed
Hydrolysis of Olive Oil of High Concentration in Reverse Phase System”
Biotechnology and Bioengineering, Vol. 33, No. 11, pp. 1469-1476.
Kim, K-H., Kwon, D.Y. and Rhee, J-S., 1984, “Effects of Organic Solvents on
Lipase for Fat Splitting,” Lipids, Vol. 19, No.12, pp. 975-977.
Phutrakul, S. and Kanasawad, P., 1992, Immobilization of Lipase on Various
Supports and Its Activity in Water Poor Media, Chiang Mai, Chiang Mai University,
pp. l-10.
Padmini, P., Rakshit, SK. and Baradarajan, A., 1994, “Kinetics of Enzymatic
Hydrolysis of Rice Bran C&i in Organic System,” Enzyme Microbial Technology,
Vol. 16, pp_ 432-435.
Gray, C-J., Narang, J.S. and Barker, S-A., 1990, “Immobiliztion of Lipase from
Candida cylindracea and Its Use in the Synthesis of Menthol Esters by
Transesterification,” Enzyme Microbial Technology, Vol. 12, pp. 800-807.
Mat-lot, C., Langrand, G., Triantaphylides, C. and Baratti, J., 1985, “Ester Synthesis
in Organic Solvent Catalyzed by Lipase Immobilized on Hydrophilic Supports,”
Biotechnology Letters, Vol. 7, No. 9, pp. 647-650.
Not-in, M., Boutelje, J., Holmberg, E. and Huh, K., 1988, “Lipase Immobilized by
Adsorption,” Applied Microbiology Biotechnology, Vol. 28, pp_ 527-530.
97
93.
94.
9s.
96.
97.
98.
99.
100.
101.
Lie, E. and Molin, G., 1991, “Hydrolysis and Esterification with Immobilized Lipase
on Hydrophobic and Hydrophilic Zeolites,” Journal of Chemistry Technology
Biotechnology, Vol. 50, pp. 549-553.
Mustranta, A., Forssell, P. and Poutanen, K., 1993, “Applications of hnmobilized
Lipases to Transesterification and Esteritication Reactions in Nonaqueous Systems,”
Enzyme Microbial Technology, Vol. 15, pp. 133-139.
Basri, M., Amporn, K., Yunus, WZ., Razak, CA. and Salleh, A.B., 1995,
“Enzymic Synthesis of Fatty Esters by Hydrophobic Lipase Derivatives Immobilized
on Organic Polymer Beads,” Journal of the American Oil Chemists’ Society,
Vol. 72, No. 4, pp. 407-411.
Bloomer, S., Adlercreutz, P. and Mattiasson, B., 1990, “Triglyceride
Interesterification by Lipase.l.Cocoa Butter Equivalents from a Fraction of Palm
Gil,” Journal of the American Gil Chemists’ Society, Vol. 67, No. 8, pp. 519-524.
Carta, G., Gainer, J.L. and Benton, A-H., 1991, “Enzymatic Synthesis of Esters
Using an Immobiiied Lipase,” Biotechnology and Bioengineering, Vol. 37, No. 11,
pp. 1004-1009.
Carta, G., Gamer, J.L. and Gibson, M-E., 1992, “Synthesis of Esters Using a
Nylon - Immobilized Lipase in Batch and Continuous Reactors,” Enzyme Microbial
Technology, Vol. 14, pp. 904-910.
Stark, M.B. and Holmberg, K., 1989, “Covalent Immobiiization of Lipase in Organic
Solvents,” Biotechnology and Bioengineering, Vol. 34, No. 7, pp_ 942-950.
Cho, S.W. and Rhee, J.S., 1993, “Immobilization of Lipase for Effective
Interesterification of Fats and Oils in Organic Solvent,” Biotechnology and
Bioengineering, Vol. 41, No. 2, pp. 204210.
Yokozeki, K., Yamanaka, S., Takinami, K., Hirose, Y., Tanaka, A., Sonomoto, K.
and Fukui, S., 1982, “Application of Immobilized Lipase to Regio-Specific
Intersterification of Triglyceride in Organic Solvent,” Europe Journal of Applied
Microbiology Biotechnology, Vol. 14, pp. l-5.
98
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
Hertzberg, S., Kvittinagen, L., Anthonsen, T. and Skjak, G., 1992, “Alginate as
Immobilization Matrix and Stabilizing Agent in a Two-Phase Liquid System :
Application in Lipase-Catalysed Reactions,” Enzyme Microbial Technology, Vol. 14,
pp. 42.-47.
Gerhardt, P., Murray, R.G.E., Costilow, R.N., Nester, E-W., Wood, W-A., Krieg,
N.R. and Phillips, G-B., 1981, Manual of Methods for General Bacteriology,
Washington, pp.358-359.
Kwon, D.Y. and Rhee, J.S., 1986, “A Simple and Rapid Calorimetric Method for
Determination of Free Fatty Acids for Lipase Assay,” Journal of the American Oil
Chemist: Society, Vol. 63, No. 1, pp. 89-92.
fl$Mgw flq&&3QX, Gas Chromatography, arol~~~n~FPaf~~w~=ooPlana;aPa~‘$,
wlai 103.
Kheok, SC. and Lim, E-E., 1982, “Mechanism of Palm Oil Bleaching by
Montmorillonite Clay Activated at Various Acid Concentrations,” Journal of the
American Oil Chemists’ Society, Vol. 59, No. 3, pp. 129-131.
Brown, H.G. and Snyder. H.E., 1985, “Adsorption of Soy Gil Phospholipids on
Silica,” Journal of the American Oil Chemist: Society, Vol. 62, No. 4, pp. 753-756.
Saleh, MI. and Adam, F., 1994, “Adsorption Isotherms of Fatty Acids on Rice Hull
Ash in a Model System,” Journal of the American Oil Chemists’ Society, Vol. 71,
No. 12, pp. 1363-1366.
Bell, G., Todd, J-R., Blain, J-A., Patterson, J.D.E. and Shaw, C.E.L., 1981,
“Hydrolysis of Triglyceride by Solid Phase Lipolytic Enzymes of Rhizopus arrtius
in Continuous Reactor Systems,” Biotechnology and Bioengineering, Vol. 23,
No. 8, pp. 1703-1719.
Linfield, W-M., O’Brien, D-J., Serota, S. and Robert, A., 1984, “Lipid-Lipase
Interactions. I _ Fat Splitting with Lipase from Candida rugosa,” Journal of the
American Gil Chemists’ Society, Vol. 61, No. 6, pp. 1067- 1071_
Tahoun, M-K., 1986, “Large Agarose-Lipase Beads for the Hydrolysis of
Triglycerides,” Food Chemistry, Vol. 22, pp_ 297-303.
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