4 !!y for thaiscience/article/61/10005800.pdfsulfuric acid. experiment on protein adsorption with...

$A/ 4____ . . . . . . _________j___ . . . . . . . .fgz s.... ____________..

i i

(war_m. ipi QbwmI)

. . . . !!Y_...& . . . . . . ____._____ . . . .fl$%BJf11$

R%%BJfIl$

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 &ltt~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.

loo

magnetic stirrer

&&lol~A4o;)

fl.1.2

0.06, 0.07, 0.08,

101

0.38

0.36

0.34

0.32

0.3

0.28

0.26

0.24

0.22

0.2

0.18

0.16

0.14

0.12

0.1

0.08

0.06

0.04

0.0;

(

I

I-

! i

I-

y = 0.0344x + 0.0213

I I I I I I I I I

0 1 2 3 4 5 6 7 8 9 10

102

hfad; OD,,, =

AoD,,, =

1. i3l~tE'tWl A: fXiWl sodium carbonate (Na,CO,) 20 k%.J %UtIlS

613i3Z~18 0.1 N sodium hydroxide (NaOH) 1 a”W3

2. 618tE'tllfJ B: tWiWJ copper (II) sulfate-S hydrate (CuSO, _ SH,O)

O S fI?I.J ~UtIl~il~ttl~ di sodium tartrate (C,H,Na,O, _ 2H,O) 1 %

(d%JlGl;ls/d?wlp15) 100 i.03.

103

2.

3.

4.

104

10s

0.4

0.36

0.32

0.28

0.24

0.2

0.16

0.12

0.08

0.04

0 I I I I I I I I I II -I

0 25 50 75 100 125 150 175 200 225 250 275

106

0.7

0.6

0.5

0.4

0.3

0.2

0.1

y = 0.1288x

R2= 0.9983

0 -

KllWWJTlUfKiFl

(%)

10

20

30

40

SO

5 ufl.

40

40

40

40

33.3

58.3

60

80

80

44.4

26.4 15.8 13.2

30.4 15.8 17

30.4 29.7 23

39.1 31.2 27.5

22.7 16.6 14

90 BJil.

9.9

13

18.7

21.8

15.6

40

40

33.3

10 %Jfl. 30 BJil. 50 aJil.

28 15.8

34.4 18.4

39.1 29.7

43.5 31.2

27.3 17.8

70 aJn.

13.2

17

23

27.5

14

10X

LIlill

(Uli)

10 46.6

2 0 SO

30 58.3

60 58.3

10% f-lB@l

46.6 54

46.6 60

63.3 I 80

68

80

80

80

6

6.5

7

7.5

8

0.1 %aJa13d

57

SO

49

50

45

46 30 33

40 44 31

35 31 27

27 26 34

31 30 31

110

25 8.8

50 16.2

75 13.3

100 12

150 9.6

ibm6

65

7

7.2

7.5

8

12.7

13

13.3

17.5

14.9

13.8

111

1s

16.7

18.5

17.6

17.4

16.1

14.5

6.8

31.4

80.4

185.2

227.5

253.5

268.1

2.5 12.5

5 16.1

10 18.5

15 15.1

20 12.7

25 10.7

9

d~xlladJlGuaJ~nafl

PJfi.>

0.02

0.05

0.1

0.2

0.3

0.5

0.7

0.9

1.1

1.3

1.2

3.6

6.7

10.7

13

14

16.1

17.3

18.5

18.5

113

60

70

13i.31

(In%)

0

10

20

30

40

SO

60

18.4 90.7

13.6 66.4

11.6 56.4

12.2 59.8

12 58.6

9.4 46.5

90 9.2 44.5

120 8.4 40.9

10 12.8 63.1

15 11.2 55.3

20 8 38.7

30 8 38.7

60 6 29.1

5 6.2 30.7

10 7.8 27.9

15 3.2 16

20 3 15.4

114

50 10 47 69.3

20 25.4 37.3

40 13.2 19.3

60 7 10.2

60 2 52.6 77.3

4 19 28

8 0.8 1.3

16 0 0