Transcript L-Arginine

2008. 11. 28
효소법을 이용한
기능성 아미노산의 상업적 생산
Topics
•
•
•
•
•
•
Chemical Feedstocks: L-Asp, L-Glu
Decarboxylation (L-Alanine, GABA)
Amino acids in Urea Cycle
ATP
NAD(P) regeneration
Transglycosylation
Bioreactor (IME)
• Batch-stirred
• CSTR
• PBR: rigid, non-adherent
Bioreactor (CO2↑)
• L-Alanine (Decarboxylase)
• GABA (Decarboxylase)
※L-Citrulline (Deiminase, NH3)
α-Ketoglutaric acid (Dehydrogenase,
NH3)
L-Aspartate
MH2 BIOCHEMICAL
GABA
A NEUROTRANSMITTER
(NATURALLY OCCURING)
FUNCTION: STRESS-RELIEVING
IMPROVE MEMORY & DIET EFFECTS
APPLICATION: Dietary supplements
ギャバ クロレラ 200mg 500粒
発売元 三井物産
メーカー希望小売価格：
\4,462
当社販売価格：\4,179(税込)
内容量：100g(1粒の重量200mg*500粒)
サイズ(外装)：幅62*奥行62*高さ118(mm)
[商品説明文]
「ギャバ クロレラ 200mg 500粒」は、クロレ
ラの細胞内にγ-アミノ酪酸(ギャバ)を代謝生産
させた、クロレラ食品です。毎日の健康のため
にお役立て下さい。
가바함유 화장품 – LG
생활건강
新개념 식물영양제의 출시
고창군과 MOU체결
Nylon-4 from GABA
L-Aspartate
• pH adjustment by substrate in
Crystallization
• Difference in solubility
maleic acid vs. fumaric acid
Urea Cycle
Amino acids in Urea cycle
• L-Arginine
• L-Ornithine: Putrescine, Agmatine
• L-Citrulline
MH2 BIOCHEMICAL
L-ORNITHINE
AN AMINO ACID IN UREA CYCLE
※CLAM SHELL
FUNCTIONS:
EASY RECOVER FROM HANG-OVER &FATIGUE
IMPROVE FACIAL WRINKLES
SECRETION OF GROWTH HORMONE
APPLICATION:
Dietary supplements
Cosmetics
MH2 BIOCHEMICAL
EFFECTS OF L-ORNITHINE ON FACIAL PROPERTIES
Improvement in Facial Properties
Placebo
L-OrnithineHCl
90
A CONSTITUENT OF
NMF IN SKIN
70
60
ORNITHINEPROLINE
COLLAGEN
50
40
30
20
10
Cr
ac
ks
G
lo
ss
in
es
s
Ro
ug
hn
es
s
sio
n
Te
n
rin
kl
es
W
Fr
e
ck
le
s
0
Co
lo
r
Improvement (%)
80
Reference: I. Toda, Food Style
21, Vol.10(No.2), 2006
오르니친의 숙취해소 효과
5시간 후 알데히드의 변화율,%
100.0
90.5
80.0
알코올+생리식염수
60.0
알코올+시트룰린
40.0
알코올+오르니친
20.0
알코올+아스파라긴산
0.0
-20.0
-8.1
-40.0
-41.8
-60.0
-80.0
-62.9
MH2 BIOCHEMICAL
L-CITRULLINE
AN AMINO ACID IN UREA CYCLE ※WATER MELON
FUNCTIONS:
HELP URINATION
DETOXIFICATION OF AMMONIA IN THE BLOOD
VIAGRA-LIKE EFFECTS (NO PRODUCTION)
APPLICATION:
Dietary supplements
Serving size: 3~6 g/day
Arginine metabolism
: Citrulline-NO cycle
L-Arginine
L-Arginine
increase the production
of cGMP
Why L-Citrulline ?
•
Extensive metabolism of L-Arginine (LA) in vivo.
•
L-Citrulline (LC) dosage increased the plasma LA more effectively
than LA.
•
LC improved the ratio of LA/ADMA (ADMA: inhibitor of NOS).
Conclusion:
L-Citrulline may be more effective
than L-Arginine
Reference: Pharmacokinetic and pharmacodynamic properties of oral
L-Citrulline and L-Arginine: impact on nitric oxide
metabolism.;Schwedehlm E. et al., Br. J. Clin. Pharmacol.
In press (2007)
MH2 BIOCHEMICAL
L-CITRULLINE
L-CITRULLINE-D,L-MALATE
ATP: IMP to GMP
Pyrophosphate instead of ATP
5′-IMP synthesis using E. coli overproducing wild-type or
mutated acid phosphatases. (Appl Environ Microbiol. 2000 July;
66(7): 2811–2816.)
GluDH/Mdh-coupled bioconversion system을
이용한 NADP+/NADPH의 재생과 이를
이용한 AKG 생산
Cofactor Regeneration
NADH oxidase
NAD(P)+
NAD(P)H
1. E.coli 에서의 E.coli gluDH 와 C.magnoliae mdh를
이용한 coupled system의 개발
1) PL promoter의 조절을 받는 E.coli gluDH cloning 및 발현.
2) Enzyme activity assay
Sample
Protein conc.
(mg/ml)
Activity
(U/ml)
Specific activity
(U/mg)
control
3.40
0
0
Recombinant
vector
4.70
0.29
0.06
⇒ PL promoter하의 gluDH와 이전에 만들어 놓았던 tac promoter하의
mdh로 coupling vector을 만들 예정.
2. E.coli 에서의 Thermus thermophilus의 gluDH와 NADH
oxidase을 이용한 coupled system 개발
1) Tac promoter의 조절을 받는 T.thermophilus gluDH cloning 및
발현
2) Tac promoter의 조절을 받는 T.thermophilus NADH oxidase cloning
및 발현
3. T.thermophilus 에서의 Thermus thermophilus의 gluDH와
NADH oxidase을 이용한 coupled system 개발
1) spl promoter의 조절을 받는 T.thermophilus gluDH cloning 및
발현
Carbohydrate Engineering
Natural
Carbohydrates
• Raw food materials
• An important constituent
in the human diet
Modified
Carbohydrates
Novel Carbohydrate
for Next Generation
• Alternative sweetner
• Functional
oligosaccharides
• Designed and Medical
Foods
• Nutraceuticals
• Glycobiology
Molecular disproportionation
Cyclization
+
Coupling
+
Disproportionation
Hydrolysis
+
H 2O +
+
Branched Oligosaccharides (BOS)
Low calorie sweetner
Humectant with low AW and viscosity
CH2OH
O
OH
OH
O
CH2OH
O
OH
OH
O
Starch (30%)
No dental caries
Liquefaction
Bifidus growth factor
CH2OH
O
OH
OH
O
CH2OH
O
OH
OH
O
CH2OH
O
OH
n= 1-2
CH2OH
O
OH
OH
MAase
O
OH CH
2
O
OH
OH
O
OH
Isopanose
CH2OH
O
OH
CH2OH
O
OH
O
OH
OH
O
CH2OH
O
OH
O
OH CH
2
OH
OH
Isomaltose
O
OH
OH
OH
OH
OH
Panose
O
O
OH
OH
OH
OH CH
2
OH CH
2
OH
OH
OH
O
OH
CH2OH
O
OH
O
CH2OH
O
OH
O
OH
Branched
maltotetraose
OH
CH2OH
O
OH
OH
OH
Transglycosylation of Natural Products
HO
OH
HO
O
OH HO
HO
O
OH
HO
H3C
HO
O
HO
HO
O
O
Properties
OH
H
HO
H
H
OH
HO
H3C
O
O
HO
O
HO
MAase
HO
O
HO
HO
OH
O
HO
O
HO
HO
OH
O HO
O
O HO
OH
H
HO
H
H
O
CH2OH
OH
H
OH
OH
CH2
O
OH
O
O
OH HO
MAase
O
O
Neutraceutical
OH
OH
HO
Genistin
OH
HO
O
HO
O
HO
HO
O
OH
O HO
O
OH
OH
O
O
HO
Antioxidant
O
OH
Maltosyl-ascorbate
HO
OH
O
HO
MAase
OH
Ascorbic acid
HO
HO
O
HO
O
OH
O HO
O
OH
O
OH
O
Bioactive
O
OH
O
+
R2
HO
O
Maltosyl-genistin
OH
HO
OH HO
Functional
Food
O
OH
HO
O
Maltosyl-sorbitol
HO
HO
Functional
Food Ingredient
Maltosyl-naringin
Sorbitol
O
O
O
O
CH2OH
OH
H
OH
OH
CH2OH
O
O
OH HO
HO
Naringin
OH HO
OH
OH
MAase
HO
O
HO
O
O HO
R1
Anthocyanin
OH
OH HO
MAase
OH
O
+
R2
HO
R1
Maltosyl-anthocyanin
OH
Transglycosylation of Naringin by BSMA
CH 2O H
O
OH
CH2O H
O
OH
OH
CH 2
O
O
O
OH
OH
OH
OH
OH
OH
CH 2OH
O
OH
O
O
O
O
OH
C H3
O
O
OH OH
OH
Major transfer product (G2-Naringin)
maltose linked a-1,6 to naringin
O O
CH 3
O
OH
OH
O
OH
OH
OH
Bacillus
stearothermophilus
Naringin (Acceptor)
maltogenic amylase
CH2OH
O
OH
OH
CH2
O
O
O
OH
OH
OH
O
OH
O O
CH3
CH 2OH
O
OH
CH 2OH
O
OH
OH
OH
O
O
OH
OH
OH
OH
OH
O
OH
CH 2OH
O
OH
OH
Minor transfer product (G1-Naringin)
glucose linked a-1,6 to naringin
Maltotriose (Donor)
CH2OH
CH2OH
O
OH
CH2OH
O
O
OH
OH
OH
CH2
O
O
OH
OH
O
OH
OH
O
O
O
OH
OH
O O
OH
CH3
OH
OH
Minor transfer product (G3-Naringin)
maltotriose linked a-1,6 to naringin
OH
O
TLC Analysis of Transfer Products
A
B
NG
NG
4
4
3
3
G2-NG
2
1
G1
G2-NG
2
1
G2
G3
1
2
A : U.V at 254nm
Std
MD
1
2
Std
B : H2SO4 dipping solusion
Developing solvent : Chloroform/Methanol/Water=65/35/10 (v/v/v)
Lane MD : Standards of Glucose, Maltose, Maltotriose
Lane 1: Transfer products Lane 2 : Purified G2- naringin
Lane Std : Standard of naringin
Solubility of G2-naringin
25℃ , in water
Neohesperidin
Naringin
G2-naringin
1.12mM
301.6mM
0.1mM
Sensory evaluation
Threshold value of bitter taste
Naringin
G2-naringin
0.0763mM
0.7102mM *
* Significantly different ( p<0.05 )
Transglycosylation of Ascorbic acid by BSMA
OH
A : H2SO4 dipping
solution
B : U.V. at 254nm
O
HO
HO
OH
OH
OH O
HO
O
O
HO
OH
O
O
OH O
HO
OH OH
OH
HO
Maltotriose
Ascorbic Acid
BSMA
OH
HO
HO
OH
HO
HO
O
OH
OH
O
HO
O
OH
+
O
OH
O
O
OH
O
O
OH
HO
OH
O
A MD 1
2
A MD 1
O
2
HO
Lane A : Ascorbic acid
Lane MD : Maltodextrin Standard
Lane 1 : a-1,6-Glucosyl ascorbic acid
Lane 2 : a-1,6-Maltosyl ascorbic acid
OH
a-1,6-Maltosyl ascorbic acid
a-1,6-Glucosyl ascorbic acid
Production
• L-Ornithine-HCl
• L-Ornithine- α -Ketoglutaric acid (2:1)
• L-Ornithine-L-Aspartic acid (1:1)
• L-Citrulline
• L-Citrulline-D,L-Malic acid (1:1)
• L-Citrulline-α-Ketoglutaric acid (2:1)
• GABA (γ-Aminobutyric acid)
• D-Glucosamine HCl
• Ricebran extract (RB90)
Thank you !