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Transcript
Bioindustri Enzim
Jurusan Teknologi Industri Pertanian, Fak Teknologi Pertanian
Universitas Brawijaya Malang
http://nurhidayat.lecture.ub.ac.id
http://ptp2007.wordpress.com
Enzim
• Enzim, dihasilkanoleh sistem hidup,
merupakan protein yg memiliki sifat
katalitik.
• Sebagai katalis, enzim efisien dan sangat
spesifik terkait keterlibatanya dalam reaksi
kimia.
• Cofactors terlibat dalam reaksi dimana
molekul dioksidasi, reduksi, dioecah
ataupun digabung.
Biotechnology
• Teknik yang melibatkan penggunaan
oragnisme hidup atau produknya
untukmembuat atau memodifikasi produk
untuk tujuan komerial.
Main Enzyme Classes
____________________________________________________
Enzyme class
Catalyzed reaction
____________________________________________________
Oxidirectadases
Oxidation-reduction reaction
Transferases
Transfer of functional group
Hydrolases
Hydrolytic reactions
Lyases
Group elimination (forming double
bonds)
Isomerases
Isomerizaion reaction
Ligases
Bond formation coupled with a
triphosphate cleavage
____________________________________________________
Enzymes in Biotechnology
• Enzymes in food and beverage production
Dairy industry
Beer industry
Wine and juice industry
Alcohol industry
Protein industry
Meat industry
Baking industry
Fat and Oil industry
• Enzymes as industrial catalysts
Starch processing industry
Antibiotic industry
Fine Chemicals industry
Enzymes in Biotechnology
• Enzymes as final products
Detergent industry
Cleaning agent industry
Pharmaceutical industry
Animal feed industry
Analytical applications
• Enzymes as processing aids
Textile industry
Leather industry
Paper and pulp industry
Sugar industry
Coffee industry
Faktor-faktor penting kenapa digunakan
enzim
• kemungkinan reaksi tidak dapat dilakukan secara kimia.
• Reaksi spesifik
• Mereduksi jumlah tahapan proses yang dibutuhkan.
• Mengeliminasi kebutuhan pelarut organik dalam proses.
• Enzim dapat digunakan ulang melalui imobilisasi.
• Dapat dikombinasikan dengan proses lain.
• Enzim dapat diperbaiki melalui rekayasa genetika.
Industrial Enzyme Market
Annual Sales: $ 1.6 billion
Food and starch processing: 45 %
Detergents:
34 %
Textiles:
11 %
Leather:
3 %
Pulp and paper:
1.2 %
Beberapa contoh enzim mikrobial
• Protease: protease netral dari Aspergillus
dan Alkali dari Bacillus
– Deterjen biologi: subtilisin dari Bacillus
licheniformis dan B. subtilis
– Penjernihan wine
– Pengolahan kulit
– Pembuatan keju
– Pengempukan daging dsb
Lipase
• Lipase terutama dari Bacillus, Aspergillus,
Rhizopus, dan Rhodotorula
–
–
–
–
Deterjen biologis
Pengolahan kulit – penghilangan lemak
Produksi senyawa flavor
Pengolahan susu dan daging
Alfa Amilase
•
•
•
•
•
•
Sumber: Aspergillus dan Bacillus
Untuk pengolahan pati menjadi sirup gula
Modifikasi tepung dalam pembuatan roti
Hidrolisis pati pada industri wine
Detergen biologis
Manufaktur tekstil
Beta Amilase dan
Amiloglukosidase
• Bacillus polymyxa, Streptomyces, Rhizopus
– Untuk produksi sirup maltosa
– Industri beer: meningkatkan gula yg dapat
difermentasi.
• Amiloglukosidase: A. niger, R. niveus
–
–
–
–
Produksi sirup glukosa
Roti,
Beer, wine
Juice buah
Production of High Fructose Corn Syrups
from Starch
Corn Starch Slurry (30-35% DS, pH 6.0-6.5, Ca2+ 50 ppm)
Liquefaction
Thermostable a-Amylase
Gelatinization (105°C, 5 min)
Dextrinization (95°C, 2h)
Liquefied Starch DE 10-15
Saccharification
Glucoamylase
(60°C, pH 4.0-4.5, 24-72 h)
Glucose Syrups DE 95-96
Isomerization
Glucose isomerase
(pH 7.5-8.0, 55-60°C, 5 mM Mg2+)
High Fructose Corn Syrups (42% fructose)
Production of Glucose from Starch
_______________________________________________________________
Liquefaction
Saccharification
DE
Glucose
_______________________________________________________________
Acid
Acid
92
85
Acid
Glucoamylase
95
91
Acid/α-amylase
Glucoamylase
96
92
α-Amylase/High pressure
cooking/ α-amylase
Glucoamylase
97
93
α-Amylase (thermostable)
Glucoamylase
97
94
α-Amylase (thermostable)
Glucoamylase
97-98.5 95-97.5
_______________________________________________________________
Conversion of Glucose to Fructose
HO
OH
O
OH
glucose
isomerase
HO
O
OH
HO
OH
HO
OH
OH
Enzim mikrobial komersial
• Enzim detergent
• Enzim dalam pengolahan Pati dan
karbohidrat
• Enzim dalam produksi keju
• Enzim dalam produksi juice
• Enzim dalam Manufaktur tekstil
• Enzim dalam manufaktur kulit
• Enzim dalam penanganan pulp kayu
• Enzim dalam sintesis bahan organik
6-Aminopenicillanic Acid (6-APA)
Penicillin:
First discovered by Fleming in 1932
19% of worldwide antibiotic market.
Superior inhibitory action on bacterial cell wall synthesis
Broad spectrum of antibacterial activity
Low toxicity
Outstanding efficacy against various bacterial strains
Excessive use has led to development of resistant pathogens
6-APA: Raw material for production of new semisynthetic penicillins
(amoxycillin and ampicillin)
Fewer side effects
Diminished toxicity
Greater selectivity against pathogens
Broader antimicrobial range
Improved pharmacological properties
Chemical and Enzymatic Deacylation
of Penicillins to 6-APA
R
C
H
N
S
O
N
CH3
CH3
Penicillin acylase
COOH
O
Penicillin V or G
[R=Ph or PhO]
Alkaline
[Enzymatic]
N
O
(6-APA)
PCl5
ROH
H2O
[Chemical]
Pyridine
Me3SiCl
R
S
NH2
C
H
N
S
O
N
O
CH3
CH3
COOSiMe3
CH3
CH3
COOH
6-Aminopenicillanic Acid (6-APA)
Chemical method:
Use of hazardous chemicals - pyridine, phosphorous
pentachloride, nitrosyl chloride
Enzymatic method:
Regio- and stereo-specific
Mild reaction conditions (pH 7.5, 37 oC)
Enzymatatic process is cheaper by 10%
Enzymes:
Penicillin G acylase (PGA)- Escherichia coli, Bacillus megaterium,
Streptomyces lavendulae
Penicillin V acylases (PVA)- Beijerinckia indica var. Penicillium,
Fusarium sp., Pseudomonas acidovorans
Immobilized Enzyme:
Life, 500-2880 hours
Enzymatic Modification of Penicillins
to 6-APA and Semisynthetic Penicillins
R
C
H
N
S
O
N
O
CH3
CH3
COOH
Penicillin V or G
Penicillin acylase
Alkaline
[Deacylation]
S
NH2
N
CH3
CH3
COOH
O
(6-APA)
Penicillin acylase
[Acylation]
Acidic
Semisynthetic Penicillins
Synthesis of Acrylamide
•
•
•
Monomeric raw material for the manufacture of polymers
and synthetic polymers
Obtained by hydration of the cyanide function of
acrylonitrile
World market, 200,000 tpa
Chemical Process:
•
•
•
Reaction of acrylonitrile with water in the presence of
H2SO4 (90 oC) or a metal catalyst (80-140 oC)
Formation of toxic waste (HCN)
The reaction must be stopped to prevent the acrylamide
itself being converted to acrylic acid
Enzymatic Process:
99.9% yield
Kg quantity product/g cells
Acrylic acid is not produced
Fewer process steps are involved
Much more environmental friendly
Nitto Chemical Industry: 6,000 tons annually
Synthesis of Acrylamide
Copper-catalysed process
Microbial process
Nitrile hyratase and amidase reactions
Aspartame (L-Asp-L-Phe-Methyl Ester)
• Aspartame is dipeptide sweetener formed by linking the
methyl ester of phenylalanine with aspartic acid
Extensively used in food and beverages
200 times as sweet as sucrose
Annual sale: 200 million Ibs, $ 850 million
Nutrasweet Corp. retains 75% of the US market
Chemical method:
The amino group of aspartic acid needs to be protected to prevent its
reacting with another molecule of aspartic acid to give unwanted
by-products
The correct single enantiomer of each of the reactants must be used
to give the required stereochemistry of aspartame (beta-aspartame is
bitter tasting)
Enzymatic method:
Thermolysin promotes reaction only at the alpha-functionality
Mild condition, pH 6-8, 40 oC
Cbz, benzyloxycarbonyl
Biocatalytic Production of Aspartame
HO2C
Ph
+
PhCH2OCNH
CO2H
H2N
CO2Me
O
N-Cbz-aspartic acid D,L-phenylalanine
Methyl ester
thermolysin
H2O
HO2C
PhCH2OCNH
O
Ph
CNH CO2Me
O
Cbz-aspartame
Cbz, benzyloxycarbonyl
L-Carnitine
Thyroid inhibitor
Slimming agent
Dietary supplement for athletes
Only one enantiomer of the compound is used
Two biocatalytic routes are available to make L-carnithine.
Saccharomyces cerevisiae
Rhizobiaceae
Synthesis of L-Carnitine
O
O
Cl
reductase
H
O
Cl
OC8H17
g-chloroacetoacetic
acid octyl ester
HO
OC8H17
(R)-g-chloro-b-hydroxybutanoic
acid octyl ester
HO
Me3N
H
O
OH
L-carnitine
O
Me3N
OH
g-butyrobetaine
hydroxylase
HO
Me3N
HO
L-carnitine
OH
Synthesis of Naproxene
CO2H
*
biocatalysts
CH3O
CH3O
CO2H
CO2H
*
multistep
*
resolution
(D/L)
CH3O
CH3O
O
C CH2CH3
CH3O
CH3O
1)
Tartaric acid
2)
Br2
3)
Hydrolysis
CH3O
CO2H
*
Synthesis of Calcium – Antagonist Drug
Diltiazem
OMe
O
OMe
O
(R,R)
esterase
MeO2C
HO2C
racemate
OMe
S
(S, S)
O
Diltiazem
N
H
O
O
Synthesis of L-malic Acid and L-Aspartic Acid
from Fumaric Acid
HO2C
fumarase
HO2C
H
CO2H
H2O
fumaric acid
HO2C
HO
CO2H
L-malic acid
aspartase
HO2C
H
CO2H
fumaric acid
NH3
HO
CO2H
L-aspartic acid
Environmentally Compatible Synthesis
of Catechol from Glucose
acetone
a
hydroquinone
b
HO
benzene
cumene
CO2H
OH
O
HO
d
OH
D-glucose
(a)
(b)
(c)
(d)
CO2H
OH
OH
OH
HO
d
d
O
c
phenol
OH
catechol
HO
OH
OH
3-dehydroshikimic protocatechuic
acid
acid
propylene, solid H3PO4 catalyst, 200-260°C, 400-600 psi.
O2, 80-130°C then SO2, 60-100°C.
70% H2O2, EDTA, Fe2+ or Co2, 70-80°C.
E. coli AB2834/pKD136/pKD9.069A, 37°C.
Draths and Frost, 1995
Debittering of Protein Hydrolyzates
•
•
•
•
•
•
Treatment with activated carbon
Extraction with alcohol
Isoelectric precipitation
Chromatographic separation
Masking of bitter taste
Enzymatic hydrolysis of bitter peptides
with aminopeptidase
with alkaline/neutral protease
with carboxypeptidase
• Condensation reactions using protease
Mill Scale Xylanase-aided Bleaching Trials
____________________________________________________
Sequence after
Pulp
Total active chlorine
Enzyme treatment
consumption
decrease (%)
____________________________________________________
(CD)EDED
Softwood kraft
21
(CD)EoDED
Pine kraft
18.4
(CD)EpDEpD
Birch kraft
18
(CD)EopDEpD
Pine kraft
12
DEopDED
Softwood kraft
15
____________________________________________________
C, elemental chlorine (Cl2), D, chlorine dioxide (ClO3), E,
alkaline extraction (NaOH), Eo/Ep, oxygen/hydrogen peroxide
reinforced alkaline extraction
•
•
•
•
•
Mannitol
Food additive
Reduces the crystallization tendency of sugars
and is used as such to increase the shelf-life of
foodstuffs
Used in chewing gum
Pharmaceutical formulation of chewable tablets
and granulated powders
Prevents moisture adsorption from the air,
exhibits excellent mechanical compressing
properties, does not interact with the active
components, and its sweet cool taste masks the
unpleasant taste of many drugs
•
•
•
•
•
•
Mannitol
Mannitol hexanitrate is a well-known vasodilator,
used in the treatment of hypertension
The complex of boric acid with mannitol is used
in the production of dry electrolytic capacitors
It is an extensively used polyol for the production
of resins and surfactants
It has low solubility in water of only 18% (w/w) at
25 oC
In alkaline solutions, it is a powerful sequestrant
of metallic ions
It is about half as sweet as sucrose
Hydrogenation of D-Fructose
H 2C
C
HO
OH
O
CH
H2, catalyst
HO
H 2C
OH
HC
OH
CH
H 2C
+
HO
CH
HO
CH
OH
HC
OH
HC
OH
HC
OH
HC
OH
HC
OH
HC
OH
H 2C
OH
H 2C
OH
H 2C
OH
D-Fructose
D-Sorbitol
D-Mannitol
Heterofermentative Conversion Pathway of Fructose into Mannitol
Fructose
2 Fructose
ATP
ADP
Fructose – 6-P
Glucose – 6-P
NADP+
NADPH + H+
6 - Phosphogluconate
NADP+
NADPH + H+
CO2
Ribulose – 5-P
Xylulose – 5-P
Glyceraldehyde - 3-P
2 ADP
NAD+
NADH + H+
2 ATP
Pyruvate
+
NADH + H
NAD+
Acetyl - P
ADP
ATP
Lactate
Acetate
2 Mannitol
Mannitol Production from Fructose
in pH-Controlled Batch Fermentation
Fructose
(g/L)
Time
(h)
Mannitol
(g/g)
Lactic Acid
(g/g)
Acetic Acid
(g/g)
150
15
0.720.00
0.17±0.00
0.12±0.00
200
40
0.69±0.03
0.17±0.00
0.13±0.00
250
64
0.70±0.02
0.16±0.00
0.12±0.00
300
136
0.66±0.03
0.15±0.01
0.11±0.00
At 37oC, 130 rpm, Initial pH 6.5, pH controlled at 5.0, 500 ml fleaker with 300 ml medium.
Fructose and Glucose (2:1) Co-Utilization and
Mannitol Production
Substrate or Product (g/L)
100
Fructose
Mannitol
O
37 C
pH 5.0
50
Lactic acid
Acetic acid
Glucose
0
0
12
36
24
Time (h)
48
Mannitol Production in pHControlled Fed-Batch Fermentation
Substrate or Product (g/L)
O
37 C
pH 5.0
200
150
Mannitol
100
Fructose
Fructose used:
300 g/L (final
concentration)
Lactic
acid
50
Acetic acid
0
0
24
48
Time (h)
72
96
Fermentation
Catalytic
Hydrogenation
All fructose converted to
mannitol
Only half of fructose
converted to mannitol
Co-product: lactic acid and
acetic acid one half of
mannitol
Co-product: sorbitol in
large excess (3)
Glucose is hydrogen source
in hydrogenation
Highly pure hydrogen gas
necessary
Nitrogen source essential for Nickel catalyst essential
growth
Ion exchanger for nickel
Electrodialysis for removing ions removal
organic acids
Highly pure substrates
to avoid catalyst
Use of less pure substrates necessary
inactivation
poses no problem
Enzymatic Conversion of Fructose to Mannitol
CH2OH
CH2OH
O
HO
H
H
Mannitol 2-Dehydrogenase
OH
NAD(P)H
H
OH
CH2OH
D-fructose
NAD(P)
HO
H
HO
H
H
OH
H
OH
CH2OH
Mannitol
Cofactor Regeneration
•
•
•
•
•
Chemical
Photochemical
Electrochemical
Biological
Enzymatic
Enzymatic Conversion of Fructose to Mannitol
with Simultaneous Cofactor Regeneration
Mannitol Dehydrogenase
Mannitol
D-Fructose
NADH
CO2 + H20
NAD
Na-Formate
Formate Dehydrogenase
Enzymatic Conversion of Fructose to Mannitol
with Simultaneous Cofactor Regeneration
Mannitol Dehydrogenase
Mannitol
D-Fructose
NADH
NAD+
Gluconic acid
Glucose Dehydrogenase
Glucose + H20