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 Glucoamylase 97 93

cooking/ α-amylase

α-Amylase (thermostable) Glucoamylase 97 94

α-Amylase (thermostable) Glucoamylase 97-98.5 95-97.5

_______________________________________________________________
 Conversion of Glucose to Fructose

HO
OH HO OH
O glucose O
isomerase
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
H
R C N S CH3 S CH3
NH2
Penicillin acylase
CH3 CH3
O
N Alkaline N
O COOH [Enzymatic] O COOH
Penicillin V or G (6-APA)
[R=Ph or PhO]

[Chemical] PCl5
Pyridine
Me3SiCl ROH
H2O
H
R C N S CH3
CH3
O
N
O COOSiMe3
 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

H
R C N S CH3 S CH3
NH2
Penicillin acylase
CH3 CH3
O
N Alkaline N
O COOH [Deacylation] O COOH

Penicillin V or G (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

+ thermolysin
PhCH2OCNH CO2H H2N CO2Me

O
H2O
N-Cbz-aspartic acid D,L-phenylalanine
Methyl ester HO2C Ph

PhCH2OCNH CNH CO2Me

O 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 reductase HO O
H
Cl OC8H17 Cl OC8H17
g-chloroacetoacetic (R)-g-chloro-b-hydroxybutanoic
acid octyl ester acid octyl ester

HO O
H
Me3N
OH
L-carnitine

O hydroxylase HO HO
Me3N Me3N
OH OH
g-butyrobetaine L-carnitine
 Synthesis of Naproxene
CO2H
biocatalysts *

CH3O CH3O
CO2H CO2H

multistep * resolution *
(D/L)
CH3O CH3O CH3O

O 1) Tartaric acid CO2H

2) Br2
C CH2CH3
3) Hydrolysis *
CH3O CH3O
Synthesis of Calcium – Antagonist Drug
Diltiazem
OMe OMe
O O
(R,R)
esterase
MeO2C HO2C
racemate

OMe

S (S, S)
O
Diltiazem
N O
H O
Synthesis of L-malic Acid and L-Aspartic Acid
from Fumaric Acid

HO2C fumarase HO2C

H

CO2H H2O HO CO2H

fumaric acid L-malic acid

HO2C aspartase HO2C

H

CO2H NH3 HO CO2H

fumaric acid L-aspartic acid

 Environmentally Compatible Synthesis
of Catechol from Glucose
acetone

hydroquinone
a b
HO c
benzene cumene phenol

OH CO2H CO2H
HO
OH
O d OH
d d catechol
HO OH O OH HO
OH OH OH
D-glucose 3-dehydroshikimic protocatechuic
acid acid
(a) propylene, solid H3PO4 catalyst, 200-260°C, 400-600 psi.
(b) O2, 80-130°C then SO2, 60-100°C.
(c) 70% H2O2, EDTA, Fe2+ or Co2, 70-80°C.
Draths and Frost, 1995
(d) E. coli AB2834/pKD136/pKD9.069A, 37°C.
 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 OH H 2C OH H 2C OH

C O HC OH HO CH

HO CH HO CH HO CH
H2, catalyst +
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+
CO2 NADPH + H+
Ribulose – 5-P

Xylulose – 5-P

Glyceraldehyde - 3-P Acetyl - P

NAD+ 2 ADP ADP
NADH + H+ 2 ATP
Pyruvate
NADH + H +

NAD+ ATP
Lactate Acetate 2 Mannitol
 Mannitol Production from Fructose
in pH-Controlled Batch Fermentation

Fructose Time Mannitol Lactic Acid Acetic Acid

(g/L) (h) (g/g) (g/g) (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

100
Fructose
Substrate or Product (g/L)
Mannitol
O
37 C
pH 5.0

50
Lactic acid
Acetic acid

Glucose
0
0 12 24 36 48
Time (h)
 Mannitol Production in pH-
Controlled Fed-Batch Fermentation
O
37 C
200
Substrate or Product (g/L)
pH 5.0

150 Mannitol

100 Fructose

Lactic Fructose used:

50 acid
300 g/L (final
Acetic acid concentration)
0
0 24 48 72 96
Time (h)
 Fermentation Catalytic
Hydrogenation
All fructose converted to Only half of fructose
mannitol converted to mannitol

Co-product: lactic acid and Co-product: sorbitol in

acetic acid one half of
mannitol
Glucose is hydrogen source
in hydrogenation
large excess (3)
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
Use of less pure substrates necessary
inactivation
to avoid catalyst
poses no problem
Enzymatic Conversion of Fructose to Mannitol
CH2OH CH2OH

O HO H

HO H Mannitol 2-Dehydrogenase HO H

H OH H OH
NAD(P)H NAD(P)
H OH H OH

CH2OH CH2OH

D-fructose Mannitol
 Cofactor Regeneration

• Chemical
• Photochemical
• Electrochemical
• Biological
• Enzymatic
Enzymatic Conversion of Fructose to Mannitol
with Simultaneous Cofactor Regeneration

Mannitol Dehydrogenase
D-Fructose Mannitol

NADH NAD

CO2 + H20 Na-Formate

Formate Dehydrogenase
Enzymatic Conversion of Fructose to Mannitol
with Simultaneous Cofactor Regeneration

Mannitol Dehydrogenase
D-Fructose Mannitol

NADH NAD+

Gluconic acid Glucose + H20

Glucose Dehydrogenase