Post on 08-Nov-2014
description
Enzyme Biotechnology
World enzyme market (2004)
USA enzyme market (2007)
• Pharmaceutical, food/beverage, research/biotech, and cleaning product markets are very important enzyme markets.
World enzyme market
USA enzyme market
• Enzyme markets increase rapidly.
Global sales of industrial enzymes
• Protease, cellulase, α-amylase, lipase are widely used.
Lipase : Triacylglycerol acylhydrolase (EC 3.1.1.3)
O C
O
O C
O
O C
O
OH
OH
OH
C
O
HO
HO C
O
C
O
HO
+
Triacylglycerol Glycerol Fatty acid
OH
OH
OH
C
O
HO
O C
O
O C
O
O C
O
OH
O C
O
OH
The role of microbial lipases
• Nutritional factor• Pathogenic factor
Microbial lipases are diverse enzymes in
1) microbial source
2) protein size/sequence
3) substrate specificity
4) position selectivity
5) physicochemical stability
O C
O
O C
O
O C
O
• We have to choose appropriate lipase for our specific application!
Industrial
Lipases
Organic
solventsExtrem
e pH
Hightemperatu
re
Detergent
Chainlength
Position
Enantiomer
Ca2+
Stability Specificity
Lipase vs. Esterase
O C
O
O C
O
O C
O
C2
C3
C4
C6
C8
C1
0
C1
2
C1
4
C1
6
C1
8
C1
8:1
Lipase
act
ivit
y (
%)
C2C3C4C6C8
C10
C12
C14
C16
C18
C18:1
C18:2
C18:3
Esterase
Lipase
chain length
Criterion 1 : Chain length specificity
• Esterase can hydrolyze short chain fatty acid esters. • Lipase can hydrolyze both short chain and long chain
esters.
Enzy
me a
ctiv
ity Lipase
1 32[Substrate]
Esterase
Criterion 2 : Interfacial activation & LidEnzy
me a
ctiv
ity
• Esterase shows Michaelis-Menten saturation kinetics. • Lipase shows interfacial
activation phenomenon.
Criterion 2 : Interfacial activation & Lid
Lipid
Water
Criterion 3 : Codon for active site serine
O O
H
N N
His
Asp
H O
Ser
R1
O CO
R2
Ser
Asp
His
Catalytic Triad
Esterases
Lipases
Ser
Asp
His
AGY
TCN
lipase
esterase
• Lipase active site consists of catalytic triad of Ser-Asp-His.
Lipases : Industrial enzymes
Lipase
Detergent
Environment
bioremediation
Drug
pharmaceutical
Chemical
fatty acidlipid
Food
flavor in cheese and
cream
Energy
biodiesel
Pulp/Paper
pitch removal
O C R1
O
O C R2
O
O C R3
O
OH OH OH
OH
O C R2
O
OH
O C R1
O
OH
O C R3
O
Glycerolysis
O C R1'
O
O C R2'
O
O C R3'
O
O C R1'
O
O C R2
O
O C R3'
O
O C R1
O
O C R2'
O
O C R3
O
Transesterification
HOOC R'
Acidolysis
O C R'
O
O C R2
O
O C R3
O
O C R'
O
O C R2
O
O C R'
O
HOOC R3
HOOC R1
HOOC R1
HOOC R2
HOOC R3
HydrolysisH2O
ROOC R1
ROOC R2
ROOC R3
ROHAlcoholysis
Lipases : Useful catalysts
• Lipase catalyze
hydrolysis
alcoholysis
acidolyasis
glycerolysis
transesterification.
Lipase-Screening Methods
Microorganisms
Genomes
Metagenomes
Intertidal flatTCN-LB
Isolation of psychrophilic microorganisms
Int. J. Syst. Evol. Microbiol. 55: 335 (2005)
Photobacterium lipolyticum nov.
Strain M37
O C
O
O C
O
O C
O
2~8 kb
M37 chromosomal DNA
Sau3A1
pUC118(3.2 kb)
Amp
ori
BamHI
BamHI/Sau3A1
Amp
ori
pUML37(4.8 kb)
BamHI/Sau3A1
Cloning of lipase M37 gene
1.6 kb
Lipase M37 coding
sequence
Appl. Microbiol. Biotechnol. 70:321-6 (2006)
1,023 bp
340 aa
Mr 38,026
→ Lipase M37 has common structures of
1) central / fold
2) catalytic triad (S174-D236-H312)
3) oxyanion hole (RG) (90-91)
4) -helix lid (?)
M T P T S P
18℃37℃
kDa
97
66
45
31
22
S
1 2 3 4 5 6 7 8 9
Production of lipase M37
His-ta
g en
zym
e
TCN-z
ymog
ram
sunflower
Palm tree Jatropha
soybeanMahua tree
rapeseed
Biodiesel
Plant oils
Plants can be used to produce oils can be used to produce biodiesel can be used as energy fuel!
OCOR1
OCOR3
OCOR2 ROH
R1COOR
R2COOR
R3COOR
OH
OH
OHlipase
+ +
OCOR1
OCOR3
OCOR2 R4COOR
R1COOR
R2COOR
R3COOR
lipase+ +
OCOR4
OCOR4
OCOR4
Oil (TAG)
Alcohol Ester(Biodiesel)
Glycerol
Oil (TAG)
Acyl-acceptor
Tri-R4
A. alcoholysis
B. interesterification
Lipases catalyze alcoholysis and interesterification reactions to convert various plant oils into biodiesels.
Oils
MeOH
Lipase
Transesterification
Separation
Upperphase
Lowerphase
Biodiesel Glycerol
Oil Transesterification Separation
Evaporation of MeOH
Alkali+
MeOH
Alkaline waste water
Biodiesel
Evaporation of MeOH
Washing
Upperphase
Lowerphase
Purification
Saponified products
Glycerol
Biodiesel can be produced by alkaline process and enzymatic processes.
Alkaline process Enzymatic process
Key issue Enzymatic process Alkaline process
Presence of FFA in the starting oil
FFA are transformed to biodiesel.
FFA are transformed to soaps.
Water content of starting oil
It is not deleterious for lipase.
Impact on the catalyst by forming soaps.
Biodiesel yield High High
Glycerol recovery Easy Complex
Catalyst recovery and reusage
Easy Difficult
Energy costs Low, 20-50℃ Medium, 60-80℃
Catalyst cost High Low
Environmental impact Low Medium
Process productivity Low High
Comparison of enzymatic process and alkaline process
• Strengths: Enzyme can be recovered and reused. Glycerol recovery is easy and environmental impact is very low. • Weakness: Catalyst cost is high
Oil Enzyme Acyl-acceptor solvent Yield (%)
Year
Sunflower Novozym-435 methanol, ethanol no, petroleum ether 79~82 1990
Tallow Lipozyme IM-60 primary alcohols hexane 95~99 1996
Soybean Novozym secondary alcohols hexane 61~84
Rapeseed Lipozyme IM methanol, ethanol no 19~65
Soybean Rhizopus oryzae lipase methanol water 4~30 80~90 1999
Palm Lipase PS-30 methanol, ethanol no 15~72 2000
Soybean Novozym-435 methanol no 97 2000
Soybean Candida antarctica B methanol no 94 2002
Soybean Novozym-435 methyl acetate no 92 2004
Triolein Novozym-435 linear, branched alcohols no ~100 2005
Soybean Various commercial lipases methanol, ethanol no 65~67 2005
Frying oils Candida sp. lipase methanol no 93~96 2006
Rapeseed Lipozyme TL IM, Novozym-435 methanol t-butanol 95 2006
Jatropha Sunflower
Novozyme-435 2-propanol hexane 93 2006
Jatropha Sunflower
Novozym-435 ethyl acetate no 91~93 2007
Microalgae Candida sp. lipase methanol hexane 98 2007
Cotton Novozym-435 methanol t-butanol 97 2007
Vegetable oils Novozym-435, Lipozyme TL IM methanol, ethanol no ~100 2008
Microalgae Various commercial lipases long chain alcohol hexane - 2006
Waste edible oil Novozym-435 methanol no ~90 2002
Acid oil Novozym-435 methanol no ~90 2007
Soybean oil Novozym-435, Lipozyme TL IM methanol t-butanol 95 2006
Research works on enzymatic production of biodiesel by transesterification
Effects of organic solvents on M37 lipase
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70 80
Solvent concentration (%)
Rela
tive a
ctiv
ity (
%)
Ethanol
MethanolDimethyl sulfoxide
Ethyl acetateAcetonitrile
Effects of methanol on M37 lipase
0
20
40
60
80
100
120
0 20 40 60 80 100Methanol concentration (%)
Rela
tive a
ctiv
ity (
%)
Pseudomonas cepacia lipase
M37 lipase
Candida rugosa lipase
Candida antarctica lipase B
0 3 6 9 12 18 24 36 480 3 6 9 12 18 24 36 48
Biodiesel
1 step 2 step 3 step
Biodiesel
Oliveoil
Wasteoil
0 3 6 9 12 18 24 36 480 3 6 9 12 18 24 36 48
0 3 6 9 12 18 24 36 48
0 3 6 9 12 18 24 36 48
0
20
40
60
80
100
0 6 12 18 24 30 36 42 48
Reaction time (h)
Bio
convers
ion (
%)
0
20
40
60
80
100
0 6 12 18 24 30 36 42 48Reaction time (h)
Bio
convers
ion (
%)
Biodiesel production using CalB lipase
Oliveoil
Wasteoil
0 3 6 9 12 18 24 36 480 3 6 9 12 18 24 36 48
Biodiesel
Biodiesel
1 step 2 step 3 step
0 3 6 9 12 18 24 36 48
0 3 6 9 12 18 24 36 48
0 3 6 9 12 18 24 36 480 3 6 9 12 18 24 36 48
0
20
40
60
80
100
0 6 12 18 24 30 36 42 48Reaction time (h)
Bio
convers
ion (
%)
0
20
40
60
80
100
0 6 12 18 24 30 36 42 48
Reaction time (h)B
ioco
nvers
ion (
%)
J. Biosci. Bioeng. 107:599-604 (2009)
Biodiesel production using M37 lipase
Table 1. Yield of CLEA preparation
Sample Activity
(U/mL)
Volume
(mL)
Total activity
(U)
Yield
(%)
Cell-free extract
(soluble M37)5.37 ± 0.39 6* 32.2 100
CLEA M37
suspension4.99 ± 0.17 6** 29.9 93.1
α9
β5
α1
α2
α3
α4
α5
α6
α7
α8 α11
α10
α12
β4
β3
β2
β1
β7β6
C
N
A
C
B
α3
M37 lipase
Lysine
Microscopy of CLEA suspensionScanning electron microscopy of CLEA precipitate
5 μm10 μm
A B
Preparation of M37 CLEA
Temperature and pH properties of M37 CLEA
Contro
l 1
Ethano
l
Meth
anol
n-Buta
nol
1-Pro
pano
l
Contro
l 1
Ethano
l
Meth
anol
n-Buta
nol
1-Pro
pano
l
B C
Biodies
el
Contro
l 2
Contro
l 1
A
Biodisel production using M37 CLEA
M37 lipase
M37 CLEA
Olive oil
Biodiesel
0 2 4 6 8 10 12
Reaction time (h)
C
Olive oil
Biodiesel
0
Retention time (min)
Retention time (min)
Sta
ndar
d (A
.U.)
Pro
duct
(A
.U.)
A
B
methyl palmitate methyl
oleate
methyl stearateBiodiesel
production using M37 CLEA
Production of Biodiesel
Before
Olive oil/MeOH
M37 lipase
Before
Waste oil/MeOH
M37 lipase
Biodiesel
After
Biodiesel
After