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, -amylase, lipase are widely used.
Lipase : Triacylglycerol acylhydrolase (EC 3.1.1.3)
O O C O O C O O COH OH OHHO
O C O
+
HO C OHO C
Triacylglycerol
Glycerol
Fatty acid
The role of microbial lipases Nutritional factor Pathogenic factorO O C O O C O O C
OH OH OH
OH O O C OH
O HO C
Microbial lipases are diverse enzymes in 1) microbial source 2) protein size/sequence 3) substrate specificity 4) position selectivity 5) physicochemical stabilityO O C O O C O O C
We have to choose appropriate lipase for our specific application!
Stability
Organic solvents
Chain length
Specificity
Extreme pH
Position
Industrial LipasesHigh temperature
Enantiomer
Detergent
Ca2+
Lipase vs. Esterase Criterion 1 : Chain length specificity EsteraseO O C O O C O O C
Lipase
Esterase can hydrolyze short chain fatty acid esters. Lipase can hydrolyze both short chain and long chain esters.
C18:1
C2 C3 C4 C6 C8 C10 C12 C14 C16 C18 C18:1 C18:2 C18:3
C8 C10
C12
C14 C16 C18
C2
C3 C4 C6
chain length
Lipase activity (%)
Criterion 2 : Interfacial activation & LidLipase Enzyme activity
Esterase shows Michaelis-Menten saturation kinetics. Lipase shows interfacial activation phenomenon.
Enzyme activity
Esterase
1
[Substrate]
2
3
Criterion 2 : Interfacial activation & Lid
Lipid
Water
Criterion 3 : Codon for active site serineCatalytic Triad SerAsp R1 O O H N His N O C R2 H O O
lipaseAGY
Asp
HisLipases
Ser
Asp
His
EsterasesSer
esteraseTCN
Lipase active site consists of catalytic triad of Ser-Asp-His.
Lipases : Industrial enzymesFoodflavor in cheese and cream
Environmentbioremediation
Pulp/Paperpitch removal
Energybiodiesel Lipase
Detergent
Drugpharmaceutical
Chemicalfatty acid lipid
Lipases : Useful catalystsHOOC R1 HOOC R2 HOOC R1 O O C O O O C O C R3 HOOC R' O O O C O O O C O C R' R2 O R' R2 O O C O C O O C R3 O C O R1' OH O R2' O C R3 R2 R1 OH OH OH OH R' HOOC R3 ROOC ROOC ROOC R1 R2 R3
Hydrolysis
H2O
ROH
Alcoholysis
OH O O C R2
Acidolysis
GlycerolysisO
O C R1
Lipase catalyze hydrolysis alcoholysis acidolyasis glycerolysis transesterification.
Trans esterification
O
C O
HOOC
R3 O O C O O C O O C R3' R2 R1'
O
C
R3' O
O
C O
R1
O
C O
R2'
O
C
R3
Lipase-Screening MethodsMicroorganisms
Genomes
Metagenomes
Int. J. Syst. Evol. Microbiol. 55: 335 (2005)
Isolation of psychrophilic microorganisms
O O C O O C O O C
TCN-LB
Intertidal flat
Photobacterium lipolyticum nov.
Strain M37
Cloning of lipase M37 geneBamHI
M37 chromosomal DNAAmp pUC118 (3.2 kb) ori
Sau3A1
2~8 kb
BamHI/Sau3A1 ori
pUML37Amp
1.6 kb
(4.8 kb)
BamHI/Sau3A1
Appl. Microbiol. Biotechnol. 70:321-6 (2006)
Lipase M37 coding sequence1,023 bp 340 aa Mr 38,026
Lipase M37 has common structures of1) central / fold 2) catalytic triad (S174-D236-H312) 3) oxyanion hole (RG) (90-91) 4) -helix lid (?)
Production of lipase M3737 M kDa 97 66 45 T S P T 18 S P
31
22
1
2
3
4
5
6
7
8
9
Plants can be used to produce oils can be used to produce biodiesel can be used as energy fuel!Plant oilsPalm tree sunflower
Jatropha
Mahua tree
rapeseed
soybean
Biodiesel
Lipases catalyze alcoholysis and interesterification reactions to convert various plant oils into biodiesels.
A. alcoholysisOCOR1 OCOR2 OCOR3 + ROH lipase R1COOR R2COOR + R3COOR OH OH OH
Oil (TAG)
Alcohol
Ester (Biodiesel)
Glycerol
B. interesterificationOCOR1 OCOR2 OCOR3 + R4COOR lipase R1COOR R2COOR + R3COOR OCOR4 OCOR4 OCOR4
Oil (TAG)
Acylacceptor
Tri-R4
Biodiesel can be produced by alkaline process and enzymatic processes. Alkaline processOil Alkali + MeOHTransesterification
Enzymatic processSeparation Upper phase Oils MeOH Lipase Separation Transesterification
Evaporation of MeOH
Alkaline waste water
Washing Lower phase Upper phase Lower phase
BiodieselEvaporation of MeOH
PurificationSaponified products Glycerol
Biodiesel
Glycerol
Comparison of enzymatic process and alkaline processKey issue Presence of FFA in the starting oil Water content of starting oil Enzymatic process FFA are transformed to biodiesel. It is not deleterious for lipase. Alkaline process FFA are transformed to soaps. Impact on the catalyst by forming soaps.
Biodiesel yieldGlycerol recovery Catalyst recovery and reusage Energy costs Catalyst cost
HighEasy Easy Low, 20-50 High
HighComplex Difficult Medium, 60-80 Low
Environmental impactProcess productivity
LowLow
MediumHigh
Strengths: Enzyme can be recovered and reused. Glycerol recovery is easy and environmental impact is very low. Weakness: Catalyst cost is high
Research works on enzymatic production of biodiesel by transesterificationOilSunflower Tallow Soybean Rapeseed Soybean Palm Soybean Soybean Soybean Triolein Soybean Frying oils Rapeseed Jatropha Sunflower Jatropha Sunflower Microalgae Cotton Vegetable oils Microalgae Waste edible oil Acid oil Soybean oil
EnzymeNovozym-435 Lipozyme IM-60 Novozym Lipozyme IM Rhizopus oryzae lipase Lipase PS-30 Novozym-435 Candida antarctica B Novozym-435 Novozym-435 Various commercial lipases Candida sp. lipase Lipozyme TL IM, Novozym-435 Novozyme-435 Novozym-435 Candida sp. lipase Novozym-435 Novozym-435, Lipozyme TL IM Various commercial lipases Novozym-435 Novozym-435 Novozym-435, Lipozyme TL IM
Acyl-acceptormethanol, ethanol primary alcohols secondary alcohols methanol, ethanol methanol methanol, ethanol methanol methanol methyl acetate linear, branched alcohols methanol, ethanol methanol methanol 2-propanol ethyl acetate methanol methanol methanol, ethanol long chain alcohol methanol methanol methanol
solventno, petroleum ether hexane hexane no water 4~30 no no no no no no no
Yield (%)79~82 95~99 61~84 19~65 80~90 15~72 97 94 92 ~100 65~67 93~96 95 93 91~93 98 97 ~100 ~90 ~90 95
Year1990 1996
1999 2000 2000 2002 2004 2005 2005 2006 2006 2006 2007 2007 2007 2008 2006 2002 2007 2006
t-butanolhexane no hexane
t-butanolno hexane no no
t-butanol
Effects of organic solvents on M37 lipase
120 Ethanol Relative activity (%) 100 Methanol Dimethyl sulfoxide Ethyl acetate Acetonitrile
8060
4020 0 0 10 20 30 40 50 60 70 80 Solvent concentration (%)
Effects of methanol on M37 lipase 120 M37 lipase 100 Relative activity (%) 80 60 40 20 0
Candida antarctica lipase B Pseudomonas cepacia lipase Candida rugosa lipase
0
20
40 60 80 Methanol concentration (%)
100
Biodiesel production using CalB lipase1 step 2 step 3 step 100 Bioconversion (%) 80 60 40 20 0 00 3 6 9 12 18 24 36048 3 6 9 12 18 24 360 48 3 6 9 12 18 24 36 48
Biodiesel
Olive oil
6 12 18 24 30 36 42 48 Reaction time (h)
100 Bioconversion (%) Biodiesel Waste oil 80 60 40 20 00 3 6 9 12 18 24 36 48 0 3 0 3 6 9 12 18 24 36 48 6 9 12 18 24 36 48
0
6 12 18 24 30 36 42 48 Reaction time (h)
J. Biosci. Bioeng. 107:599-604 (2009)
Biodiesel production using M37 lipase1 step 2 step 3 step Bioconversion (%) 100 80 60 40 20 0 00 3 6 9 12 18 24 36 0 48 3 69 12 18 24 36 48 0 3 6 9 12 18 24 36 48
Biodiesel Olive oil
6
12 18 24 30 36 42 48 Reaction time (h)
100 Bioconversion (%) 80 60 40 20 00 3 6 9 12 18 24 36 0 483 0 3 6 9 12 18 24 36 48 6 9 12 18 24 36 48
Biodiesel
Waste oil
0
6
12 18 24 30 36 42 48
Reaction time (h)
Table 1. Yield of CLEA preparation Sample Activity (U/mL) Cell-free extract (soluble M37) CLEA M37 suspension 5.37 0.39 Volume (mL) 6* Total activity (U) 32.2 Yield (%) 100
4.99 0.17
6**
29.9
93.1
A
M37 lipase93 8 11 10 C 12
B
32
Lysine
5 4 7 4
6 1
1 2 5 7 3 6
N
C
Preparation of M37 CLEAA B
10 m Microscopy of CLEA suspension
5 m Scanning electron microscopy of CLEA precipitate
Temperature and pH properties of M37 CLEA
Biodisel production using M37 CLEAM37 lipaseA B C
M37 CLEA
Biodiesel Olive oil
A
Biodiesel production using M37 CLEA
Standard (A.U.)
methyl palmitate
methyl oleate
methyl stearat e
BProduct (A.U.)
Retention time (min)
Retention time (min)
CBiodiesel Olive oil
0
2
4
6
8
10 12 0
Reaction time (h)
