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 C
OH OH OH
HO

O C O

HO C O
HO C

Triacylglycerol

Glycerol

Fatty acid

The role of microbial lipases

Nutritional factor Pathogenic factor
O 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 stability
O 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 Lipases
High temperature

Enantiomer

Detergent

Ca2+

Lipase vs. Esterase Criterion 1 : Chain length specificity Esterase

O 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 & Lid

Lipase Enzyme activity

Esterase shows Michaelis-Menten saturation kinetics. Lipase shows interfacial activation phenomenon.

Enzyme activity

Esterase

[Substrate]

Criterion 2 : Interfacial activation & Lid

Lipid

Water

Criterion 3 : Codon for active site serine

Catalytic Triad Ser
Asp R1 O O H N His N O C R2 H O O

lipase
AGY

Asp

His
Lipases

Ser

Asp

His

Esterases
Ser

esterase
TCN

Lipase active site consists of catalytic triad of Ser-Asp-His.

Lipases : Industrial enzymes

Food
flavor in cheese and cream

Environment
bioremediation

Pulp/Paper
pitch removal

Energy
biodiesel Lipase

Detergent

Drug
pharmaceutical

Chemical
fatty acid lipid

Lipases : Useful catalysts

HOOC 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

Glycerolysis
O

O C R1

Lipase catalyze hydrolysis alcoholysis acidolyasis glycerolysis transesterification.

Trans esterification

C O

HOOC

R3 O O C O O C O O C R3' R2 R1'

R3' O

C O

R1

C O

R2'

R3

Lipase-Screening Methods
Microorganisms

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 gene

BamHI

M37 chromosomal DNA

Amp pUC118 (3.2 kb) ori

Sau3A1

2~8 kb

BamHI/Sau3A1 ori

pUML37
Amp

1.6 kb

(4.8 kb)

BamHI/Sau3A1

Appl. Microbiol. Biotechnol. 70:321-6 (2006)

Lipase M37 coding sequence

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 (?)

Production of lipase M37

37 M kDa 97 66 45 T S P T 18 S P

31

22

Plants can be used to produce oils can be used to produce biodiesel can be used as energy fuel!
Plant oils
Palm tree sunflower

Jatropha

Mahua tree

rapeseed

soybean

Biodiesel

Lipases catalyze alcoholysis and interesterification reactions to convert various plant oils into biodiesels.

A. alcoholysis
OCOR1 OCOR2 OCOR3 + ROH lipase R1COOR R2COOR + R3COOR OH OH OH

Oil (TAG)

Alcohol

Ester (Biodiesel)

Glycerol

B. interesterification
OCOR1 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 process
Oil Alkali + MeOH
Transesterification

Enzymatic process
Separation Upper phase Oils MeOH Lipase Separation Transesterification

Evaporation of MeOH

Alkaline waste water

Washing Lower phase Upper phase Lower phase

Biodiesel
Evaporation of MeOH

Purification
Saponified products Glycerol

Biodiesel

Glycerol

Comparison of enzymatic process and alkaline process

Key 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 yield
Glycerol recovery Catalyst recovery and reusage Energy costs Catalyst cost

High
Easy Easy Low, 20-50 High

High
Complex Difficult Medium, 60-80 Low

Environmental impact
Process productivity

Low
Low

Medium
High

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 transesterification

Oil
Sunflower 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

Enzyme
Novozym-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-acceptor
methanol, 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

solvent
no, 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

Year
1990 1996

1999 2000 2000 2002 2004 2005 2005 2006 2006 2006 2007 2007 2007 2008 2006 2002 2007 2006

t-butanol
hexane no hexane

t-butanol
no hexane no no

t-butanol

Effects of organic solvents on M37 lipase

120 Ethanol Relative activity (%) 100 Methanol Dimethyl sulfoxide Ethyl acetate Acetonitrile

80
60

40
20 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

20

40 60 80 Methanol concentration (%)

100

Biodiesel production using CalB lipase

1 step 2 step 3 step 100 Bioconversion (%) 80 60 40 20 0 0
0 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 0

0 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

6 12 18 24 30 36 42 48 Reaction time (h)

J. Biosci. Bioeng. 107:599-604 (2009)

Biodiesel production using M37 lipase

1 step 2 step 3 step Bioconversion (%) 100 80 60 40 20 0 0
0 3 6 9 12 18 24 36 0 48 3 6
9 12 18 24 36 48 0 3 6 9 12 18 24 36 48

Biodiesel Olive oil

12 18 24 30 36 42 48 Reaction time (h)

100 Bioconversion (%) 80 60 40 20 0

0 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

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

M37 lipase
9
3 8 11 10 C 12

3
2

Lysine

5 4 7 4

6 1

1 2 5 7 3 6

Preparation of M37 CLEA

A 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 CLEA

M37 lipase
A B C

M37 CLEA

Biodiesel Olive oil

Biodiesel production using M37 CLEA

Standard (A.U.)

methyl palmitate

methyl oleate

methyl stearat e

B
Product (A.U.)

Retention time (min)

Retention time (min)

C
Biodiesel Olive oil

10 12 0

Reaction time (h)

Production of Biodiesel

Before

After

Before

After

M37 lipase Biodiesel

M37 lipase

Biodiesel Olive oil /MeOH Waste oil /MeOH