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