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Materi kuliah pemanfaatan biokimia (lipase prof korea edited).ppt

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BIOKIMIA

Text of Materi kuliah pemanfaatan biokimia (lipase prof korea edited).ppt

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)

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