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Recombinant Lipase- Catalyzed Biodiesel Production. 蕭介夫 講座教授兼校長 Jei-Fu Shaw, Ph.D. Department of Food Science and Biotechnology National Chung Hsing University Tel: 04-22840201 e-mail: Boplshaw@gate.sinica.edu.tw. Global Warming. 全球 CO 2 總量收支狀況,圖中數值單位為十億噸

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Recombinant Lipase- Catalyzed Biodiesel Production

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Recombinant Lipase- Catalyzed Biodiesel Production

蕭介夫 講座教授兼校長

Jei-Fu Shaw, Ph.D.

Department of Food Science and Biotechnology

National Chung Hsing University

Tel: 04-22840201

e-mail: Boplshaw@gate.sinica.edu.tw


Global Warming

全球CO2總量收支狀況,圖中數值單位為十億噸

(Source: Stowe, K. 1996, "Exploring Ocean Science", 2th ed.)


Global Warming

因人為原因造成全球暖化現象之預估發展趨勢

[Source: Stowe 1996, "Exploring Ocean Science", 2th ed.]


能源現況

  • 國際原油價格走勢(西元2003~2007年;美金/桶)

(資料來源: 經濟部能源局)


Clean-Burning Fuel

  • Biodiesel

    • Renewable resource

    • Better for environment

    • Grown and refined domestically

    • Substantially decreasing harmful emissions

(Source: http:// www.propelbiofuels.com/site/aboutbiodiesel.html)


市場分析

(資料來源: 自由時報 2007/7/1)


What is Biodiesel?

  • Biodiesel

    • Alkyl ester of long chain fatty acid (C16-C18)

      • FAME (Fatty acid methyl ester)

      • FAEE (Fatty acid ethyl ester)

(TAG)

(Biodiesel)

Catalysts: Two of the most commonly used catalysts for transesterification are NaOH and KOH.

R1, R2, and R3 are long chains of carbons and hydrogen atoms, sometimes called fatty acid chains.


Sources of Renewable Oils and Fats

  • Plant-derived oils (TAG)

    • Green plants grow through the photosynthesis process with CO2 as a carbon source

    • The combustion of plant-derived oils will release CO2 which has previously been fixed through photosynthesis

    • Advantages

      • Renewable

      • Inexhaustible

      • Nontoxic

      • Biodegradable

      • Similar energy content to fossil diesel fuel

(Source: http: www.fengyuan.gov.tw)


Fuel Ingredients Comparison

Fuel ingredientsFossildieselBiodiesel

Fuel Standard ASTM D975ASTM PS121

Fuel Composition C10-C21 HCC16-C18 FAME

Lower Heating Value, Btu/gal. 131,295117,093

Kin. Viscosity, at 40oC 1.3-4.11.9-6.0

Water, ppm by wt. 1610.05% max.

Carbon, wt % 8777

Hydrogen, wt % 1312

Oxygen, wt % 011

Sulfur, wt % 0.5 max.0.0-0.0024

Boiling Point, oC 188-343182-338

Flash Point, oC 60-80100-170

Pour Point, oC -35 to –15-15 to 10

Cetane Number 40-55 48-70

BOCLE Scuff, grams 3,6007,000

HFRR, microns 685314

(Source: National Renewable Energy Laboratory, Sept. 2001)


Sources of Renewable Oils and Fats

  • Waste oils and fats

    • Frying oils, lard, beef tallow, yellow grease, and other hard stock fats

    • Advantage

      • Cheap

    • Disadvantages

      • High polymerization products

      • High free fatty acid contents

      • Susceptibility to oxidation

      • High viscosity

      • Poor-quality oils may inactivate the basic or even enzyme catalysts

    • Solve strategy

      • Preliminary treatment

        • Such as the use of adsorbent materials (magnesium silicates)

        • Reduce free fatty acid content and polar containminants


Sources of Renewable Oils and Fats

  • Microbial oils—Algal oils

    • Largely produced through substrate feeding and heterotrophic fermentation

    • Another cheap source of renewable new materials

(Source: Biotechnol. Bioeng. 2007, DOI: 10.1002/bit.21489. In press; Appl. Microbiol. Biotechnol. 2006, 73, 349-355)


Different alcohol and different fatty acid produce different biodiesel of different properties

  • Source of acyl acceptors

  • Main purpose of alcoholysis (transesterification)

    • Reduce the viscosity of the fat

    • Increase volatility and FA ester combustion in a diesel engine

(Source: Akoh et al., J. Agric. Food Chem., 2007, 55, 8995-9005)


杏核仁油

月桂葉油

玻璃苣油

椰子油

玉米油

棉花子油

海甘藍油

落花生油

榛果油

麻瘋樹

水黃皮籽油

亞麻子油

橄欖油

棕櫚油

花生油

罌粟籽油

葡萄籽油

米糠油

葵花籽油

芝麻油

大豆油

葵花油

烏桕

核桃仁油

小麥油

Different alcohol and different fatty acid produce different biodiesel of different properties

(Source: Akoh et al., J. Agric. Food Chem., 2007, 55, 8995-9005)


Fatty Acid Characterizations

TABLE 1 Some naturally occuring fatty acids: structure, properties, and nomenclature

[Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]


Fatty Acids Extended Conformations

Saturated Fatty Acid (SFA) Unsaturated Fatty Acid (UFA)

[Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]


Fatty Acids Biosynthesis Pathway

(Source: http:// www.chori.org)


Routes of Fatty Acid synthesis

[Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]


Lipid Biosynthesis

DAGAT

Diacylglycerol

[Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]


Glycerol and Triacylglycerol

[Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]


Chemical Catalyst Transesterification

  • Initial reaction

  • Transesterification (Alcoholysis) reaction


Chemical Catalyst Transesterification

  • Proton exchange reaction


Chemical Catalyst Transesterification

  • Other unfavorable reactions


Chemical Catalyst Transesterification

  • Reaction condition

(Sources: NoureddiniH et al.75(12), 1775-1783, 1998; DarnokoD. and CheryanM. JAOCS, 77, 1269–1272, 2000.;

He et al. Transactions of the ASAE, 48, 2237-2243, 2005; He et al. Transactions of the ASABE, 49, 107-112, 2006)


Biodiesel Production

  • Two available approaches

    • Chemical catalyst

    • Enzyme catalyst

(Source:Bioresour. Technol. 1999, 79, 1-15.;J. Mol. Catal. B: Enzymatic 2002, 17, 133-142.)


Lipase-Catalyzed Reactions

(Source: Lipid 2004, 39, 513-526)


Enzymatic Biodiesel

  • Reaction mechanism

  • Materials

    • Oil resources: soy bean, rapeseed, palm, and jatropha etc.

    • Alcohol types: Ethanol, Methanol, and Isopropanol etc.

    • Solvent systems: n-hexane, t-butanol, and solvent-free etc.

RCOOR1

COOR1

OH

OH

COOR1

Lipase

ROH

+

COOR2

OH

COOR2

OH

RCOOR2

+

+

+

COOR3

OH

COOR3

OH

RCOOR3

Alcohol Oil Alkyl ester DG MG Glycerol

(Triacylglycerol) (Biodiesel)

R1, R2, and R3 are long chains of carbons and hydrogen atoms, sometimes called fatty acid chains.

DG: Diacylglycerol; MG: Monoacylglycerol


Lipases

  • Advantages

    • Mild reaction conditions

    • Specificity

    • Reuse

    • Enzymes or whole cells can be immobilized

    • Can be genetically engineered to improve

      • Their efficiency

      • Accept new substrates

      • More thermostable

      • The reactions they catalyze are considered as “natural” and “green”


CRL isozymes

  • Similarity

    • High-identity gene family (lip1 to lip7)

    • Consisting of 534 amino acids

    • An evident MW of 60 kDa

    • Conserved at a catalytic triad

      • Ser-209, His-449 and Glu-341

    • Disulphide bond formation sites

      • Cys-60/Cys-97

      • Cys-268/Cys-277


United States Patent


Related Publications

  • Recombinant LIP1

    • Chang, S. W., C. J. Shieh, G. C. Lee and J. F. Shaw*, 2005. Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris. Appl. Microbiol. Biotechnol. 67: 215-224. (SCI)

    • Chang, S. W., G. C. Lee, J. F. Shaw*, 2006. Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J. Agric. Food Chem. 54, 815-822. (SCI)

    • Chang, S. W., G. C. Lee, C. C. Akoh, J. F. Shaw*, 2006. Optimized growth kinetics of Pichia pastoris and recombinant Candida rugosa LIP1 production by RSM, J. Mol. Microbiol. Biotechnol. 11, 28-40. (SCI)

  • Recombinant LIP2 and LIP4

    • Lee, L. C., Y. T. Chen, C. C. Yen, T. C. Y. Chiang, S. J. Tang, G. C. Lee*, and J. F. Shaw*, 2007. Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites. J. Agric. Food Chem. 55: 5103−5108. (SCI)

    • Lee, G. C., L. C. Lee, and J. F. Shaw*, 2002. Multiple mutagenesis of nonuniversal serine codons of Candida rugosaLIP2 gene and its functional expression in Pichia pastoris. Biochem. J. 366:603-611. (SCI)

    • Tang, S.J., J.F. Shaw, K.H. Sun, G.H. Sun, T.Y. Chang, C.K. Lin, Y.C. Lo and G.C. Lee. 2001. Recombinant expression and characterization of the Candida Rugosa Lip4 lipase in Pichia pastoris:Comparison of glycosylation, activity and stability. Archives Biochem. Biophys. 387: 93-98. (SCI)

  • Recombinant LIP3

    • Chang, S. W., G. C. Lee, J. F. Shaw*, 2006. Efficient production of active recombinant Candida rugosa LIP3 Lipase in Pichia pastoris and biochemical characterization of the purified enzyme. J. Agric. Food Chem. 54: 5831-5838. (SCI)

  • Review

    • Akoh, C. C., G. C. Lee, and J. F. Shaw*, 2004. Protein Engineering and Applications of Candida rugosa Lipase Isoforms. Lipids 39 (6):513-526. (SCI)

    • Akoh, C. C., Chang, S. W., G. C. Lee, and J. F. Shaw*, 2007.Enzymatic Approach to Biodiesel Production. J. Agric. Food Chem. 55: 8995-9005.


Recombinant CRL Isoform for Biodiesel Production

Figure Effect of methanol addition times on biodiesel conversion catalyzed by LIP2. The reactioncondition was subject to a loading of 0.5 g soybean oil, oil/methanol molar ratio = 1/4, 20% water content, and 12-h reaction time at 35 ºC. The enzyme solution (LIP2) used in this work was 70 μL. The time interval between the two methanol additions was 1 h.


Protein Engineering Technology

  • Computer modeling

    • prediction

      • Protein structure

      • Catalytic triad

      • Active site

      • Substrate binding site

  • Develop functional enzyme

    • Directed evolution

    • Point mutation

    • Error prone PCR

    • DNA shuffling

Docking for Candida rugosa lipase


Table Important amino acid changes producing structural differences among C. rugosa LIP 1, LIP 2, LIP 3 and LIP 4

Protein Engineering Technology

Residue LIP1 LIP2 LIP3 LIP4

69 Tyr Phe Phe Trp

127 Val Leu Ile Val

132 Thr Leu Ile Leu

296 Phe Val Phe Ala

344 Phe Leu Ile Val

450 Ser Gly Ala Ala

( Source: Mancheno, J.M. et al. 2003 )


Protein Engineering Technology

(kb)

M 1 2 3 4 5

Lane 1: Represent the WT lip4

Lane 2:Represent the A296I

Lane 3: Represent the V344Q

Lane 4: Represent the V344H

Lane 5: Represent the H448S

3.0

1.5

2.0

Figure PCR analysis of P. pastoris transformants. Using the genomic DNA as templates, and rector specific 5’ α-factor primer and 3’ AOX1 primer.


(A)

wild-type Candida rugosa lip4

A296I

(B)

wild-type Candida rugosa lip4

296

V344Q

V344H

(C)

wild-type Candida rugosa lip4

344

H448S

448

Figure Genomic DNA-sequence comparisons between wild-type Candida rugosa lip4(CRL4)and A296(A),CRL4, V344Q and V344H(B)CRL4, and H448S(C). The difference locations between mutated codons and wild-type are cased in red squre.


Protein Engineering Technology

Negative control

(P. pastoris KM 71)

Wild-type

A296I

V344Q

V344H

H448S

Figure Lipase plate(C4) assay. Thewild-type, A296I, V344Q, V344H, H448S and negative control ( P. pastoris KM 71) were transferred on the YPD agar plate containing 100 μg/ml Zeocin and 1% tributyrin, and cultured for 48 hours at 30 ℃.


Protein Engineering Technology

1 2 3 4 5 6 M

(kDa)

97.4

84.0

66.0

55.4

Figure 13 SDS-PAGE of the wild-type, A296I, V344Q, V344H, H448S and Negative control .

Lane 1: Represent the wild-type; Lane 2:Represent the A296I

Lane 3: Represent the V344Q ; Lane 4: Represent the V344H

Lane 5: Represent the H448S ; Lane 6: Represent the Negative control(KM 71)


Protein Engineering Technology

Specific activity (U/mg)

Figure The substrate specific activity of C. rugosa LIP4 wild-type , A296I, V344Q, V344H and a H448Swith p-nitrophenyl (p-NP) esters of various chain-length fatty acids. The lipase sample was added to a reaction mixture containing 5 mM p-nitrophenyl ester(such as acetate、butyrate、caproate and caprylate)and 2.5 mM p-nitrophenyl ester(such as caprate、laurate、myristate、palmitate and stearate)at pH 7.0.


Protein Engineering Technology

Specific activity (U/mg)

FigureThe substrate specific activity of C. rugosa LIP4 wild-type , A296I, V344Q, V344H and a H448S with triglyceride of various chain-length fatty acid. The lipase sample was added to a reaction mixture containing 50 mM triglyceride (such as tributyrin、tricaprin) and 10 mM triglyceride (such as trilaurin、ripalmitin) and the activity was measured by pH stat at pH 7.0.


Enzyme Immobilization

  • Advantages

    • Easy to control enzyme concentration

    • Easy separation of the immobilized enzyme

    • Easy to control micro-environment

    • Easy separation of enzyme from product

    • Reuse of the enzyme


Enzyme Immobilization

  • Principal Methods

    • Adsorption

    • Covalent binding

    • Encapsulation

    • Entrapment

    • Cross-linking

(Source: Gordon F. Bickerstaff ,1997)


Lipolytic Activities of a Lipase Immobilized on Six Selected Supporting Materials

(Source: Biotechnology and Bioengineering, 1990, 35, 132-137)


Lipolytic Activities of a Lipase Immobilized on Six Selected Supporting Materials

(Source: Biotechnology and Bioengineering, 1990, 35, 132-137)


Continuous Bioreactor Systems

  • Types

    • Stirred-tank bioreactor

    • Membrane bioreactor

    • Fluidized bed reactor

    • Packed-bed bioreactor

  • Advantages

    • Easily used

    • Continuous process with automatic control

    • Long term reaction time

    • High product concentration

(Source: Lipid biotechnology. 2002. p. 387–398.; 生物固定化技術與產業應用。2000。 第121–155頁)


(c)

(b)

(d)

(a)

(e)

Continuous Packed-Bed Bioreactor

(a) Substrate mixture

(b) Pump

(c) Incubation chamber

(d) Packed bed reactor

(e) Product collector


How to increase oil content of plants?

  • Fatty acid biosynthesis pathway

    • Locations

      • ER: Polyunsaturated fatty acids (PUFAs)

      • Plastid: primary saturated and monounsaturated fatty acids

      • Oil body: triglyceride

    • First step: Acetyl-CoA + CO2 Malonyl-CoA

    • First enzyme: Acetyl-CoA carboxylase (ACCase)


How to increase oil content of plants?

  • Fatty acid biosynthesis pathway in plant

Acetyl-CoA carboxylase (ACCase)

(Source: http://www.uky.edu)


How to increase oil content of plants?

  • Genetic modified technology for ACCase in plants

    • Functional promoter region construct

    • Functional gene expression

[Source: Ohlrogge and Browse (1995) The Plant Cell 7, 957-970]


Conclusion

  • Advantages of Biodiesel from vegetable oils or their blends

    • Renewable

    • Biodegradable

    • oxygenated

    • Less or nontoxic

    • Low sulfur content and higher cetane numbers

    • Produces less smoke and particulates

    • Produces lower carbon monoxide and hydrocarbon emissions

    • Low aromatic content

    • Higher heat content of about 88% of number 2 diesel fuel

    • Readily available


Conclusion

  • Future trend for fuel production—Biotechnology

    • Protein (enzyme) engineering

      • Catalytic efficiency improvement

      • High specific activity

      • Novel substrate specificity

      • Different regio-selectivity

      • Enantioselectivity improvement

      • High stabilities

        • pH

        • Temperature

        • Organic solvents etc.


Conclusion

  • Future trend for fuel production—Biotechnology

    • Genetic modified technology

      • Increase oil content in various plants

        • Functional promoter development

        • High level expression of key enzyme

    • Microbial engineering--Microdiesel

      • Recombinant E. coli host for ethanol production

      • Coexpression of the ethanol production genes

        • Pyruvate decarboxylase (pdc gene product)

        • Alcohol dehydrogenase (adhB gene product)

        • Unspecific acyltransferase WS/DGAT gene

From Zymomonas mobilis

From Acinetobacter baylyi strain ADP1

(Source: Microbiology, 2006, 152, 2529-2536)


Conclusion

  • Microbial engineering--Microdiesel

Figure Pathway of FAEE biosynthesis in recombinant E. coli. FAEE formation was achieved by

Coexpression of the ethanolic enzymes pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB)

From Z. mobilis and the unspecific acyltransferase WS/DGAT from A. baylyi strain ADP1.

(Source: Microbiology, 2006, 152, 2529-2536)


References

  • Li, X.; Xu, H.; Wu, Q. Large-scale biodiesel production from microalga Chlorella protothecoids through heterotrophic cultivation in bioreactors. Biotechnol. Bioeng. 2007, DOI:10.1002/bit. 21489(in press).

  • Luo, Y.; Zheng, Y.; Jiang, Z.; Ma, Y.; Wei, D. A novel psychrophilic lipase from Pseudomonas fluorescens with unique property in chiral resolution and biodiesel production via transesterification. Appl. Microbiol. Biotechnol. 2006, 73, 349-355.

  • Kalscheuer, R.; Stölting, T.; Steinbüchel, A. Microdiesel: Escherichia coli engineered for fuel production. Microbiology 2006, 152, 2529-2536.

  • Stowe, K. Exploring Ocean Science, 2nd Ed., Wiley, New York, 1996.

  • Nelson, D. L; Cox, M. M. “Lehninger Principles of Biochemistry, 4th ed. W.H. Freeman and Company, New York, 2005.

  • Kuo, T. M. and Gardner, H. W. Lipid biotechnology. p. 387–398. Marcel dekker. New York. USA, 2002.

  • Ma, F.; Hanna M. A. Biodiesel production: a review. Bioresour. Technol.1999, 70, 1–15.

  • Shimada, Y.; Watanable, Y.; Sugihara, A.; Tominaga, Y. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J. Mol. Catal. B: Enzymatic2002, 17, 133–142.


Contact Information

ISBB Symposium Secretariat

250, KuoKuang Rd., 

Taichung, 40227, Taiwan

Tel: +886-4-2284-0550 ext304

Fax:+886-4-2285-0177

E-Mail: tlko@dragon.nchu.edu.tw

Welcome to Taichung, Taiwan.

The main theme of the symposium is Agricultural Biotechnology. The following lists the main areas that will be focused in the meeting:

1). Functional Food and Industry Products

2). Improvement of Agronomic and Microbial Traits

3). Biofuel

4). Nanobiotechnology


Acknowledgement

  • Shu-Wei Chang, Ph. D.

    • Department of Nutrition and Health Science

    • Chung-Chou University of Technology

  • Prof. Casimir C. Akoh

    • Department of Food Science and Technology

    • The University of Georgia

  • Prof. Chwen-Jen Shieh

    • Department of Bioindustry Technology

    • Dayeh University

  • Mr. Chih-Chung Yen

    • Institute of Agricultural Biotechnology

    • National Chiayi University


THANK YOU !!!


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