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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.

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Presentation on theme: "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."— Presentation transcript:

1 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:

2 2 Global Warming 全球 CO 2 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.)

3 3 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.]

4 4 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 )

5 5 Clean-Burning Fuel Biodiesel Renewable resource Better for environment Grown and refined domestically Substantially decreasing harmful emissions (Source:

6 6 市場分析 國內市場國際市場 量需求中油估算三年後全面供應 B2 油品, 所需生質柴油的量高達 14 萬公秉 1. 德國最先研發並推行, 其年產量超過 130 萬噸 2. 英國規定所有進入該城 市的公車,均需使用至 少 B5 的生質柴油為燃料 。今年 6 月,全球第一列 使用生化柴油的火車也 在英國上路,英國維京 航空計畫二年後推出使 用生質柴油的環保客 機。 生產成本棕欖油 : 23~24 元 / 公升 大豆油 : 27 元 / 公升 油菜籽油 : 30 元 / 公升 年產量最大可生產 33 萬公秉的生質柴油 產能承德油脂公司利用回收食用油及積 勝利用棕櫚油,合計產能約為 5,000 公秉 ( 資料來源 : 自由時報 2007/7/1)

7 7 What is Biodiesel? Biodiesel Alkyl ester of long chain fatty acid (C16-C18) FAME (Fatty acid methyl ester) FAEE (Fatty acid ethyl ester) Catalysts: Two of the most commonly used catalysts for transesterification are NaOH and KOH. R 1, R 2, and R 3 are long chains of carbons and hydrogen atoms, sometimes called fatty acid chains. (Biodiesel) (TAG)

8 8 Sources of Renewable Oils and Fats Plant-derived oils (TAG) Green plants grow through the photosynthesis process with CO 2 as a carbon source The combustion of plant-derived oils will release CO 2 which has previously been fixed through photosynthesis Advantages Renewable Inexhaustible Nontoxic Biodegradable Similar energy content to fossil diesel fuel (Source: http:

9 9 Fuel Ingredients Comparison Fuel ingredients Fossil diesel Biodiesel Fuel Standard ASTM D975 ASTM PS121 Fuel Composition C10-C21 HC C16-C18 FAME Lower Heating Value, Btu/gal. 131, ,093 Kin. Viscosity, at 40 o C Water, ppm by wt % max. Carbon, wt % Hydrogen, wt % Oxygen, wt % 0 11 Sulfur, wt % 0.5 max Boiling Point, o C Flash Point, o C Pour Point, o C -35 to – to 10 Cetane Number BOCLE Scuff, grams 3,600 7,000 HFRR, microns (Source: National Renewable Energy Laboratory, Sept. 2001)

10 10 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

11 11 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 StrainLipids content (%) EnzymeBiodiesel yield (%) Reaction conditions Chlorella protothecoids44-48%Immobilized Candida sp. lipase 98% of FAEEMolar ratio 3:1 Reaction time 12 h ––Pseudomonas fluorescens lipase (1,3-specific) 92%20 ºC in 12 h (Source: Biotechnol. Bioeng. 2007, DOI: /bit In press; Appl. Microbiol. Biotechnol. 2006, 73, )

12 12 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 Acyl acceptorsProductsAdvantagesDisadvantages MethanolFAME 1. Cheaper than ethanol 2. More reactive 3. More volatile 1.Toxic 2.Mostly nonrenewable EthanolFAEE 1. More renewable 2. Environmental friendly 1. More expensive 2. Less reactive Others: propanol, isopropanol, butanol, branched-chain alcohols, t-butanol, octanol, methyl and ethyl acetate (Source: Akoh et al., J. Agric. Food Chem., 2007, 55, )

13 13 Different alcohol and different fatty acid produce different biodiesel of different properties 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 (Source: Akoh et al., J. Agric. Food Chem., 2007, 55, )

14 14 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.]

15 15 Fatty Acids Extended Conformations Saturated Fatty Acid (SFA) Unsaturated Fatty Acid (UFA) [Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]

16 16 Fatty Acids Biosynthesis Pathway (Source:

17 17 Routes of Fatty Acid synthesis [Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]

18 18 Lipid Biosynthesis [Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.] DAGAT Diacylglycerol

19 19 Glycerol and Triacylglycerol [Source: Nelson and Cox 2005, “Lehninger Principles of Biochemistry, 4th ed.]

20 20 Chemical Catalyst Transesterification Initial reaction Transesterification (Alcoholysis) reaction

21 21 Chemical Catalyst Transesterification Proton exchange reaction

22 22 Chemical Catalyst Transesterification Other unfavorable reactions

23 23 Chemical Catalyst Transesterification Reaction condition (Sources: NoureddiniH et al.75(12), , 1998; DarnokoD. and CheryanM. JAOCS, 77, 1269–1272, 2000.; He et al. Transactions of the ASAE, 48, , 2005; He et al. Transactions of the ASABE, 49, , 2006)

24 24 Biodiesel Production Two available approaches Chemical catalyst Enzyme catalyst Approach Parameters ChemicalEnzyme CatalystSodium methoxideLipase TemperatureHighMild SeparationDifficultEasy Reusable  PuritySide reactionNature (Source: Bioresour. Technol. 1999, 79, ; J. Mol. Catal. B: Enzymatic 2002, 17, )

25 25 Lipase-Catalyzed Reactions (Source: Lipid 2004, 39, )

26 26 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. COOR 1 COOR 2 COOR 3 Lipase RCOOR 2 COOR 1 COOR 2 COOR 3 OH ROH Alcohol Oil Alkyl ester DG MG Glycerol (Triacylglycerol) (Biodiesel) RCOOR 1 RCOOR 3 R 1, R 2, and R 3 are long chains of carbons and hydrogen atoms, sometimes called fatty acid chains. DG: Diacylglycerol; MG: Monoacylglycerol

27 27 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”

28 28 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

29 29 United States Patent

30 30 Related Publications Recombinant LIP1 Chang, S. W., C. J. Shieh, G. C. Lee and J. F. Shaw*, Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris. Appl. Microbiol. Biotechnol. 67: (SCI) Chang, S. W., G. C. Lee, J. F. Shaw*, 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, (SCI) Chang, S. W., G. C. Lee, C. C. Akoh, J. F. Shaw*, Optimized growth kinetics of Pichia pastoris and recombinant Candida rugosa LIP1 production by RSM, J. Mol. Microbiol. Biotechnol. 11, (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*, 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*, Multiple mutagenesis of nonuniversal serine codons of Candida rugosa LIP2 gene and its functional expression in Pichia pastoris. Biochem. J. 366: (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 Recombinant expression and characterization of the Candida Rugosa Lip4 lipase in Pichia pastoris : Comparison of glycosylation, activity and stability. Archives Biochem. Biophys. 387: (SCI) Recombinant LIP3 Chang, S. W., G. C. Lee, J. F. Shaw*, Efficient production of active recombinant Candida rugosa LIP3 Lipase in Pichia pastoris and biochemical characterization of the purified enzyme. J. Agric. Food Chem. 54: (SCI) Review Akoh, C. C., G. C. Lee, and J. F. Shaw*, Protein Engineering and Applications of Candida rugosa Lipase Isoforms. Lipids 39 (6): (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:

31 31 Figure Effect of methanol addition times on biodiesel conversion catalyzed by LIP2. The reaction condition 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. Recombinant CRL Isoform for Biodiesel Production

32 32 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

33 33 Table Important amino acid changes producing structural differences among C. rugosa LIP 1, LIP 2, LIP 3 and LIP 4 ( Source: Mancheno, J.M. et al ) 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 Protein Engineering Technology

34 34 Figure PCR analysis of P. pastoris transformants. Using the genomic DNA as templates, and rector specific 5’ α-factor primer and 3’ AOX1 primer. M 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 (kb) Protein Engineering Technology

35 35 ( A ) wild-type Candida rugosa lip4 ( B ) wild-type Candida rugosa lip4 ( C ) wild-type Candida rugosa lip4 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. A296I V344Q V344H H448S

36 36 Figure Lipase plate(C4) assay. The wild-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 ℃. V344QV344HH448SWild-type Negative control (P. pastoris KM 71) A296I Protein Engineering Technology

37 37 (kDa) M 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

38 38 Figure The substrate specific activity of C. rugosa LIP4 wild-type, A296I, V344Q, V344H and a H448S with 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. Specific activity (U/mg) Protein Engineering Technology

39 39 Figure The 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. Specific activity (U/mg) Protein Engineering Technology

40 40 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

41 41 Enzyme Immobilization Principal Methods Adsorption Covalent binding Encapsulation Entrapment Cross-linking (Source: Gordon F. Bickerstaff,1997)

42 42 Lipolytic Activities of a Lipase Immobilized on Six Selected Supporting Materials (Source: Biotechnology and Bioengineering, 1990, 35, )

43 43 Lipolytic Activities of a Lipase Immobilized on Six Selected Supporting Materials (Source: Biotechnology and Bioengineering, 1990, 35, )

44 44 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 p. 387–398.; 生物固定化技術與產業應用。 2000 。 第 121–155 頁 )

45 45 Continuous Packed-Bed Bioreactor (b) (c) (d) (a) (e) (a) Substrate mixture (b) Pump (c) Incubation chamber (d) Packed bed reactor (e) Product collector

46 46 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 + CO 2  Malonyl-CoA First enzyme: Acetyl-CoA carboxylase (ACCase)

47 47 How to increase oil content of plants? Fatty acid biosynthesis pathway in plant (Source: Acetyl-CoA carboxylase (ACCase)

48 48 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, ]

49 49 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

50 50 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.

51 51 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, )

52 52 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, )

53 53 References Li, X.; Xu, H.; Wu, Q. Large-scale biodiesel production from microalga Chlorella protothecoids through heterotrophic cultivation in bioreactors. Biotechnol. Bioeng. 2007, DOI: /bit (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, Kalscheuer, R.; Stölting, T.; Steinbüchel, A. Microdiesel: Escherichia coli engineered for fuel production. Microbiology 2006, 152, Stowe, K. Exploring Ocean Science, 2nd Ed., Wiley, New York, Nelson, D. L; Cox, M. M. “Lehninger Principles of Biochemistry, 4th ed. W.H. Freeman and Company, New York, Kuo, T. M. and Gardner, H. W. Lipid biotechnology. p. 387–398. Marcel dekker. New York. USA, 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: Enzymatic 2002, 17, 133–142.

54 54 Contact Information ISBB Symposium Secretariat 250, KuoKuang Rd., Taichung, 40227, Taiwan Tel: ext304 Fax: 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

55 55 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

56 THANK YOU !!!


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