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 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 生產成本 棕欖油: 23~24元/公升 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)

What is Biodiesel? Biodiesel FAME (Fatty acid methyl ester) 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 ingredients Fossil diesel Biodiesel Fuel Standard ASTM D975 ASTM PS121 Fuel Composition C10-C21 HC C16-C18 FAME Lower Heating Value, Btu/gal. 131,295 117,093 Kin. Viscosity, at 40oC 1.3-4.1 1.9-6.0 Water, ppm by wt. 161 0.05% max. Carbon, wt % 87 77 Hydrogen, wt % 13 12 Oxygen, wt % 0 11 Sulfur, wt % 0.5 max. 0.0-0.0024 Boiling Point, oC 188-343 182-338 Flash Point, oC 60-80 100-170 Pour Point, oC -35 to –15 -15 to 10 Cetane Number 40-55 48-70 BOCLE Scuff, grams 3,600 7,000 HFRR, microns 685 314 (Source: National Renewable Energy Laboratory, Sept. 2001) -閃火點(FLASH POINT) 並非燃點, 它是燃料在自然揮發下其油氣得以點燃卻不足以維持燃料燃燒的最低溫度 -流動點(Pour Point)為測定潤滑油在規定條件下，將油料冷卻至不可流動之最低溫度。 -凝結的溫度則叫做「雲點(cloud point)」

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 Strain Lipids content (%) Enzyme Biodiesel yield Reaction conditions Chlorella protothecoids 44-48% Immobilized Candida sp. lipase 98% of FAEE Molar ratio 3:1 Reaction time 12 h – Pseudomonas fluorescens lipase (1,3-specific) 92% 20 ºC in 12 h (Source: Biotechnol. Bioeng. 2007, DOI: 10.1002/bit.21489. In press; Appl. Microbiol. Biotechnol. 2006, 73, 349-355)

Source of acyl acceptors 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 acceptors Products Advantages Disadvantages Methanol FAME 1. Cheaper than ethanol 2. More reactive 3. More volatile 1.Toxic 2.Mostly nonrenewable Ethanol FAEE 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, 8995-9005)

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, 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 Approach Parameters Chemical Enzyme Catalyst Sodium methoxide Lipase Temperature High Mild Separation Difficult Easy Reusable   Purity Side reaction Nature (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. COOR1 RCOOR1 COOR1 OH OH Lipase ROH + COOR2 RCOOR2 + COOR2 + OH + OH 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 Recombinant LIP2 and LIP4 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 rugosa LIP2 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 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.

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

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

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 Wild-type A296I V344Q V344H H448S Negative control (P. pastoris KM 71) 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 ℃.

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 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.

Protein Engineering Technology Specific activity (U/mg) 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.

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 )

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) KASs=3-ketoacyl-ACP synthases TEs=thioesterases DEs=Desaturases ATs=Acyltransferases (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: Enzymatic 2002, 17, 133–142.

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Acknowledgement Shu-Wei Chang, Ph. D. Prof. Casimir C. Akoh 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

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