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. 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.)

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

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 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)

7. 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.

8. 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:

9. Fuel Ingredients Comparison
Fuel ingredients Fossil diesel Biodiesel
Fuel Standard ASTM D ASTM PS121
Fuel Composition C10-C21 HC C16-C18 FAME
Lower Heating Value, Btu/gal , ,093
Kin. Viscosity, at 40oC
Water, ppm by wt % max.
Carbon, wt %
Hydrogen, wt %
Oxygen, wt %
Sulfur, wt % max
Boiling Point, oC
Flash Point, oC
Pour Point, oC to – to 10
Cetane Number
BOCLE Scuff, grams , ,000
HFRR, microns
(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)」

10. 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

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
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: /bit In press; Appl. Microbiol. Biotechnol. 2006, 73, )

12. 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, )

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

16. Fatty Acids Biosynthesis Pathway
(Source:

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

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

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

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

21. Chemical Catalyst Transesterification
Proton exchange reaction

22. Chemical Catalyst Transesterification
Other unfavorable reactions

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. 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, ; J. Mol. Catal. B: Enzymatic 2002, 17, )

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

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.
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

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. 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. United States Patent

30. Related Publications Recombinant LIP1 Recombinant LIP2 and LIP4
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. 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.

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. Protein Engineering Technology
Table Important amino acid changes producing structural differences among C. rugosa LIP 1, LIP 2, LIP 3 and LIP 4
Residue LIP LIP LIP LIP4
Tyr Phe Phe Trp
Val Leu Ile Val
Thr Leu Ile Leu
Phe Val Phe Ala
Phe Leu Ile Val
Ser Gly Ala Ala
( Source: Mancheno, J.M. et al )

34. Protein Engineering Technology
(kb) 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
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.

35. 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.

36. 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 ℃.

37. Protein Engineering TechnologyM
(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）

38. 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.

39. 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.

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

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

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

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

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 + CO2  Malonyl-CoA
First enzyme: Acetyl-CoA carboxylase (ACCase)

47. 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:

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. 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. 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. 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. 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. 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, 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.

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. 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

56. THANK YOU !!!