Transcript Biocatalysis Overview

Overview of Biocatalysis in Green
Chemistry
Steve S.-F. Yu
Institute of Chemistry, Academia Sinica
俞聖法，中央研究院化學研究所
B601室
Tel: 02-27898650
[email protected]
Sow Choo University
March 25th, 2009

The Definition of Biocatalysis
Green Chemistry vs. Biocatalysis
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Whole Cell Biocatalysis: Fermentation
Bio-related Energy Conversion vs.
Biofuel
The Definition of Biocatalysis

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The employment of enzymes and whole cells have
been important for many industries for centuries. The
most obvious usages have been in the food and drink
businesses where the production of wine, beer,
cheese etc. is dependent on the effects of the
microorganisms.
More than one hundred years ago, biocatalysis was
employed to do chemical transformations on nonnatural man-made organic compounds, and the last
30 years have seen a substantial increase in the
application of biocatalysis to produce fine chemicals,
especially for the pharmaceutical industry.
Levels of Organization
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Atoms
Molecules and macromolecules
Cells
Tissues
Organs
Organism
Population
Community
Ecosystem
Biosphere
Figure 1.8
Molecular organization
in the cell is a hierarchy.
Central Dogma
複製
Replication
DNA
轉 錄 Transcription
Reverse
逆轉錄 Transcription
RNA
轉 譯 Translation
Protein
Juang RH (2004) BCbasics
Seven Characteristics of Life
1.
2.
3.
4.
5.
6.
7.
Cells and organization
Energy use and metabolism
Response to environmental changes
Regulation and homeostasis
Growth and development
Reproduction
Biological evolution
Whole cells
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Many complicated chemical conversion
process.
Many side reaction may occurred.
It is not required with cofactor recycling and
usually exhibit relatively higher activities.
However, it may require expensive equipment,
tedious workup, delicate control of the
metabolism details.
Isolated Enzymes
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Fewer steps for chemical conversion
Less side reaction
Isolated enzymes can behave in any form
such as in aqueous solution, organic solvents
(reduction of activity low) and immobilied
(hard to maintain its activity via
immobilization).
However, it is required the cofactor recycling,
less reactivity towards lipophilic substrates.
Advantages of Biocatalysts
using Enzymes
Enzymes are very efficient Bocatalysis
 Enzymes are environmentally acceptable.
 Enzymes act under mild conditions.
pH 5-8, 20-40C
 Enzymes are compatible with each other. (no
side reaction)
 The conversions are carried out in aqueous
solution

Disadvantage

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Enzymes are provided by Nature in only one
enantiomeric form.
Enzyme require narrow operation parameters.
Enzymes display their highest catalytic
activity in water.
Enzymes are bound to their natural cofactors.
Enzymes are prone to inhibition phenomena.
Enzymes may cause allergies.
12 Principles of Green Chemistry
1. Prevention
It is better to prevent waste than to treat or clean
up waste after it has been created.
2. Atom Economy
Synthetic methods should be designed to
maximize the incorporation of all materials used in
the process into the final product.
3. Less Hazardous Chemical Syntheses
Wherever practicable, synthetic methods should
be designed to use and generate substances that
possess little or no toxicity to human health and
the environment.
12 Principles of Green Chemistry
4. Designing Safer Chemicals
Chemical products should be designed to effect their
desired function while minimizing their toxicity.
5. Safer Solvents and Auxiliaries
The use of auxiliary substances (e.g., solvents,
separation agents, etc.) should be made unnecessary
wherever possible and innocuous when used.
6. Design for Energy Efficiency
Energy requirements of chemical processes should be
recognized for their environmental and economic
impacts and should be minimized. If possible,
synthetic methods should be conducted at ambient
temperature and pressure.
12 Principles of Green Chemistry
7. Use of Renewable Feedstocks
A raw material or feedstock should be renewable
rather than depleting whenever technically and
economically practicable.
8. Reduce Derivatives
Unnecessary derivatization (use of blocking groups,
protection/ deprotection, temporary modification of
physical/chemical processes) should be minimized or
avoided if possible, because such steps require
additional reagents and can generate waste.
9. Catalysis
Catalytic reagents (as selective as possible) are
superior to stoichiometric reagents.
12 Principles of Green Chemistry
10. Design for Degradation
Chemical products should be designed so that at the
end of their function they break down into innocuous
degradation products and do not persist in the
environment.
11. Real-time analysis for Pollution Prevention
Analytical methodologies need to be further developed
to allow for real-time, in-process monitoring and control
prior to the formation of hazardous substances.
12. Inherently Safer Chemistry for Accident
Prevention
Substances and the form of a substance used in a
chemical process should be chosen to minimize the
potential for chemical accidents, including releases,
explosions, and fires.
The Sun is Energy for Life

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Phototrophs use light to drive synthesis of
organic molecules
Heterotrophs use these as building blocks
CO2, O2, and H2O are recycled
See Figure 17.3
Figure 17.3
The flow of energy in the biosphere is coupled primarily to the carbon and oxygen cycles.
How Do Anabolic and Catabolic Processes
Form the Core of Metabolism Pathways?
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Metabolism consists of catabolism and
anabolism
Catabolism: degradative pathways
 Usually energy-yielding!
Anabolism: biosynthetic pathways
 energy-requiring!
Comparison of the state of reduction of carbon atoms in biomolecules: -CH2- (fats) > CHOH- (carbohydrates) C=O (carbonyls) > -COOH (carboxyls) >CO2 (carbon dioxide,
the final products of catabolism).
A comparison of state of reduction of
carbon atoms in biomolecules.
Fermentation

French
chemist Louis
Pasteur was
the first
zymologist,
when in 1857
he connected
yeast to
fermentation.
Fermentation

The German
Eduard Buchner,
winner of the
1907 Nobel Prize
in chemistry, later
determined that
fermentation was
actually caused by
a yeast secretion
that he termed
zymase.
The research efforts
undertaken by the
Danish Carlsberg
scientists greatly
accelerated the gain
of knowledge about
yeast and brewing.
Beer fermenting at a brewery.
Fermentor is easy to be used to
control the bioorganism growth
and fermentation process.
Industrial fermentation
Though fermentation can have stricter definitions, when speaking of it
in Industrial fermentation, it more loosely refers to the breakdown of
organic substances and re-assembley into other substances.
Commercially Important
Fermentation
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Microbial cells or Biomass as the product: Eg. Bakers
Yeast, Lactic acid bacillus, Bacillus sp.
Microbial Enzymes: Catalase, Amylase, Protease,
Pectinase, Glucose isomerase, Cellulase,
Hemicellulase, Lipase, Lactase, Streptokinase etc.
Microbial metabolites :

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Primary metabolites – Ethanol, Citric acid, Glutamic acid,
Lysine, Vitamins, Polysaccharides etc.
Secondary metabolites: All antibiotic fermentation
Recombinant products : Insulin, HBV, Interferon,
GCSF, Streptokinase
Biotransformations: Eg. Phenyl acetyl carbinol,Steroid
Biotransformation
酒精醱酵（alcoholic fermentation）
許多真菌和一些細菌、藻類、原生蟲可醱酵糖類產生酒精和二氧化碳，稱
為酒精醱酵。
C6H12O6 → 2C2H5OH + 2CO2 + 2 ATP (Energy Released:118 kJ mol−1)
Below the sugar will be glucose (C6H12O6) the simplest sugar,
and the product will be ethanol (2C2H5OH). This is one of the
fermentation reactions carried out by yeast, and is used in food
production.
The Structure of Glucose and
Ethanol
Glucose
Ethanol
Anaerobes, Faculative anaerobes
and obligate aerobes
Faculative anaerobes (organisms that can survive in either
oxygenated or deoxygenated environments and can switch
between cellular respiration or fermentation, respectively)
and obligate (strict) aerobes (organisms that can survive
only with oxygen) have special enzymes (superoxide
dimutase and catalase) that can safely handle these
products and transform them into harmless water and
diatomic oxygen in the following reactions:
1. 2O2- + 2H+ ---Superoxide Dismutase--> H2O2 (hydrogen
peroxide) + O2
The hydrogen peroxide produced is then transferred to a
second reaction...
2. 2H2O2 ---Catalase--> 2H2O + O2
Aerobic respiration
C6H12O6 (aq) + 6O2 (g) → 6CO2 (g) + 6H2O (l)
ΔHc = -2880 kJ
Figure 18.1
The glycolytic pathway.
Figure 19.4
The tricarboxylic acid cycle.
Step
coenzyme
ATP yield
yield
Glycolysis
-2
preparatory phase
4
Glycolysis pay-off
phase
2 NADH
4 (6)
Source of ATP
Phosphorylation of glucose and fructose 6-phosphate uses
two ATP from the cytoplasm.
Substrate-level phosphorylation
Oxidative phosphorylation. Only 2 ATP per NADH since
the coenzyme must feed into the electron transport chain
from the cytoplasm rather than the mitochondrial matrix. If
the malate shuttle is used to move NADH into the
mitochondria this might count as 3 ATP per NADH.
Oxidative
decarboxylation of
pyruvate
Krebs cycle
Total yield
2 NADH
6
Oxidative phosphorylation
2
Substrate-level phosphorylation
6 NADH
18
Oxidative phosphorylation
2 FADH2
4
Oxidative phosphorylation
36(38)ATP
From the complete oxidation of one glucose molecule to
carbon dioxide and oxidation of all the reduced coenzymes.
Anaerobic respiration
In organisms which use glycolysis, the
absence of oxygen prevents pyruvate from
being metabolised to CO2 and water via the
citric acid cycle and the electron transport
chain (which relies on O2) does not function.
Fermentation does not yield more energy
than that already obtained from glycolysis (2
ATPs) but serves to regenerate NAD+ so
glycolysis can continue. Various end products
can also be created, such as lactate or
ethanol.
Pyruvate reduction to ethanol in yeast provides a means
for regenerating NAD+ consumed in the glyceraldehyde3-P dehydrogenase reaction.
Nature Biotechnology
Discovering Enzyme
古埃及人用麥粉醱酵製造啤酒
現代科技防止啤酒氧化變質
直鏈澱粉
α(1→4)糖苷鍵導致直鏈澱粉應承螺旋狀結構
澱粉是一種多醣。製造澱粉是
植物貯存能量的一種方式。分
子式(C6H10O5)n。 多個葡萄糖
分子以α-1，4-糖苷鍵首尾相
連而成，在支鏈處為α-1，6
糖苷鍵。
Carbohydrate
Sugar (sucrose)
saccharide (monosaccharide, disaccharide, oligosaccharide, polysaccharide)
Fischer projection formula
CHO
H
OH
OH
CHO
H C OH
OH
O
1
2
OH
(HCOH)n
2+n OH
OH
O
(HCOH)n
2+n OH
Aldoses
Ketoses
1
O
1
O
2
OH
OH
OH
2
OH
D-Aldoses
1
2
HO
OH
L-Aldoses
1
2
OH
O
OH
OH
D-Ketoses
1
2
OH
O
HO
OH
L-Ketoses
cis
trans
HO
OH
OH
HO
HO
OH
O
OH
O
-D
-D
-L
OH
OH
2
3
HO
4
O
OH
5
90o
OH
OH
OH
OH
O
OH
1
trans
HO
OH
HO
HOH2C
6
cis
HO
OH
OH
O
OH
6
CH2OH
5 O
4 OH 1
2
HO
OH
3
OH
OH
-L
OH
OH
HO
HO
OH
HO
O
OH
OH
-D
O
OH
OH
-D
生質能源
生質能是利用生質物， 經轉換所獲得的電與熱等可用的能源， 是一種兼顧環保
並可永續經營的能量來源。
當植物吸收陽光、二氧化碳（CO2）及水分之後，進行光合作用，產生氧氣，並促
進了植物本身的成長；而後我們將植物轉換為燃料，產生能源加以利用，在利用
過程所產生的二氧化碳回歸到大氣中，所以是一種潔淨的再生能源，稱之為生質
能。
過去在遠古蠻荒時期，人類鑽木取火，以摩擦木頭產生熱能，獲得火源，而其燃
燒所產生的二氧化碳則再度回歸到大氣中，與陽光進行光合作用，促進數目之生
長，不造成環境的負擔，是人類最早的生質能利用。
生質物則泛指由生物產生的有機物質，例如木材與林業廢棄物如木屑等；農作物
與農業廢棄物如黃豆莢、玉米穗軸、稻殼、蔗渣等；畜牧業廢棄物如動物屍體；
廢水處理所產生的沼氣；都市垃圾與垃圾掩埋場與下水道污泥處理廠所產生的沼
氣；工業有機廢棄物如有機污泥、廢塑橡膠、廢紙、造紙黑液等。
http://www.besc.org.tw/biomass/m201.htm
蒸氣火車，它是利用燃燒木
柴和煤炭等燃料，將水加熱
以產生蒸氣，來推動輪子使
車子前進。
煤的燃燒與污染
C + O2 → CO2 每莫耳放熱94千卡
石油的成因與成份
遠古時代之動植物死亡後，與泥沙沈積在
地底部，因細菌的生物作用，經過長期的
變化漸漸分解，氧、氮元素消失，剩下的
碳和氫鍵結而成碳氫化合物。再經幾百萬
年來，地下的高溫、高壓作用，進行複雜
而緩慢的化學作用，逐漸形成了黏稠的液
態石油及氣態的天然氣，其成份大部分為
碳氫化合物。從地底下抽出的石油稱為原
油，為一烴類混合物。
Free Energy
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
Hypothetical quantity - allows chemists to
asses whether reactions will occur
G = H - TS
For any process at constant P and T:
G = H - TS
If G = 0, reaction is at equilibrium
If G < 0, reaction proceeds as written
Figure 3.9
The triphosphate
chain of ATP
contains two
pyrophosphate
linkages, both of
which release large
amounts of energy
upon hydrolysis.
Figure 3.8 The activation
energies for phosphoryl grouptransfer reactions (200 to 400
kJ/mol) are substantially larger
than the free energy of
hydrolysis of ATP (-30.5
kJ/mol).
E. D. Larson, Biofuel Technologies Overview (2007)
First generation biofuels
'First-generation fuels' refer to biofuels
made from sugar, starch, vegetable oil,
or animal fats using conventional
technology.
Bioalcohols

Alcohol fuels are produced by
fermentation of sugars derived from
wheat, corn, sugar beets, sugar cane,
molasses and any sugar or starch that
alcoholic beverages can be made from
(like potato and fruit waste, etc.).
酒精汽油
酒精學名為乙醇，可作為替代汽油的一種生質燃料，其
主要利用甘蔗甘藷含糖或澱粉的作物經發酵而得。此
外，雖然技術上以機可以利用稻或蔗渣等木質纖維製
造酒精，但因為成本仍高，目前尚無實際商業化生產。
酒精可與汽油混合使用，所得到的混合燃料可稱為酒
精汽油，添加 3％vol 酒精稱為 E3，添加 10％vol
酒精稱為 E10，由於酒精容
易吸水而會與汽油形成相分離（如油水混合時會分成
二層），因此需要使用無水酒精，使用過程也需防止
水氣吸收，可當作汽油的燃料使用。
生化柴油

生化柴油的原料來自植物，具有可再生
的特色。植物在生長過程中，可以固定
空氣中的二氧化碳作為碳源，兼有再生
能源及減廢的優點。生化柴油除具備可
再生特色之外，由於所含雜質少，且燃
燒後所產生的微細固體顆粒量低，既可
降低空氣污染，又能保護、延長內燃機
的引擎壽命。
國科會網頁
植物油脂

黃豆、油棕櫚、油菜籽、向日葵
籽、棉花籽與花生等六種作物的
產油脂能力都很高，產量占全世
界植物油脂的百分之八十四。植
物所產油脂約有百分之九十是供
人類食用，僅有約百分之十應用
於非食用品。
雖然油脂作物含油脂量高，但由
於可耕作土地及年收成次數有限，
近年來紛紛改以微生物生產油脂。
Biodiesel
It is produced from oils or fats using
transesterification and is a liquid similar
in composition to mineral diesel. Its
chemical name is fatty acid methyl (or
ethyl) ester (FAME). Oils are mixed with
sodium hydroxide and methanol (or
ethanol) and the chemical reaction
produces biodiesel (FAME) and glycerol.
Transesterification
Fat Triglycerides
沼氣利用技術

沼氣的產生主要是藉由細菌把廢棄物中的有機物質分解以得到可燃性氣
體，主要成分是甲烷、二氧化碳及少量硫化氫。分解有機物的細菌可分
為好氣菌與厭氣菌兩種，當氧氣充足時，好氣菌會把有機物分解，所產
生氣體大都是二氧化碳，稱之為好氣發酵；相反地，若在缺氧狀態時，
則由厭氣菌負責把有機物分解，產生沼氣，稱之為厭氣發酵。
沼氣是一種相當好的能源，甲烷含量約在 50 ~ 80％ 之間，所含的熱值
通常在 5,000 千卡／立方公尺以上，屬中熱值氣體，且有抗爆等特性，
極適合於燃燒或引擎的使用。
目前臺灣的沼氣來源以廢棄物為大宗，其種類包括畜牧廢水、家庭污水、
城鎮垃圾及各行業廢水等四大類，其中，畜牧廢水以豬隻糞尿廢水為大
宗；家庭污水以都市污水處理場為主；城鎮垃圾主要以垃圾掩埋場為主；
各行業廢水（物）則來自食品業、紡織業、膠帶業等。
Microbial Methane
Biogenic Natural Gas

Natural gas can also be formed through
the transformation of organic matter by
tiny microorganisms.
such as
Methanogens, tiny methane producing
microorganisms, chemically break down
organic matter to produce methane.
Methanogens

are archaea that produce methane as a
metabolic byproduct in anoxic conditions.
Microbial Methane
Biogenic Natural Gas


Livestock, paddy rice farming, and
covered vented landfill emissions
leading to the production of
atmospheric methane.
In certain circumstances, however, this
methane can be trapped underground,
recoverable as natural gas.
Landfill Methane
http://www.epa.gov/methane/sources.html
Second generation biofuels

Second generation (2G) biofuels use
biomass to liquid technology,
including cellulosic biofuels from non
food crops.
纖維素

纖維素是自然界中分佈最廣、含量最多
的一種多糖。無論一年生或多年生植物，
尤其是各種木材都含布大量的纖維素。
自然界中，植物體內約有50％的碳存在
於纖維素的形式。棉花、亞麻、芋麻和
黃麻部含有大量優質的纖維素。棉花中
的纖維素含量最高，達90%以上。木材中
的纖維素則常與半纖維素和木質素共同
存在。
纖維素

纖維素是一種複雜的多糖，有8.0×103—
1.0×104個葡萄糖殘基通過β—1，4—糖
苷鍵連接而成。天然纖維素為無臭、無
味的白色絲狀物。纖維素在水中有高度
的不溶性，同時也不溶於稀酸、稀鹼和
有機溶劑，主要的生物學功能是構成植
物的支持組織。
纖維素結構式, 葡萄糖通過β—1，4—糖苷鍵連接而成
由木質纖維素轉化成生物酒精
稻草桿
甘蔗渣
木屑
纖維素
醣
加入
酵母菌
低濃度
酒精
能源作物: 狼尾草
Third generation biofuel

Algae fuel, also called oilgae or third
generation biofuel, is a biofuel from
algae. Algae are low-input/high-yield
(30 times more energy per acre than
land) feedstocks to produce biofuels
and algae fuel are biodegradable.
藻類的油脂生產

綠藻具有使用太陽能及不與現有耕地競爭的優點，故有學者提出
以綠藻生產三酸甘油脂，作為生化柴油原料來源的構想。增加細
胞累積三酸甘油脂程度的方法可以分為二大類，分別是以環境營
養源短缺，造成藻類累積大量三酸甘油脂，及以基因調控方式，
使藻類大量生產合成三酸甘油脂的酵素，大量累積三酸甘油脂。
未來除了開發新型的高效率培養系統外，將會同時朝這兩個方向
研究。
有別於其他菌體培養，培養藻類的反應器需要能提供充足的光線，
該類反應器稱為光反應器。於一九四○年起，便有光反應器的相
關研究。
Diatoms
Cyanobacteria
Inside the tube photosynthetic bacteria
are making ethanol more efficiently
than other forms of biomass because
the cyanobacteria are natural
fermentators.
http://newsinfo.wustl.edu/asset/page/normal/4
954.html