Overview of Biocatalysis in Green Chemistry

Steve S.-F. YuInstitute of Chemistry, Academia Sinica

俞聖法，中央研究院化學研究所B601 室

Tel: 02-27898650sfyu@chem.sinica.edu.tw

Sow Choo UniversityMarch 25th, 2009

The Definition of Biocatalysis Green Chemistry vs. Biocatalysis

Whole Cell Biocatalysis: Fermentation

Bio-related Energy Conversion vs. Biofuel

The Definition of Biocatalysis The employment of enzymes and whole cells have b

een 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 non-natural 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. Atoms2. Molecules and macromolecules3. Cells4. Tissues5. Organs6. Organism7. Population8. Community9. Ecosystem10. Biosphere

Figure 1.8Molecular organization in the cell is a hierarchy.

RNA

DNA

Protein

轉 錄

轉 譯

Transcription

Translation

Replication複 製

逆轉錄 ReverseTranscription

Central Dogma

Juang RH (2004) BCbasics

Seven Characteristics of Life

1. Cells and organization2. Energy use and metabolism3. Response to environmental changes4. Regulation and homeostasis5. Growth and development6. Reproduction7. Biological evolution

Whole cells 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 Fewer steps for chemical conversion Less side reaction Isolated enzymes can behave in any form su

ch 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, 20-40C Enzymes are compatible with each other.

(no side reaction) The conversions are carried out in aqueous

solution

Disadvantage Enzymes are provided by Nature in only one e

nantiomeric form. Enzyme require narrow operation parameter

s. Enzymes display their highest catalytic activit

y 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. PreventionIt is better to prevent waste than to treat or clean up waste after it has been created.

2. Atom EconomySynthetic methods should be designed to maximize the incorporation of all materials used in the process into the final product.

3. Less Hazardous Chemical SynthesesWherever practicable, synthetic methods should be designed to use and generate substances that possess little or no toxicity to human health and the environment.

4. Designing Safer ChemicalsChemical products should be designed to effect their desired function while minimizing their toxicity.

5. Safer Solvents and AuxiliariesThe use of auxiliary substances (e.g., solvents, separation agents, etc.) should be made unnecessary wherever possible and innocuous when used.

6. Design for Energy EfficiencyEnergy 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 FeedstocksA raw material or feedstock should be renewable rather than depleting whenever technically and economically practicable.

8. Reduce DerivativesUnnecessary 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. CatalysisCatalytic reagents (as selective as possible) are superior to stoichiometric reagents.

12 Principles of Green Chemistry

10. Design for DegradationChemical 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 PreventionAnalytical 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.

12 Principles of Green Chemistry

The Sun is Energy for Life

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 17.3The 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?

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) CO (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

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 : 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 18.1The glycolytic pathway.

Figure 19.4The tricarboxylic acid cycle.

Step coenzyme

yield ATP yield Source of ATP

Glycolysis

preparatory phase -2

Phosphorylation of glucose and fructose 6-phosphate uses

two ATP from the cytoplasm.

4 Substrate-level phosphorylation

Glycolysis pay-off

phase 2 NADH 4 (6)

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

2 NADH 6 Oxidative phosphorylation

2 Substrate-level phosphorylation

6 NADH 18 Oxidative phosphorylation Krebs cycle

2 FADH2 4 Oxidative phosphorylation

Total yield 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 glyceraldehyde-3-P dehydrogenase reaction.

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

Na

ture

Bio

tech

no

log

y

Dis

cove

ring

En

zym

e

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

H OH

CHO

OH

H C OH

CHO

OH

O

OH

(HCOH)nOH2+n

O

(HCOH)nOH2+n

1

2

1

2

Aldoses Ketoses

OH

O

OH

OH

O

OH

1

2

1

2

D-Aldoses D-Ketoses

OH

OH

O

OH

OH

1

2

L-Aldoses

HO OH

O

OH

1

2

L-Ketoses

OH

HO

O

OH

OH

OH

HO

OH

OH

O

OH

HO

OH

OH

OH

O

OH

HO

HOOH

O

OH

OH

OH

HO

HO

OH

OH

OH

HO

OH

O

OH

OH

OH

HO

O

HO

-D -D -L-L

cis trans cis trans

-D -D

OH

OH

HO

OH

HOH2C

O

90o

O

1

2

3

4

5

CH2OH

HO

OH

OHOH

6

5

6

4

3

21

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

Hypothetical quantity - allows chemists to asses whether reactions will occur

G = H - TS For any process at constant P and T:

G = H - TS If G = 0, reaction is at equilibrium If G < 0, reaction proceeds as written

Figure 3.9The 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 group-transfer 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 MethaneBiogenic Natural Gas

Natural gas can also be formed through the transformation of organic matter by tiny microorganisms.

such as Methanogens, tiny methane producin

g microorganisms, chemically break down organic matter to produce methane.

Methanogens

are archaea that produce methane as a metabolic byproduct in anoxic conditions.

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.

Microbial MethaneBiogenic 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。

DiatomsCyanobacteria

Inside the tube photosynthetic bacteria are making ethanol more efficiently than other forms of biomass because the cyanobacteria are natural fermentators.

http://news-info.wustl.edu/asset/page/normal/4954.html