Bio- catalysis in Green Chemistry.

1. BIO-CATALYSIS
Nitin Pandey
M-Pharm 1st year [Pharmaceutical Chemistry]

2. INTRODUCTION
The catalyst is derived from renewable resource and is bio-compatible [some time
even edible],bio-degradable and essentially non- hazardous ;that is fulfill the criteria
of sustainability remarkably well.
Bio-catalysis avoids the use of , contamination of products by ,scarce precious metal
such as palladium ,platinum and rhodium .the long term commercial viability of
many ‘’endangered’’ element such as various noble metal .
Reaction are performed in an environmentally compatible solvents [water under mild
condition and physiological pH and ambient temperature and pressure]
Reaction of multifunctional molecule proceed with activities and chemo-,
regioselective and generally without the need for functional group activation ,
protection and deprotection ,steps required in traditional organic syntheses .
As a direct result of the higher selective and milder reaction condition ;biocatalytic
processes often afford products in high purity than traditional chemical process .

3. Contd.
 It can be conducted in standard multipurpose batch reactor and
,hence do not require any extra investment .

4. Example of Green Bio-Catalytic Processes
I. A Chemoenzymatic Process for Pregabalin
Pfizer scientists have described a second –generation chemo – enzymatic
process for the manufacture of Pregabalin ,the active ingredient of the CNS
drug [LYRICA] .
2 .A Three –Enzyme Process for Atorvastatin Intermediate
Codexis scientists developed and commercialized a green –by –designed three
enzyme of process for the synthesis of a key intermediate in manufacturing of
Atorvastatin, the active ingredient of Cholesterol –lowering drug [Lipitor].

5. Type of Bio-catalysts
• Oxidoreductase : Catalyze oxidation /reduction reaction .
For example ,alcohol dehydrogenase converts primary alcohols to
aldehydes.
In the reaction ,ethanol is converted to acetaldehyde ,and the cofactor NAD is
converted to NADH, In other words, ethanol is oxidized and NAD is reduced .

6. Cont.
• Transferases : transfer a functional group .alanine aminotransferase
shuffles the alpha – amino group between alanine and aspartate .
Hydrolases : catalyze the hydrolysis of various bonds .
• For example ,phosphates break the oxygen –phosphorous bond of
phosphate ester .

7. • Lyases : Formation or removal of a double bond with group transferase.
• For example Dehydratases remove of water ,as in fumarase [fumarate
hydratase ]
• Isomerases :catalyze isomerization changes within a single molecule
[Rearrangement] For example triose phosphate isomerase ,carry out the
rearrangements.

8. • Ligases : removing the elements of water from two functional group to form a
single bond .
• Kinase : This enzyme in the body attaches a phosphate group to a high energy
bond .It is a very important enzyme required for ATP production and activation
of certain enzyme.

9. Advantage & of Disadvantage of bio- Biocatalyst
Advantage
1. Similar to chemical catalyst ,biocatalyst increase the speed of chemical
reaction but do not affect the thermodynamics of the reaction .
2. The most important advantage of a bio-catalyst is its high selectivity.
3. High catalytic efficiency and mild operational condition .
4. Almost all of the bio-catalysts characteristics can be tailored with protein
engineering and metabolic engineering method to meet the desired
process condition .
Disadvantages
1. The characteristics of limited operating region ,substrate or product
inhibition ,and reaction in only aqueous solution have often been
considered as the most serious drawback of bio-catalysts .

10. • Bio- catalysts is very important tool in organic synthesis because of
following reason ;
• Single step in organic synthesis can be accomplished .
• Preservation of stereochemical centers ,which can be important for drugs.
• Elimination of the nee for protection or deprotection groups .
• Can be done in an aqueous environment –Green chemistry .
Factor affecting Bio- catalysts
• Concentration of bio-catalysts
• Concentration of reactant
• Temperature
• pH
• Activators
• Light and radiation

11. Enzyme Production
• Bacteria and fungi are the most popular hosts for industrial enzyme ,due to
easy handling and high productivity .They can be readily genetically
engineered their performance ; for example ,by incorporating secretion
system to facilitate enzyme isolation an purification .
• Some of the most popular expression host are Escherichia coli ,Pichia pastoris,
Pseudomonas fluorescens ,Aspergillus and bacillus sp. Mammalian or plants
cells are used in special cases .By regulation ,the production host should have
GRAS status .
• On a large scale production ,a computer – controlled Bio- reactor is used to
maintain an appropriate control of pH , O2, NH3 ,AND CO2 to maximize cell
density .the cells are harvested by centrifugation in a batch or continuous
fashion .

12. Cont.
• At a scale of over 5-10 L , a homogenizer is usually used .After
centrifugation to remove cell debris ,the crude enzyme remain in the
supernatant and can be concentrated through precipitation by adding
either inorganic salts that is ammonium sulphate or organic solvents
[acetone ]
• The crude enzyme are then purified by dialysis or variety of
chromatographic method .The dry powder is usually obtained after
lyophilization under freeze –drying condition .

13. Use of Enzyme in Organic Synthesis
• The use of enzymes in organic synthesis is increasing and has been
reported for a diverse set of chemical reactions. (Urlacher & Schmid, 2006
.
• With help of deeper knowledge about directed evolution of enzyme
functions and improved biotechnology the scope of useful biocatalyst is
likely to expand in the coming years.
• Stereochemistry is an important section in both biochemistry and organic
chemistry. Before we discuss this we will start by defining two fundamental
concepts.
• First, enantiomers: two structures that are not identical, but are mirror
images of each other. Second, chiral: structures that are not
superimposable on their mirror image, and can therefore exist as two
enantiomers Clayden et al., 2005).

14. In order to acquire enzymes with the appropriate function wild-type enzymes
will be structurally engineered by different directed evolution approaches.
The stereo specific production of hydroxy carbonyl compounds is the focus.
Chiral epoxides will be converted to vicinal diols, catalyzed by a structurally
engineered epoxide hydrolase (Elfström & Widersten 2005). Subsequently, the
diol will be oxidized by a diol dehydrogenase to form a chiral end product (Fig.
1).

15. • Microbes – Yeast ,and other anaerobic bacteria.
• Lipases – These are the most widely used class of enzymes in organic
synthesis ,they are preferred widely because of their better stability as
compared to others.
• Protease – Enzymes which break down proteins.
• Cellulose- Enzyme which break down cellulose.
• Amylases- which break down into simple sugar.
Bio-catalyst enzyme in human body –Digestive enzyme
Our food is made up of –
1.Carbohydrates- Bread Pasta ,Potato
2.Protein – Nuts , lentils ,Meat
3. Fats - Butter ,Milk

16. Immobilized enzyme / cells in Organic reaction
• An enzyme is immobilized by attaching it into an insoluble support which allows its reuse
and continuous usage ,thus eliminating the tedious recovery process .
• Immobilization stabilizes the enzyme ;moreover two or more enzyme catalyzing a series of
reactions may be in placed in close proximity to one another .
• Adsorption ,covalent linkage ,cross linking ,matrix entrapment or encapsulation are
different method for making immobilized enzyme .
• Immobilization offers an elegant solution to these problems. Since the 1916 report by
Nelson and Griffin chemists have sought efficient methods for enzyme immobilization ,
Which offers several potential improvements,
1. Easier handling;
2. Minimized protein contamination in the final product;
3. Efficient enzyme recovery and recycling;
4. Enhanced enzyme stability;
5. Low allergenicity;
6. Higher productivities and consequently lower costs.

17. Immobilization techniques
• Protocols for enzyme immobilization include entrapment, cross-linking,
and adsorption or linkage to porous or nano support .
• It should also be noted that enzyme immobilization can be used as a step
in enzyme isolation and purification in many cases.
• The immobilization technique are as follows.
 Enzyme Entrapment.
 Cross – linked Enzyme Aggregates [CLEAs].
 Enzyme Immobilization of porous supports.
 Nano-structured support .

18. Enzyme Entrapment
The methods in which the enzyme is confined inside the inner cavities of a
solid organic or inorganic matrix.
 Enzyme entrapment is one of the simplest immobilization strategies and
is least aggressive with regard to changing enzyme structure since the
protein interactions are weak and the conditions during the preparation
are usually mild.
Entrapment promotes enzyme insoluble and thereby enables easy
recovery of the biocatalyst from the reaction media.
 Depending on the gel material and its modification, the matrix can also
provide an advantageous microenvironment.

20. Cross-Linked Enzyme Aggregates (CLEAs)
• Cross-linked enzyme aggregates (CLEAs) are a type of enzyme
immobilization that does not require exogenous solid supports.
• It Compared with the earlier cross-linked enzyme crystals (CLECs)
methodology, CLEAs are far simpler to prepare since no prior enzyme
crystallization is required.
• CLEAs are formed by adding both protein precipitating and cross-
linking reagents to produce stabilized aggregates.
• The cross-linker prevents the aggregate from redissolving after the
precipitant is removed.
• The precipitation step utilizes conventional techniques such as
salting-out, solvent, or polymer precipitation.

22. Enzyme Immobilization on Porous Supports
• Adsorbing or linking enzymes to the surfaces of porous materials is one
of the most common methodologies for enzyme immobilization. The
interactions can be reversible, for example, by hydrophobic and ionic
interactions or irreversible if covalent bonds are formed.

23. Nanostructured Supports
• Enzymes immobilized on nanostructures have many applications in bio -
catalysis, biosensors and for biomedical purposes.
• Nanostructures for enzyme immobilization can be particles with spherical
shapes, fibers, or tubes. Although most nanostructures used for enzyme
immobilization are nonporous materials, their very high surface-to-volume
ratios still allows them to incorporate significant quantities of enzyme per unit
mass of carrier.
• Moreover, because all enzymes are exposed on the surface, nanostructures
offer several advantages over porous macro beads such as minimum diffusion
limitation, less mass transfer problems as well as higher mechanical strength.

24. Reference
• https://www.researchgate.net/publication/230852070_The_use_of_
enzymes_in_organic_synthesis_and_the_life_sciences_Perspectives_f
rom_the_Swiss_Industrial_Biocatalysis_Consortium_SIBC
• https://oaktrust.library.tamu.edu/handle/1969.1/DISSERTATIONS-
745804
• https://www.slideshare.net/aayushikushwaha/organic-synthesis-
using-enzymes
• https://www.hansrajcollege.ac.in/hCPanel/uploads/elearning/elearni
ng_document/Biocat_Biochem.pdf

25. THANK YOU