BIOCATALYSIS

Enzymes are involved in life processes, including digestion and getting energy
from digested food. Therefore, a lot of chemical reactions catalyzed by enzymes
have a biologically-related function. Possibly the oldest example of biocatalysis is
brewing, where microorganisms are used to convert sugars into alcohol; historical
records date back 6,000 years.

Biocatalysis refers to the use of living (biological) systems or their parts to speed

up (catalyze) chemical reactions. "Biocatalyst" is a broad term used for an
industrial catalyst produced biologically. This term is used synonymously for:

 pure enzymes
 enzyme mixtures
 formulated enzyme products
 crude enzymes
 cell fragments
 and whole cells (viable or non-viable).

The enzymes can be taken from the cell, either from the original cell or from a
different cell that was modified to produce the enzyme. They can be either those
which are isolated from the cell or those which are still residing inside living cells. 
Utilizing natural or modified enzymes to perform organic synthesis is termed 
chemoenzymatic synthesis and similarly the reactions performed by them are
classified as chemoenzymatic reactions.

Benefits over Chemical Catalysis

 Enzymes are environmentally benign, being completely degraded in the
environment so toxic by-products of chemical catalysis are bypassed. This makes
it cleaner and removes the need to clean up the toxins.
 Most enzymes typically function under mild or biological conditions which
minimizes problems of undesired side-reactions such as decomposition, 
isomerization, racemization and rearrangement, which often plague traditional
methodology.
 Enzymes are larger than traditional chemical catalysts therefore there are more
contact points between the substrate and the enzyme.
 Also, modifications can easily be made to the enzyme via protein engineering,
so that it can work with a different substrate. Modifications can also be done to
enhance reaction rate or catalyst turnover.
 Enzymes selected for chemoenzymatic synthesis can be immobilized on a solid
support. These immobilized enzymes demonstrate very high stability and re-
usability and can be used to conduct reactions in continuous mode in
microreactors.
 Enzymes exhibit extreme selectivity towards their substrates. Typically enzymes
display three major types of selectivity:

 Chemoselectivity: Since the purpose of an enzyme is to act on a single type

of functional group, other sensitive functionalities, which would normally react
to a certain extent under chemical catalysis, survive. As a result, biocatalytic
reactions tend to be "cleaner" and laborious purification of product(s) from
impurities emerging through side-reactions can largely be omitted.
 Regioselectivity and diastereoselectivity: Due to their complex three-
dimensional structure, enzymes may distinguish between functional groups
which are chemically situated in different regions of the substrate molecule.
 Enantioselectivity: Since almost all enzymes are made from L-amino acids,
enzymes are chiral catalysts. As a consequence, any type of chirality present in
the substrate molecule is "recognized" upon the formation of the enzyme-
substrate complex. Thus a prochiral substrate may be transformed into an
optically active product and both enantiomers of a racemic substrate may react
at different rates. This is the major reason of interest of synthetic chemists in
biocatalysis because enantiopure  compounds are required in Pharmaceutical
drugs and agrochemicals.
In short only one type of product is formed faster, yet controllable at lower cost with
higher yield and is also economically sustainable, renewable, degradable, non-toxic
and easier to handle.

However, there are some disadvantages too

 They may be insufficiently stable for commercial applications

 Separation of the enzyme from the aqueous reaction mixture for
subsequent reuse is often difficult.
  Recovery of the product (usually present in low concentrations in the
aqueous solution) is sometimes difficult.

Methods
Immobilized enzymes
When utilizing isolated enzymes for organic synthesis, a typical approach is to
keep the enzymes immobilized on the surface of an insoluble material. This is
preferred to using soluble enzymes due to both technical and economic benefits.
The immobilization will allow for easier recycling, manipulation and separation of
the enzyme, as well as an increase in its thermal and operational stability.
Additionally, immobilization of the enzyme will ensure minimized protein
contamination during the experimental procedure, and improved control over the
overall reaction process.

Rotating bed reactors

A rotating bed reactor (RBR) is designed to prevent disintegration of solid phase
particles by retaining them inside a rotating cylinder during the reaction span.
This minimizes solid phase debris even at high speeds, which would normally
occur due to mechanical forces when using stirred tank reactors. Due to these
properties of the RBR, reaction rates can be improved by increasing the speed at
which an RBR is rotated, without shortening the lifespan of the solid phase
particles. This ensures easy in-line monitoring of the process, as well as a more
efficient recycling of the solid phase particles, making the RBR a cost and time
efficient alternative to conventional methods for immobilized enzyme
biocatalysis.

BIOCATALYSTS IN CHEMICAL INDUSTRY

Both enzymes and whole cells can be used to catalyse organic reactions, with the
most general example of biocatalysis being the yeast-mediated transformation of
sugars into alcohols, acids or gases. However, the most frequently employed
group of biocatalysts is lipases. These enzymes are very versatile, facilitating the
hydrolysis of lipids into fatty acids as well as the synthesis of esters and amides.
Areas of lipase-catalysed reactions include food processing, synthesis of fine
chemicals and production of biofuel. Other regularly used enzymatic biocatalysts
are proteases, cellulases and amylases, used for the hydrolysis of proteins,
cellulose and starch, respectively.
Bulk industry
(The chemical industry can be simplified into two main parts – fine
chemicals, which are pure and complex, and bulk chemicals, which are
made in huge quantities. Fine chemicals provide the means for things like
drugs, fragrances, and additives in food. Bulk chemicals are like ammonia,
sulfuric acid, and sodium hydroxide, which are all made at large chemical
plants via a variety of different processes.)
The main challenge in bulk chemistry is chemoselectivity. Typically relatively small
molecules with a limited number of functional groups need to be converted. If the
starting material contains just one type of functional group, the challenge is to
avoid side reactions, i.e. polymerisations or isomerisations that might be induced
by acid or base catalysts. When more functional groups are present, protecting
groups need to be avoided. In both cases, the superior selectivity of enzymes
helps to reduce side reactions and thus purification steps. For eg. ‘Nitrilases’ have
selective application in nitrile conversion from the nitrile directly to the acid
without the amide intermediate.
Glycolic acid is the smallest molecule of the hydroxy acids containing both an
alcohol and a carboxyl moiety. It has applications in the food and flavour
industries and used in industrial cleaners and for the preparation of polyglycolic
acid.
The chemical production routes to glycolic acid utilise drastic reaction conditions
which come with significant amounts of impurities leading to separation
problems.
A biocatalytic process has thus been developed for its production. The method
comprises of the reaction of formaldehyde and cyanide in sodium hydroxide to
produce an aqueous solution of glycolonitrile in high yield and purity, which is
followed by enzyme-catalysed hydrolysis by a nitrilase to ammonium glycolate.
The nitrilase is produced in E. coli and is used in the immobilised whole cells of E.
coli. The acid is then released from its ammonium salt via ion exchange
chromatography (IEC). This chemo-enzymatic process yields more than 1 kg of
GLA per g dry cell weight without the need for expensive distillation or
crystallization steps. Overall, the chemoselectivity of the nitrilase ensures a clean
conversion of the nitrile directly to the acid, avoiding the amide intermediate, all
this under mild conditions and with a straightforward purification via IEC .
Fine chemical industry
Today, there is a huge demand for sweeteners. A straightforward manner to
increase the sweetness of glucose is its isomerisation to fructose. The resulting
high-fructose corn syrup (HFCS) is a fine chemical that is used on bulk scale in our
everyday food. Glucose isomerase (GI) is an interesting industrial enzyme that
catalyzes the isomerization of d-glucose to the sweeter d-fructose. Although the
chemical conversion of glucose to fructose was known for more than 100 years,
the industrial processes suffered from shortcomings like the high pH and
temperature of this base-catalysed process. The reaction produced non-
metabolisable sugars and the fructose concentration could not exceed more than
40%. The enzymatic conversion of glucose to fructose produces an equilibrium
mixture of glucose and fructose (practically 42% fructose, 52% glucose and 6%
dextrin). This mixture is sweeter than glucose and has the same sweetness as
sucrose. The regioselective mechanism of this reaction is based on the basic
center of the enzyme that catalyses the intramolecular hydrogen transfer from C-
2 of glucose to C-1 of fructose and to no other carbon atom.

Application of Biocatalysis in the Pharmaceutical Industry

Despite the advantages that enzymes can bring, their use in the pharmaceutical
industry was not wide-spread earlier. It used to take time to identify and optimize
enzymes for reactions. Improvements in access to enzymes and protein
engineering have led to the possibility of using enzymes in pharmaceutical
industry.
One of the most successful examples in the practical application of enzymes in the
pharmaceutical industry is the synthesis of simvastatin, a cholesterol-lowering
drug marketed by Merck as Zocor. Simvastatin was chemically synthesised
starting with the hydrolysis of the natural product lovastatin to give monacolin J,
which can be converted to simvastatin by the lactonization of the acid.
Subsequent protection of the hydroxyl group followed by acylation to install the
dimethylbutyryl side chain yields the protected form of simvastatin, which is then
deprotected to yield simvastatin. The overall process needs six steps which are
technically and economically demanding. On the other hand, the biocatalytic
approach towards simvastatin requires just two steps. With the development of a
whole cell acyltransferase LovD which enables the regioselective acylation of the
C-8 hydroxyl group of monacolin J with α-dimethylbutyryl-S-N-acetylcysteamine
(DMB-S-NAC), simvastatin is afforded directly. It is noteworthy that a hydrolase
selectively cleaves the ester bond and that an acyltransferase catalyses the ester
formation in water with both enzymes using very similar mechanisms.

Comparison of chemical and biocatalytic synthesis of simvastatin.

Cosmetic industry
Among all the classes of organic compounds used in cosmetic products, esters
have a wide range of applications in the cosmetic industry. For example, wax
esters have a wide range of applications in a huge number of cosmetic
formulations as cleansers, conditioning agents and emollients. Natural wax esters
can be extracted from natural sources which are very expensive such as jojoba oil
and sperm whale. Emollient esters (used in cosmetic emulsions which improve
the smoothness and overall appearance of the skin) can be obtained by direct
esterification or by transesterification. The traditional chemical method required
high temperatures and used an acid or base as a catalyst with high pressure.
However, under these conditions, poor quality products were generated that
needed more treatment and therefore caused additional costs. On the other
hand, many enzymes can be used as biocatalysts for these reactions. For the
formation of esters, for example, hydrolases and in particular lipases are again the
enzymes of choice. Emollient esters produced by enzymes are formed particularly
pure and colour- and odourless thereby saving greatly on downstream processing.
BIOCATALYSIS AND GREEN CHEMISTRY
Biocatalytic transformations can potentially satisfy ten out of the twelve
principles of green chemistry. They often result in shorter, less wasteful,
environmentally and economically appealing processes when compared to
conventional chemical syntheses.
1. Prevention of waste generation: Biocatalysis can allow for more sustainable
routes for generation of products and reducing waste.
2. Atom Economy: Biocatalysis allows for better atom economy.
3. Less hazardous chemical synthesis: Biocatalysts are produced by living
organisms thus these processes have low toxicity and are Generally
Recognized As Safe (GRAS).
4. Safer Solvents and Auxiliaries: Biocatalysis is generally performed in water,
solvent of low toxicity or mostly on the surface of an insoluble material.
5. Design for Energy Efficiency: Biocatalysis is carried out at mild conditions so it
is energy efficient.
6. Use of Renewable Feedstocks: Biocatalysts are renewable.
7. Reduce Derivatives: Since biocatalysts are regio-, chemo- and enantioselective
so unnecessary derivatization doesnot take place.
8. Catalysis: Enzymes are generally used as catalysts.
9. Real-time analysis for Pollution Prevention: Biocatalysis is environmentally
safe.
10.Inherently Safer Chemistry for Accident Prevention: Biocatalysis takes place
under mild conditions so it is safer than chemically catalyzed processes.