10.542 – Biochemica l Engineerin g

Spring 2005

Applicati o n s o f Enz y m e Catalysi s – Bi ocatalysis

 Working definition – the use of an enzym e -cataly zed reaction to convert a single starting com pound to a single product

o distinguished from the use of whole cells for m u l ti-step synthetic pathways, which also use enzym e s to catalyze each conversion

W h y us e enzym e catalysi s ove r trad ition a l (u su . solvent-base d ) organi c s ynthesis ?

( R o zze l l , J . D. " C o mme r c ia l s c a le b i o c a t a l ys is : myt h s a n d r e a lit i e s . " Bioorg Med Chem 7, no. 10 (Octob er 1999 ): 225 3- 61. )

 Catalys t sele ctiv ity/specif i city

 Mild reaction conditions

 Environm entally friendly, “green chem istry”

 High catalytic efficiency

 Greatest interest industrially is for production of chiral compounds (see handout for exam ples)

Substrat e se lectiv it y versu s substrat e range

 Industrial emphasis on selectivity is m o st often in the context of stereo selectivity, i.e. , selective conversion or produc tion of one enantiom e r, but

 The best ind u strial en zy m e s will hav e a broad su bstra t e r a ng e, i.e. , th e ab ility the catalyze the sam e type of reaction (att ack the same functional group) with a variety of substrates.

Exam ple – Subtilisi n (se r in e pro t ease )

Natural rea ction : peptide bond hydrolysis, though ac tivity against es ters was known

R 1

O R 2

O

O R 3

R 1

O

OH

R 2

H 2 N

O

NH

O R 3

Unnatural reaction : resolution of a racem ic ester m i xture for productio n of a pharm aceutical in term ediate (Courtesy of Merck & Co. Used with perm ission.)

F F F

Me O

F

O

N

N O

OM e

F

O H

HO N

N O

OM e

+

Me O

F

O H

N

N O

OMe

DH P Me t h y l E s t e r

R- D H P A c i d

S- D H P M et h y l E s t e r

See “Survey of Biocatalytic Reactio ns” handout for addition a l exam ples. Quantif yin g enantio sele c tivit y (s ter e o s elec tiv ity)

 The “enantiom e ric excess (EE)” is d e term ined relative to one enantiom e r.

EE ( S )  S  R  100 %

S  R

 Range of values are from -100% (all unde sired enantiom e r) to +100% (all desired enantiom e r). A racem ic m i xture has an EE of ‘0’ for both enantiom e rs.

 Typical EE target is >95%, or >97.5 % yield of the desired enantiom e r.

Selectin g a n appropriat e catalyst

 Typically, more than on e enzym e will perf orm a desired conv ersion (spec i es diversity)

 Derivation of rate equations is not ef fi cient as a m eans of evaluating catalyst perform a nce in a screen

 Determ ine the f o llowing inste a d, f r om a set of near-identical re ac tions (ca t alys t is variable), fixed volum e, fixed tim e:

(1)

Conversion  X  S 0  S f

S 0

 100 %   P i ( usu . R , S )  100 %

S 0

(2)

Yield

i

 P i

S

 100 %

for each product, and if the products are chiral,

(3)

0

EE ( R )  R  S  100 %

R  S

 Conversion is the proxy for a reaction rate.

 For a “decen t” conversio n, EE should be m o st important factor

o Process developm ent could im prove conversion (How do you increase V ma x ?)

o EE is m o re often an inherent property of the enzym e .

 Conversion vs yield = substrat e consum ed vs product obtained

o distin ction is im portant f o r rea c tion s in which m u ltip le produ c t s are m a de, includ ing en antiom e rs and by-products

Biocatalysis, p. 2