Enzyme

Activity enhancement

1. Turnover

(a) Solubilization of enzyme

Subtilisin (isooctane + AOT)

K_cat/ KM, 10³ x in suspended enzyme, 0.1 x aq medium; stability, 10³ x more in aq medium

Chymotrypsin + subtilisin (biocatalyst plastics)

Increased reaction rate (10⁴ x); higher stability in organic solvents

(b) Molecular imprinting

(i) Imprinting of subtilisin with sucrose, thymidine and other nucleosides

Reaction rate (50 x); substrate specificity (50-180 x)

(ii) Lyophilization of papain and lactoglobin (imprinting with transition-state analog)

Reaction rate (3 x); β-elimination

2.Altered enantioselectivity

Directed evolution (mutants)

Lipases,

P. aeruginosa lipase; 2% ee to 81% ee*

Hydantoinase,

Arthrobacter sp. hydantoinase 5 x increase; D- to L-selectivity 

Esterases

P. fluorescens esterase; 25% ee*

3. Altered functionality

Rational design

New isomerase

Conversion of indol-3-glycerol-P- synthase into phosphoribosylanthranilate isomerase (K_cat and K_M, lower)

Desaturase altered to Hydroxylase Modified β-galactosidase

Oleate Desaturase to unrelated hydroxylases DNA shuffling 10 x more β-galactosidase; 40 x less galactosidase activity

4. Substrate specificity

Recombination to alter substrate specificity

Biphenyl oxygenase activity with substrate specificity

Two genes form Pseudomonas sp and Burkholderia sp recombine to give dioxygenase activity with altered specificity

5. Increased enzyme stability and activity

(a) Mutation

Esterase, subtilisin E, and protease (six cycles of mutations)

Increase in Tm (improved thermostability

(b) Covalent immobilization (dextran dialdehyde)

Thermophilic esterase

Thermostability increased (amino groups linked to glyoxyl agarose)

(c) Crosslinking

Dextran aldehyde for crosslinking

Penicillin G acylase (9 x increased in half life; peroxidase activity of subtilisin (stabiligy 10x)

(d) Lipid coating

Glycoside hydrolases in organic medium

Mannosidase, glucosidse and glucosaminidase became hydrophobic – (transglycosylation activity 10 x)

(e) Modification with reagent

Catalase modified with Brij 35 surfactant

Catalase activity increased 200 x in trichloracetylene

6. Modified reaction milieu

Directed evolution

Esterase in aq/organic solvent

Esterase activity of subtilisin in 30% DMF increased 50 x using random mutagenesis