Food Biochemistry and Food Processing

8 Enzyme Engineering and Technology 203

more efficient catalysts (Chen et al. 1999, Born-
scheuer and Pohl 2001, Zaks 2001, Panke and
Wubbolts 2002).

RATIONALENZYMEDESIGN

The rational protein design approach is mainly used
for the identification and evaluation of functionally
important residues or sites in proteins. Although the
protein sequence contains all the information
required for protein folding and functions, today’s
state of technology does not allow for efficient pro-
tein design by simple knowledge of the amino acid
sequence alone. For example, there are 10^325 ways
of rearranging amino acids in a 250-amino-acid-
long protein, and prediction of the number of
changes required to achieve a desired effect is an
obstacle that initially appears impossible. For this
reason, a successful rational design cycle requires
substantial planning and could be repeated several

times before the desired result is achieved. A ration-

al protein design cycle requires the following:

1.Knowledge of the amino acid sequence of the

enzyme of interest and availability of an expression

system that allows for the production of active

enzyme.

Isolation and characterization (annotation) of

cDNAs encoding proteins with novel or preobserved

properties has been significantly facilitated by ad-

vances in genomics (Schena et al. 1995, Zweiger

and Scott 1997, Schena et al. 1998) and proteomics

(Anderson and Anderson 1998, Anderson et al.

2000, Steiner and Anderson 2000) and is increasing

rapidly. These cDNA sequences are stored in gene

(NCBI) and protein databanks (Swiss-Prot; Release

44.3 of 16-Aug-04 contains 156,998 protein se-

quence entries; Apweiler 2001, Gasteiger et al.

2001, Apweiler et al. 2004). However, before the

protein design cycle begins, a protein expression

Figure 8.16.Comparison of rational design and directed evolution.