Hydrolases Part I: Enzyme Mechanism, Selectivity and Control in the Synthesis of Well-Defined Polymers - Enzymatic Polymerisation - page 56

This chapter will deal with the mechanistic features of lipases and how these
affect the polymerizability of monomers. Although several lipases are capable of
catalyzing polymerization reactions, Novozym 435 - an immobilized preparation
of Candida antarctica Lipase B (CALB) on a crosslinked porous resin - is to date
by far the most active biocatalyst. As a result, we will mainly focus on the use of
CALB or Novozym 435 unless otherwise noted. Polyesters are especially interesting
for future biomaterials and, as a result, approaches using lipase catalysis have been
investigated in great detail over the past decade. The synthesis of other highly in-
teresting polymers such as polycarbonates, polyamides, and polythioesters has been
pursued with less rigor but a number of exciting results have appeared in literature.
Excellent reviews have recently focused on the details of the different types of poly-
mers that can be accessed via lipase catalysis [ 11 - 13 ] . In this chapter, we review the
intrinsic possibilities of commercially available and easy-to-handle Novozym 435 in
the field of enzymatic polymerizations, concentrating on results of the last decade.
Moreover, we will also address the limitations that arise from applying lipases in
the preparation of well-defined polymers.

2 Reaction Mechanism of Lipases and Implications

for Monomer Acceptance in the Acylation
and Deacylation Step

Lipases belong to the subclass of serine hydrolases, and their structure and reac-
tion mechanism are well understood. Their common α / β -hydrolase enzyme fold is

characterized by an -helix that is connected with a sharp turn, referred to as the

nucleophilic elbow, to the middle of a

α

-sheet array. All lipases possess an identical
catalytic triad consisting of an Asp or Glu residue, a His and a nucleophilic Ser [ 14 ] .
The latter residue is located at the nucleophilic elbow and is found in the middle of
the highly conserved Gly

β

Gly sequence in which amino acids
AA1 and AA2 can vary. The His residue is spatially located at one side of the Ser
residue, whereas at the opposite side of the Ser a negative charge can be stabilized in
the so-called oxyanion hole by a series of hydrogen bond interactions. The catalytic
mechanism of the class of

− AA1 − Ser − AA2 −

-hydrolases is briefly discussed below using CALB as
a typical example, since this is the most commonly applied lipase in polymerization
reactions [ 15 ] .
The catalytic triad of CALB consists of Asp187, His224, and Ser105 while the
oxyanion hole is formed by the backbone amide protons of Thr40 and Gln106 and
the side chain of Thr40 [ 16 - 18 ] . First, a substrate reversibly complexes to the free
enzyme (Fig. 1 top left), thereby forming a Michaelis-Menten complex. After cor-
rect positioning of the substrate, nucleophilic attack of Ser105 onto the substrate
carbonyl group occurs and a first tetrahedral intermediate is formed (Fig. 1 top
right). In this tetrahedral intermediate, the negative charge on the former substrate
carbonyl oxygen is stabilized by threefold hydrogen bonding interactions with the
oxyanion hole, whereas the positive charge on His224 is stabilized by interaction

α / β