Industrial Biotransformations

Thermolysin
Bacillus thermoproteolyticus
2
D,L-1
1) Reaction conditions
pH: 7.0–7.5
T: 50 °C
medium: aqueous
reaction type: homogeneous
catalyst: solubilized enzyme
enzyme: thermolysin (thermoase)
strain: Bacillus proteolicus / thermoproteolyticus
CAS (enzyme): [9073–78–3]
2) Remarks
NH2 HN
+
COOMe HOOC
COOH
X = protecting group
X
L-2
E
EC 3.4.24.27
● The main problem in chemical synthesis is the formation of by-product β-aspartame. This isomer
is of bitter taste and has to be completely removed from the α-isomer.
● The advantages of the enzymatic route are:
1) no β-isomer is produced,
2) the enzyme is completely stereoselective, so that racemic mixtures of the substrate or the
appropriate enantiomer of the amino acid can be used,
3) no racemization occurs during synthesis, and
4) the reaction takes place in aqueous media under mild conditions.
● The bacterial strain was found in the Rokko Hot Spring in central Japan. Consequently it is
very stable up to temperatures of 60 °C.
● The enzyme contains a zinc ion which is responsible for its activity and 4 calcium ions which
play an important role in its stability.
● Since the reaction is limited by the equilibrium the products have to be removed from the reaction
mixture to achieve high yields.
D-1
NH2
+
COOMe
H
N
COOMe O
HN X
1 = phenylalanine methylester
2 = aspartic acid (protected)
3 = α-aspartame (protected) DSM
Fig. 3.4.24.27 – 1
L-3
COOH
373