Industrial Biotransformations

Penicillin acylase
Escherichia coli
EC 3.5.1.11
● The separation of d-(–)PGA can be done by addition of benzaldehyde and formation of the
poorly soluble Schiff base which can be filtered off subsequent to separation of enzyme and
solid products by filtration.
● Also the d-(–)-PGA is almost insoluble in aqueous solution, so that at the end of the reaction
three solids (d-(–)-PGA, cefalexin, d-(–)-HPGA) have to be separated.
● One solution of this problem was the use of a special immobilized enzyme, which floats after
stopping of the stirrer (Novo Nordisk, Denmark). The reaction solution containing the solid
products can be removed from the bottom of the reactor.
● A better technique uses enzyme immobilizates with a defined diameter. At the end the reaction
solution and solid substances can be removed from the reactor using a special sieve that is
not permeable to the immobilized enzyme. This technique is shown in the flow scheme (Fig.
3.5.1.11–4).
● 7-ACCA (= 7-aminodeacetoxymethyl-3-chlorocephalosporanic acid) can be obtained by ozonolysis
and chlorination of 3-methylene cephams. It is the precursor for the synthesis of cefaclor.
Cefaclor is unstable at pH values above 6.5 while the solubility of 7-ACCA is very low at pH
values under 6.5.
● One strategy, also established for cefalexin, is to add a complexing agent (β-naphthol). The
complex crystallizes out and yields above 90 % are possible.
● Using this technique the concentration of product in the reaction mixture is very low, so that
succeeding reactions can be suppressed and the mother liquor can be discarded. The disadvantage
is the necessity of an organic solvent to yield a two phase system in which the decomplexation
at low pH is possible.
● Using the same synthetic pathway alternatively to 7-ADCA and 7-ACCA 6-APA derivatives can
also be synthesized:
R 1
NH 2
D-(-)-1
O
R 2
+
H 2N
1a = phenylglycineamide (R1 =H, R2 =NH2) = PGA
1b = phenylglycinmethyl ester (R1 =H, R2 =OMe) = PGM
1c = hydroxyphenylglycineamide (R1 =OH, R2 =NH2) = HPGA
1d = hydroxyphenylglycinemethyl ester (R1 =OH, R2 =OMe) = HPGM
2 = 6-APA
3a = ampicillin (R1 =H)
3b = amoxicillin (R1 =OH)
Fig. 3.5.1.11 – 3
O
N
2
S
COOH
pencillin acylase
NH2 H
N
- H2O
R
O
O
N
1
S
COOH
● In contrast to cefalexin, ampicillin has a higher solubility, so that by using the recovery strategy
of cefalexin too much product would be lost.
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