Industrial Biotransformations

Subtilisin
Bacillus licheniformis
O O
O O
S
COOEt
i) + NaOH; - EtOH
ii) + H2SO4, - Na2SO4
S
(R/S)-1
1 = 2-benzyl-3-(tert-butylsulfonyl)propionic acid ethyl ester
2 = 2-benzyl-3-(tert-butylsulfonyl)propionic acid
Fig. 3.4.21.62 – 1
1) Reaction conditions
[1]: 0.733 M, 228.25 g · L –1 [311.39 g · mol –1 ]
pH: 7.5–8.5
T: 39–40 °C
medium: two-phase: aqueous / organic
reaction type: ester bond hydrolysis
catalyst: solubilized enzyme
enzyme: subtilisin Carlsberg (hydrolase)
strain: Bacillus licheniformis
CAS (enzyme): [9014-01–1]
2) Remarks
● The EC number was formerly 3.4.4.16 and 3.4.21.14.
E
COOH
EC 3.4.21.62
● On a 150 kg scale, (R,S)-1 is reacted under non-optimized conditions as a 22 % aqueous emulsion
using solid Optimase ® M 440 (5 % with respect to (R,S)-1, pH 7.5, 40 °C, 70 h) to yield
41 % of (S)-2.
● Under optimized conditions (R,S)-1 is reacted as a 50 % emulsion in a 130 mM borax buffer
using liquid Protease ® L 660 (10 % with respect to (R,S)-1, pH 8.5, 40 °C, 42.3 h) to give 42 %
of (S)-2 (space-time yield of 550 g · L –1 ·d –1 ).
● Optimase ® M 440 is a granular subtilisin preparation used in detergent formulations.
● Protease ® L 660 is a stabilized, food-grade, water-miscible, liquid subtilisin preparation used
in detergent formulations. It has a much higher activity than Optimase ® M 440 based on
weight.
● Substilin Carlsberg is extremely tolerant to high concentrations of substrate, salt and organic
solvent, to elevated temperature and to a combination of all these factors.
● Reduction of the reaction volume is crucial for lowering production costs. This can be achieved
either by increasing the initial concentration of substrate (to about 20 %; higher concentrations
lead to emulsification problems) or by using more concentrated sodium hydroxide solutions
(2 M NaOH; higher concentrations affect hydrolysis rate) for pH control during reaction.
364
+
O O
S
(R)-2 (S)-1
COOEt
Hoffmann La-Roche AG