Catalysis Science & Technology
Paper
Calculation of the equilibrium constant
condensed by rotary evaporation to recover the ethanol. The
residual solution mixture (5 L) was separated by using an NF
membrane (DK 1812, MWCO of 150 Da). A volume-constant
pattern was employed during the NF process using water as a
leachate solvent at an osmosis rate of 5 L h−1, till the relative
mass content of L-Car increased to over 95%.
After nanofiltration, the retentate was collected and 3 g
L−1 activated carbon was added and stirred at 60 °C for 0.5 h
for decolourisation. After cooling, the mixture was filtered
and 10 g L−1 artificial zeolite powder was added into the
filtrate and stirred at 30 °C for 0.5 h to adsorb the undesired
Mn2+ ions. The mixture was filtered again and the filtrate was
vacuum concentrated till a solid sediment was observed.
Then a 10-fold volume of ethanol was added slowly into the
saturated solution followed by gentle stirring. The mixture
was filtered and the filter cake was dried to constant weight.
Enzymatic hydrolysis and synthesis of L-Car were performed
simultaneously in a 10 mL system that was magnetically stirred
at 600 rpm. Both reactions were conducted under the same
conditions except for the substrate concentration. For the
hydrolytic reaction, 1.9 M β-Ala and 100 mM L-Car were used,
whereas for the synthetic reaction, 2.0 M β-Ala and 100 mM
L-His were used. 0.1 g of lyophilized cell free extract (15 U) was
added to start the reaction, and extra catalyst was supplemented
if necessary, till the concentration of L-Car in the reactions of
both directions became identical, that is, the reaction reached
equilibrium. Concentrations of β-Ala, L-His and L-Car at
equilibrium were determined by HPLC, and the equilibrium
constant (Keq) was calculated according to eqn (1).
Keq. = CCar × CH O/CAla × CHis
(1)
2
Conflicts of interest
where CCar, CH O, CAla and CHis represent the concentrations
2
of L-Car, water, β-Ala and L-His, respectively, at reaction
equilibrium.
There are no conflicts to declare.
Acknowledgements
Batch reaction for the preparation of L-Car
This work was financially sponsored by the National Key
The batch reaction was performed in a 15 L glass-jacked
stirred tank reactor. In the reactor, 5.874 kg β-Ala (6.6 M) and
387 g L-His (250 mM) were added, and water was added to
dissolve the substrates β-Ala and L-His at 40 °C and 200 rpm,
till the volume of the reaction mixture reached 10 L. The
reaction was started by adding 5 g lyophilized cell free extract
of SmPepD (750 U) and 198 mg MnCl2 (1.0 mmol), stirred at
40 °C and 200 rpm continuously. The samples were
withdrawn intermittently for the detection of the L-Car
concentration.
Research
and
Development
Program
of
China
(2016YFA0204300), the National Natural Science Foundation
of China (No. 21536004, 21776085 & 21871085), and the
Fundamental Research Funds for the Central Universities
(222201714026 & WF1714026).
Notes and references
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Continuous flow reaction based on a UF membrane for the
preparation of L-Car
A
continuous reaction was performed in a membrane
filtration device equipped with the UF membrane PT 1812
(MWCO 10 000 Da), as shown in Scheme S1.† The reaction
volume was 5 L. A batch reaction was performed initially in a
mixture containing saturated β-Ala and L-His, 0.1 mM MnCl2
and lyophilized cell free extract of SmPepD (750 U). When the
L-Car concentration reached 50 mM, the continuous flow
reaction began by continuously feeding the saturated
solution of β-Ala and L-His (containing 0.1 mM MnCl2), and
discharging the reaction mixtures at the same space velocity
(0.5 L h−1). The whole reaction process lasted for 4 d.
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Product isolation and purification
Portions of the continuous reaction mixture (50 L) were
combined with a relative mass content of only 2.3% L-Car.
Every 10 L of the reaction mixture was mixed with an equal
weight (11.7 kg) of ethanol. Major contents of β-Ala and L-His
were precipitated because of their relatively low solubility in
an ethanol solution. After filtration, the filtrate was
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Catal. Sci. Technol., 2019, 9, 5971–5978 | 5977