Now, I would tell you about the results

The frist is enzyme screening

Various lipases were firstly selected to catalyze the enantioselective hydrolysis of 3-DFG in phosphate
buffer containing 10% acetonitrile as co-solvent. The results were listed in Table 1. Of the 9 lipases
employed, Novozym 435 showed the best activity and enantioselectivity, with (R)-enantiomer being
preferentially formed, which has the correct configuration to complete the synthesis of (−)-paroxetine
with yields is 53,9 % and ee values is 91.8%. In the consequent, Novezyme 435 was chosen as enzyme
for the enantioselective hydrolysis of 3-DFG.

Next, is effect of reaction medium

Since the prochiral substrate 3-DFG has a poor solubility in aqueous medium, organic solvent was used
as co-solvent in order to enhance the solubility of 3-DFG. However, the organic co-solvent may influence
the activity and enantioselectivity of Novozym 435, so we had to choose solvent phù hợp ??.

Choice of organic co-solvent

As shown in Fig. 1,Đây là yield , đay là ee, đây là các dmoi khảo sát ………. the use of hydrophobic
solvents (i-Pr2O, MTBE) gave better results. The addition of MTBE profoundly enhanced the activity and
enantioselectivity of Novozym 435 compared to that in phosphate buffer only. The high yield (around-
khoảng 55%) and ee value (around 93.%) were obtained when MTBE was used, this may be attributed to
that the addition of MTBE, the reaction equilibrium occurs in the forward direction as MTBE don’t
produce byproduct in the hydrolysis of 3-DFG. And, there seems to be no correlation between the
activity and enantioselectivity of Novozym 435 and the solvent parameters such as log P, dielectric
constant, dipole moment. In the consequent experiments MTBE was chosen as co-solvent for the
enantioselective hydrolysis of 3-DFG.

AND The concentration of a co-solvent also has profound influence on the enzyme activity and
enantioselectivity. the influence of MTBE concentration on the yield and ee value of (R)-3-MFG at 24 h
was investigated (Fig. 2). Đây là yield , đay là ee, đây là nồng độ

At a lower concentration (≤20%), the yield increased obviously with the addition of MTBE,

up to a yield of 69.1% at 20% MTBE, probably due to that all substrate can dissolve in MTBE until its
volume ratio reaches 20%.

However, an excess of MTBE (>20% ) resulted in a loss of enzymatic activity. This may be attributed to
the mass action effect as further increase of MTBE concentration will dilute the substrate concentration
in the organic phase and increase the enzyme concentration based on the aqueous phase.

At the same time, the influence of MTBE concentration on the ee value of (R)-3- MFG was similar to that
on the yield (Fig. 2). The highest ee value was obtained when the MTBE concentration was 20% .

The results clearly indicated that 20% (v/v) of MTBE produced the best effect on the activity and
enantioselectivity of Novozym 435.
3.2.3 PH
In addition, the activity and enantioselectivity of enzymes are highly influenced by buffer pH since the
conformation of an enzyme depends on its ionization state.

The pH range of different buffered solutions from 4.0 to 10.0 was selected to investigate the effect of
buffer pH on the activity and enantioselectivity of Novozym 435.

As shown in Fig. 3, đây là yield, đây là ee values……..the activity of Novozym 435 exhibited high
dependence on pH while the enantioselectivity was less vulnerable to pH changes.

As pH was elevated from 4.0 to 8.0, the yield increased from 26.8% to 68.7%,

after which it decreased sharply as pH increased,

and the yield declined to 35.9% at pH 10.0, indicating that the enzymatic activity was partially
inhibited in acidic and strong basic solutions.

However, ee value slightly increased with the pH range from 4.0 to 9.0,

and decreased to 91.0% as pH increased up to 10.0.

According to the data obtained in this experiment, pH 8.0 was the optimum for this hydrolytic reaction.

3.3 nhiệt độ

Following, is Effect of reaction temperature

In enzymatic reaction, temperature significantly influences the activity, enantioselectivity and stability of
a biocatalyst and the thermodynamic equilibrium of a reaction as well .

Effect of reaction temperature on enantioselective hydrolysis of 3-DFG was investigated at different

temperatures and the results were displayed in Fig. 5. đây là yield, đây là ee values……..

It was observed that with the rise of reaction temperature from 20 ◦C to 60 ◦C, yield of (R)-3-MFG
increased, indicating that Novozym showed a good thermostability. The highest yield was obtained at
60 ◦C, and the decrease of enantioselectivity at the temperature exceeding 30 ◦C was observed.

This may be due to the conformational change of the active site of Novozym caused by excessive high
temperature. Similar behaviors for the hydrolysis of diethyl 3-hydroxyglutarate using Novozym 435 were
reported [19]. However, low temperature seems to be in favor for enhancement of the
enantioselectivity of Novozym [38,39]. Thus, 30 ◦C was selected as the optimum temperature by
considering a compromise between the yield and ee value of (R)-3-MFG. Especially, enantioselectivity
was preferentially taken into accountant in the preparation of optical pure compounds