Bioproduction of l-2-Aminobutyric Acid by a Newly-Isolated Strain of Aspergillus tamarii…
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1 g wet cell were washed and suspended in 5 mL 0.1 M,
pH 7.0 potassium phosphate buffer (KPB). Cell suspen-
sions were mixed with 50 μL substrate solution (N-Boc-2-
aminobutyrate:acetone=1:4,V/V) and kept at 200 rpm, 30°C
for 6 h reaction, the enantioselective hydrolysis of the sub-
strate was determined by GC-MS. The screening medium
contained (per liter, pH 7) 0.5 g KCl, 0.5 g MgSO4∙7H2O,
1 g K2HPO4∙3H2O, 3 g NaNO3, 0.01 g FeSO4, 0.25 g Bro-
mocresol purple, 20 g agar. Fermentation medium con-
tained (per liter, pH 7) 0.5 g KCl, 0.5 g MgSO4∙7H2O, 1 g
K2HPO4∙3H2O, 3 g NaNO3, 0.01 g FeSO4, 20 g sucrose.
1 H), 1.67 (dt, J=14.2, 7.3 Hz, 1 H), 1.44 (s, 9 H), 0.92 (t,
J=7.5 Hz, 3 H).
Compound 2: Light yellow liquid (67.1% yield),1 H
NMR (500 MHz, CDCl3) δ 5.06 (d, J = 7.0 Hz, 1 H),
4.27–4.19 (m, 2 H), 1.94–1.79 (m, 1 H), 1.70 (dd,
J = 14.2, 7.1 Hz, 1 H), 1.28 (dd, J = 13.8, 6.6 Hz, 10 H),
0.98–0.81 (m, 6 H).
Compound 3: Light yellow liquid (73.4% yield),1 H
NMR (500 MHz, CDCl3) δ 5.09 (d, J = 7.6 Hz, 1 H),
4.23 (d, J = 13.1 Hz, 1 H), 4.11–4.01 (m, 3 H), 2.23 (s,
1 H), 1.81 (dd, J = 17.2, 10.1 Hz, 2 H), 1.65 (dt, J = 14.2,
7.0 Hz, 4 H), 1.42 (s, 9 H), 1.22 (s, 2 H).
Compound 4: Light yellow liquid (71.8% yield),1 H
NMR (500 MHz, CDCl3) δ5.10 (d, J = 7.7 Hz, 1 H),
4.14–4.09 (m, 2 H), 3.62 (t, J = 6.7 Hz, 2 H), 2.99 (s,
3 H), 2.18 (d, J = 6.0 Hz, 2 H), 1.64 (dq, J = 13.8, 6.8 Hz,
2 H), 1.55 (d, J = 4.3 Hz, 2 H), 1.44 (s, 9 H), 1.34 (d,
J = 3.3 Hz, 3 H).
2.4 Identification of the Aspergillus tamarii ZJUT
ZQ013
The isolated strain was preliminarily identified by mor-
phological and microscopic observation. Furthermore, its
18 S-internal transcribed spacer (ITS) regions of the rDNA
were obtained through gene sequencing. The 18 S-ITS
region sequence was deposited in the GenBank database.
Related sequences were obtained from GenBank database
(National Center for Biotechnology Information) using
the BLAST system. The 18 S-ITS regions determined
GenBank databases using ClustalW ver.1.81 [14]. MEGA
ver.5.1 was applied for the calculation of evolutionary dis-
tance and finally a phylogenetic tree was constructed using
the neighbor-joining method [15, 16].
Compound 5: Light yellow liquid (64.2% yield),1 H
NMR (500 MHz, CDCl3) δ 4.13–4.08 (m, 1 H), 3.61
(t, J = 6.7 Hz, 3 H), 2.03 (d, J = 6.4 Hz, 2 H), 1.63 (t,
J = 11.0 Hz, 1 H), 1.58–1.50 (m, 3 H), 1.43 (s, 4 H),
1.37–1.33 (m, 1 H), 1.28 (d, J = 6.9 Hz, 9 H), 0.98–0.78
(m, 7 H).
Compound 6: Light yellow liquid (76.8% yield),1 H
NMR (500 MHz, CDCl3) ä 5.10 (s, 1 H), 4.38–4.25 (m,
1 H), 3.73 (s, 3 H), 1.43 (s, 9 H), 1.37 (d, J = 7.2 Hz, 3 H).
Compound 7: Light yellow liquid (74.2% yield),1 H
NMR (500 MHz, CDCl3) ä 5.03 (d, J = 7.5 Hz, 1 H),
4.29 (d, J = 5.5 Hz, 1 H), 4.11 (q, J = 7.1 Hz, 1 H), 3.73
(m, 4 H), 3.70 (s, 3 H), 1.44 (s, 9 H), 0.93 (d, J = 7.3 Hz,
3 H).
Compound 8: Light yellow liquid (79.1% yield),1 H
NMR (500 MHz, CDCl3) ä 5.04 (d, J = 8.3 Hz, 1 H),
4.21 (dd, J = 9.0, 4.8 Hz, 1 H), 3.73 (s, 3 H), 2.12 (dd,
J = 12.2, 6.6 Hz, 1 H), 1.44 (s, 9 H), 0.94 (s, 3 H), 0.88
(d, J = 6.9 Hz, 3 H).
2.5 Preparation of Derivatives of Methyl
rac-N-Boc-2-aminobutyrate
As described in synthesis of methyl rac-N-Boc-2-aminobu-
tyric acid, compounds 1, 6, 7, 8 were synthesized. Com-
pounds 9–12 are purchased from Aladdin. The structure of
compounds 1–12 were showed in Table 1.
2.6 Analytical Methods
Scheme 2. 5 mmol (1 eq.) N-Boc-2-amino butyric acid was
dissolved in 30 mL dry dichloromethane, adding 0.5 mmol
4-DMAP (0.1 eq.), 6 mmol alcohols (R–OH, 1.2 eq.) and
6 mol DCC (1.2 eq.) at 0 °C. Then the reaction was allowed
to warm slowly to room temperature and stirred overnight.
The precipitate was removed by filtration, then washed with
saturated sodium chloride solution (30mL×2), dried over
anhydrous sodium sulfate and concentrated under vacuum
to get the product. Products have been purified by thin layer
chromatography.
N-Boc-2-aminobutyric acid and its ester were detected
and separated by GC (6890 N of Agilent) with chiral cap-
illary column BGB-175. The initial temperature of oven
was 120 °C for 2 min, 4 °C/min to 195 °C for 2 min, and
the flow rate was 1mL/min. The value of e.e.s is expressed
as enantiomeric excess of the remaining ester, which was
calculated using the following equation: e.e.s=([R]−[S])/
([R]+[S]). E value was enantiomeric ratios of esters,
which was calculated using the following equation:
E = ln[(1−c)(1−e.e.s)]/ln[(1−c)(1 + e.e.s)] (c conversion,
e.e.s ee of the remaining epoxide) [17].
Compound 1: Light yellow solid (78.7% yield),1 H
NMR (500 MHz, CDCl3) δ 5.06 (d, J=6.9 Hz, 1 H), 4.26
(dd, J=13.3, 7.2 Hz, 1 H), 3.73 (s, 3 H), 1.83 (q, J=6.6 Hz,
1 3