Cyanobacterium Nitrilase as Catalyst for Nitrile Hydrolysis
FULL PAPER
Table 4. Details of the chiral HPLC and GC analysis of β-hydroxy carboxylic acids.
Entry
β-Hydroxy carboxylic acid
Method[a]
Retention time
t(S) [min]
T(R) [min]
1
2
3
4
5
6
7
8
9
3-hydroxy-3-phenylpropionic acid
A
A
A
A
B
B
B
C
A
12.5
11.1
12.1
12.4
17.4
13.8
20.6
34.3
14.3
17.3
14.7
16.5
16.9
20.9
20.9
32.5
34.9
15.1
3-(p-fluorophenyl)-3-hydroxypropionic acid
3-(p-chlorophenyl)-3-hydroxypropionic acid
3-(p-bromophenyl)-3-hydroxypropionic acid
3-(p-acetylphenyl)-3-hydroxypropionic acid
3-hydroxy-3-(p-methoxyphenyl)propionic acid
3-hydroxy-3-naphthylpropionic acid[b]
3-hydroxy-4,4-dimethylpentanoic acid
4-hydroxy-4-phenylbutyric acid[b]
[a] (A) HPLC, column: Whelk O-1; flow rate: 1 mL/min; solvent: hexane (0.1% HOAc): 2-propanol(0.1% HOAc) = 95:5. (B) HPLC,
column: Whelk O-1; flow rate: 1 mL/min; solvent: hexane (0.1% HOAc)/2-propanol (0.1% HOAc) = 90:10. (C) GC, column: Chirasil
DEX-CB; temperature: 60 °C for 2 min, 1 °C/min, 100 °C for 10 min. The acid was converted to methyl ester prior to GC analysis. [b]
The low ee of the samples resulted in difficulty in determining their absolute configurations, so they were assigned on the basis of
correlation of the retention time with other samples in the Table.
Expression of Nitrilase Gene in E. coli: The nitrilase gene from cya-
nobacterium Synechocystis sp. strain PCC 6803 (NCBI accession
number D64005) was cloned by following the literature pro-
cedure.[16] This nitrilase gene was cloned into pET15b expression
vector at the Nco I/BamH I sites and the plasmid DNA containing
this nitrilase gene was transformed into Rosetta2(DE3)pLysS E.
coli strain (Novagen). Overnight pre-cultures were diluted into LB
containing 100 µg/mL of ampicillin and 34 µg/mL of chloramphen-
icol, the cells were induced with 0.1 m of IPTG when optical den-
sity at 595 nm was 0.6. The bacterial cultures were incubated at
30 °C on an orbital shaker at 180 rpm for another 4 h. The cells
were harvested.
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Preparation of Cell-free Extract and Purification of Nitrilase En-
zyme: The cultures of E. coli Rosetta2(DE3)pLysS were harvested
by centrifugation. The cell pellet was re-suspended in potassium
phosphate lysis buffer (10 m, pH 7.2, 1 m DTT), and the cell
was lyzed by homogenizer. The cell-free extract was mixed with
equal volume of PEI solution (0.25% polyethyleneimine MW 40K-
60K, 6% NaCl, 100 m Borax, pH 7.4) to remove lipids.[27] After
centrifugation the supernatant was precipitated with 30% ammo-
nium sulfate. The resulting precipitate was collected after centrifu-
gation and dissolved in potassium phosphate buffer (10 m, pH
7.2, 1 m DTT). The lysate was desalted by gel filtration into po-
tassium phosphate buffer (10 m, pH 7.2, 1 m DTT), and re-
sulting enzyme solution was lyophilized and used for enzymatic
reactions.
[8]
[9]
[10]
[11]
[12]
[13]
Typical Procedure for the Enzymatic Hydrolysis of Nitriles: To a
solution of nitriles (0.5–1.0 mmol) in potassium phosphate buffer
(5 mL, 100 m, pH 7.2), the enzyme from Synechocystis sp. PCC
6803 (2–3 mg) was added. The reaction mixture was incubated at
30 °C for 12 h or the specified period. The mixture was acidified
with hydrochloric acid (1 ) to pH ca. 5, saturated with NaCl and
then extracted with ethyl acetate. The organic layer was separated
and dried with anhydrous sodium sulfate. Removal of solvent pro-
vided the crude products that were further purified by preparative
TLC using ethyl acetate/hexane as eluting solvents. The products
were characterized by comparing the 1H and 13C NMR spectro-
scopic data with those in the literature or standard sam-
ples.[8,9,23,24,26,28] For β-hydroxy carboxylic acids, the ee value was
measured by chiral HPLC or GC analysis (Table 4) and the abso-
lute configuration was determined by comparing the retention time
with those of standard samples.[23,26] For 3-hydroxy-4,4-dimeth-
ylpentanoic acid, the acid was converted to methyl ester by treating
with freshly prepared diazomethane, and the resulting methyl ester
was subjected to chiral GC analysis.
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[19]
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Eur. J. Org. Chem. 2006, 5238–5242
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