122
Y.-P. Xue et al. / Catalysis Communications 66 (2015) 121–125
O
COOH
HN
COOH
NH2
CN
NC
Whole cells of
recombinant E. coli
harboring nitrilase
NC
1) HCl
2) Na2CO3
Raney Ni
Direct
Hydrogenation
Regioselective
hydrolysis
Hydrolysis
2
1
4
3
Scheme 1. Chemoenzymatic route to gabapentin.
(5% Palladium on carbon), FTH-Ni 011 (50% nickel on alumina), RTH-
3110 (Molybdenum-promoted Raney-nickel), RTH-4110 (Chromium-
promoted Raney-nickel) and RTH-6110 (Raney-cobalt) were purchased
from Dalian Tongyong Chemical Co. Ltd. (Dalian, China).
starting material 4 was recovered. The aqueous phase was cooled to
0–4 °C. After 1 h, a white crystalline solid was collected by filtration.
The solid was dried at 40 °C to obtain gabapentin hydrochloride. The
mother liquors were recovered and reused in next reaction.
36.4 g of gabapentin hydrochloride and 50 mL water were added to a
250-mL round bottomed flask. The mixture was stirred at 40 °C to dis-
solve the gabapentin hydrochloride, 12.5 mL of methylbenzene was
added and the pH was adjusted to 7.5 with 200 g/L sodium carbonate
aqueous solution. After 30 min, the mixture was cooled to 4 °C for a
few hours. The solid was separated, and flushed with methylbenzene.
The crude gabapentin was obtained. After crystallization by methanol/
isopropanol, the pure 1 was obtained. The mother liquors were reused
in next reaction.
2.2. Strain, gene cloning and mutagenesis of nitrilase
E. coli JM109 and E. coli BL21(DE3) were used as hosts for cloning and
expression, respectively. The gene (GenBank No. KJ001820) encoding
A. facilis ZJB09122 nitrilase was cloned, mutated and expressed in E. coli
BL21(DE3) as described previously [17].
2.3. Fermentation of nitrilase in a 500-L fermentor
The optimized medium composition was as follows: peptone,
15 g/L; yeast extract, 12 g/L; NaCl, 10 g/L; glycerol, 15 g/L; (NH4)2SO4,
5 g/L; K2HPO4·3H2O 4.1 g/L, KH2PO4, 6.8 g/L; MgSO4·7H2O, 1.125 g/L
(pH 7.0). Cells were firstly transferred to 750-mL flasks containing
150 mL of LB medium from the colony and incubated at 37 °C and
150 rpm. Kanamycin (50 mg/L) was added to the medium at the begin-
ning of inoculation. When cells were grown to the end of exponential
growth phase, 900 mL of the culture broth was transferred to a 50-L
fermentator containing 30 L of LB medium. Cells were cultivated at
37 °C for 3 h with aeration at 1.1 vvm and agitation at 500 rpm. 15 L
of the culture broth was then transferred to 500-L fermentator contain-
ing 300 L of optimized fermentation medium. Fermentation was carried
out at 37 °C with aeration at 1.4 vvm and agitation at 240 rpm for 4 h.
The fermentation temperature was then decreased to 28 °C, and lactose
(12.5 g/L) was added to induce the nitrilase activity. After an 8 h fer-
mentation, whole cells were harvested by centrifugation.
2.6. Analytical methods
Biomass was measured by dry cell weight (DCW) [18]. The concen-
trations of gabapentin 1, cyanocarboxylic acid 2, and lactam 4 were de-
termined by HPLC as described previously [17]. Cell-specific activity was
measured at 40 °C using 6.79 g DCW/L in sodium phosphate buffer
(0.2 M, pH 7.0) containing 0.2 M substrate. The regioselectivity was
the molar ratio of the desired product to the total amount of carboxylic
acid products formed.
3. Results and discussion
3.1. Improvement of biocatalyst specific activity and nitrilase fermentation
In the initial experiment, we evaluated the activity of the regioselec-
tive nitrilase in E. coli transformant that expresses wild type nitrilase
[E. coli BL21(DE3)/pET28b(+)-WT]. The cell-specific activity was deter-
mined to be 30.9 U/g DCW. Although it was superior to the native strain
(17.7 U/g DCW), the biocatalyst specific activity was still too low to
achieve the yield and productivity targets. Based on homology modeling
and “hot spot” mutation analysis [17], a key amino acid Phe168 was
2.4. Preparation of 4 by hydrogenation
Into a 500-mL stirred autoclave was added 150 mL of aqueous solu-
tion containing 2 and 10 wt.% catalyst (based on the weight of 2). After
flushing the reactor with nitrogen, the mixture was stirred at 1000 rpm,
110 °C and 290 psig of hydrogen for 9 h. After hydrogenation, the pH of
the mixture was increased due to the released ammonia. The mixture
was cooled to 60 °C, and filtered to remove the catalyst. The filtrate
was adjusted to pH 7.0 with 6 M HCl and sodium chloride was added
to saturate the solution. The resulting solution was extracted with
equivoluminal dichloromethane for three times. The organic phases
were combined and then evaporated to obtain yellowish liquid. After
cooling to −20 °C for a few hours, a white crystalline solid was collected
and dried at 40 °C, to obtain the compound 4.
10000
Cell-specific activity
Regioselectivity
100
80
60
40
20
0
1000
100
10
2.5. Preparation of 1
Lactam 4 (15.3 g), water (50 mL) and hydrochloric acid (50 mL)
were added to a 250-mL round bottomed flask with a mechanical stir-
rer, and refluxed for 4 h at 150 rpm. The mixture was then cooled to
room temperature, and washed twice with dichloromethane (50 mL
each). The organic phases were combined, dried by CaCl2 and filtered.
The resulting filtrate was evaporated to remove the organic, and the
A
B
C
D
A: A. facilis ZJB09122;
C: E.coli BL21(DE3)/pET28b(+)-F168G; D: E.coli BL21(DE3)/pET28b(+)-F168V
B: E.coli BL21(DE3)/pET28b(+)-WT;
Fig. 1. Cell-specific activity and regioselectivity for the hydrolysis of dinitrile 3 by microbial
catalysts.