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K. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 250–255
were collected by centrifuging at 20,000 × g for 10 min, dried at
2.7. Continuous use of cells for bioconversion
60 ◦C and weighted.
The first reaction was carried as follows: 0.5 g (dry weight) of
fresh cell mass, 6.25 mg 7-XDT, 25 mL 50 mM PBS buffer (pH 6.0)
in a 100-mL flask (7-XDT final concentration: 250 mg/L), 7 days’
incubation at 120 rpm at 26 ◦C. After first run, the cell mass was
collected by centrifuging at 10,000 × g at 4 ◦C, washed by the same
buffer (3× 5 mL), and re-suspended in the same buffer for the next
run. The pooled supernatant of each run was extracted, analyzed
by HPLC. The same process was performed three times.
2.5. Bioconversion with growing cells
The two-stage procedure was performed, the cultivation and
bioconversion were carried out in 250-mL flask with 50 mL wheat
bran medium as described previously. The final concentration of
substrate was 2.0 mg/50 mL (final concentration: 40.0 mg/L), and
the reaction was quenched by adding 50 mL ethyl acetate and
extracted three times totally. The pooled extract was evaporated
under reduced pressure, then dissolved in 10 mL MeOH and fil-
tered through 0.25 M-pore-sized membranes just prior to HPLC
analysis.
2.8. Bioconversion of ethanol extract of T. yunnanensis
The content of 7-XDT in ethanol extract of T. yunnanensis was
2.98% by HPLC analysis. The bioconversion were performed as fol-
lows: 0.5 g (dry weight) of fresh cell mass, 210 mg extract (7-XDT
final concentration: 250 mg/L), 25 mL 50 mM PBS buffer (pH 6.0)
in a 100-mL flask, 7 days’ incubation at 120 rpm at 26 ◦C. The pro-
cedures of incubation, extraction and analysis were performed as
described before.
+
2.5.1. Effects of NH4 on the conversion
Into basic medium, were added 0, 3.0, 6.0, 13.0, and 20.0 mM
(NH4)2SO4, respectively. After the above procedure, the resulting
samples were analyzed by HPLC.
2.5.2. Effects of oat xylan on the bioconversion
3. Results and discussion
0.04%, 0.06%, 0.08%, and 0.16% (w/v) of oat xylan were added
into basic medium, respectively. After the above procedure, the
resulting samples were analyzed by HPLC.
Plants release enormous amounts of chemicals through their
roots, at a significant carbon cost, to combat pathogenic microor-
ganisms and attract beneficial ones [13]. Considering these
rhizosphere interactions, soil samples over 5 cm depth from ground
surface around T. cuspidata plants were collected for the strain
screening. Following the procedure described in Section 2.2, 34
strains with xylosidase activity as indicated by hydrolysis of
4-methyl-umbelliferyl--d-xyloside to the fluorescent 4-methyl-
umbelliferone were isolated. Then, these strains were further
subjected to identify their effectiveness with 7-XDT. By the com-
bined analyses of TLC, HPLC, LC/UV, LC/MS, four strains of them have
been determined to possess the ability to convert 7-XDT to 10-DT.
Especially, in the LC–MS spectrum, the quasi molecular ion peak of
the product at m/z 812 [M+H]+, 834 [M+Na]+and 850 [M+K]+ were
observed, indicating the loss of xyloxyl moiety (mass 132 unit). The
strain which gave the highest yield of 10-DT was identified as Enter-
obacter sp. by morphologic and molecular characteristics, and the
designated number was CGMCC 2487. This strain was selected for
the further investigation.
2.6. Bioconversion with resting cells
The cell cultured procedures were carried out as previously out-
lined, and the cells were collected by centrifuging at 10,000 × g at
4 ◦C, and then re-suspended in PBS buffer (50 mM) prior to biocon-
version use. The procedures of incubation, extraction and analysis
were performed as described before.
2.6.1. Effects of PBS buffer pH values on the bioconversion
A 0.1 g (dry weight) of fresh cells were suspended in 100 mL
flask containing 25 mL PBS buffer with different pH values (2.0, 4.0,
5.0, 6.0, 7.0, 8.0, 9.0, and 10.0), and 1.25 mg of 7-XDT (final con-
centration: 50 mg/L) in 50 L DMF was added into each flask. After
7 days’ incubation, the reactions were quenched by adding 25 mL
ethyl acetate, and extracted thrice, then analyzed by HPLC.
Moreover, the other two 7-d-xylosyltaxanes, 7-d-xylosyl-
10-deacetyl-cephalomannine ([M+H]+ m/z 922) and 7-d-xylosyl-
10-deacetyltaxol C ([M+H]+ m/z 938) were used as substrates for
examining the substrate specificity of this xylosidase. It was ver-
ified by the TLC, HPLC and LC–MS analyses that this xylosidase
can remove the xyloxyl moiety from these two molecules. In the
LC–ESIMS spectra, were observed the quasi ion peaks of [M+H]+
at m/z 790 and 806 responsible for the products 10-deacetyl-
cephalomannine and 10-deacetyltaxol C with the loss of xyloxyl
moiety. Thus, the xylosidase of this strain may take various 7-d-
xylosyltaxanes as its substrates.
Based upon the above results, for the case of further identifi-
cation of the desired product and by-products, the transformation
of 7-XDT by Enterobacter sp. was scaled up to 12 L incubation by
two-stage fermentation procedure outlined in Section 2.3. Finally,
along with 10-DT, the other three by-products (7-epi-10-DT, 10-
DAB, 7-XDB) have been obtained in the yields of about 29%, 6.9%,
1.7%, and 1.2%, respectively (Fig. 1). All the structures of products
were determined by 1H NMR, 13C NMR and MS spectroscopic data.
In this biotransformation process, the undesired reactions, 7-OH
epimerization and 13-side chain hydrolysis were also observed. For
the purpose of further examining substrate-spectrum of the xylosi-
dase of this strain, one by-product, 7-XDB was used as a substrate,
2.6.2. The kinetics of bioconversion with resting cells
The experiments were performed on a 25-mL scale (100-mL
flask, 50 mM pH 6.0 PBS buffer solution), shaking at 120 rpm at
26 ◦C. Every 24 h, three flasks were sampled randomly. The pro-
cess of extract, concentration and HPLC analysis was followed as
outlined before.
2.6.3. Effects of cell density on the bioconversion
In this experiment, the different cell densities (2.0, 4.0, 10.0,
20.0, and 40 g/L dry weight) were used for bioconversion in 50 mM
PBS buffer (pH 6.0), the final added 7-XDT concentration was
250 mg/L (6.25 mg/25 mL reaction solution). The processes of incu-
bation, extraction and analysis were performed as described before.
2.6.4. Effects of substrate concentration on the bioconversion
In this experiment, the cell density used for bioconversion
was 40 g/L cells (dry weight) in 50 mM PBS buffer (pH 6.0), the
final added substrate concentrations were designated to 250 mg/L,
500 mg/L, 1000 mg/L, 2000 mg/L, 5000 mg/L, and 10,000 mg/L. The
procedures of incubation, extraction and analysis were performed
as described before.