92
E.C. De Benedetti et al. / Journal of Molecular Catalysis B: Enzymatic 121 (2015) 90–95
Table 1
2.6.2. Immobilized biocatalyst characterization
Bed shape was quantified using the sphericity factor (SF), study-
ing the bead diameter after polymerization. SF was calculated using
the following equation:
Screening of thermophilic microorganisms for ribavirin biosynthesis.
Genera
Evaluatedstrains Positivestrains Ribavirinbiosynthesisa
Geobacillus
Streptomyces
Thermomonospora
4
5
4
3
4
2
++
+
+
(dmax − dmin)
SF =
(1)
(dmax + dmin)
Total
13
9
a
where dmax is the largest diameter and dmin is the smallest diam-
eter, perpendicular to dmax.
Conversion of 10–40% (+) and conversion higher than 40% (++).
Moreover, gel beads having uniform diameters were placed in
an excess of sodium phosphate buffer (30 mM, pH 7.0) at 60 ◦C for
swelling ratio (SR) determination. Changes in bead volume during
the course of swelling were monitored by measuring the weight
using an analytical balance. SR was determined using the following
equation:
2.9. Environmental parameters
Green chemical parameters of the aforementioned bioprocesses
were calculated to demonstrate how bioreactor development
increases the mass utilization efficiency.
Environment-factor (E-Factor) is a measurement of the indus-
trial environmental impact. Carbon efficiency (C-Efficiency) and
atom economy (A-Economy) are designed as parameters to eval-
uate the efficiency of synthetic reactions. All the abovementioned
parameters were calculated as previously described [21].
(Mt − Md)
SR=100 ×
(2)
Md
where Mt is the mass of swollen thermogel beads at a given time
during swelling and Md is the dry mass of the thermogel. These
parameters for matrix characterization were previously described
by Cappa et al. [20].
3. Results and discussion
Mechanical stability of immobilized biocatalysts was tested sub-
jecting spheres to extreme agitation conditions (Multimatic 9N,
Selecta). Shear stress was measured by mass loss, comparing initial
and final weight of the spheres.
3.1. Screening
The ability of extremophiles to produce a wide variety of com-
pounds of industrial interest has been demonstrated in previous
reports [22]. Thus, several genera of thermophilic microorgan-
isms were tested for ribavirin biosynthesis. Of the thirteen strains
studied, nine were active, three of which belong to the genus
Geobacillus, four to the genus Streptomyces and two to the genus
Thermomonospora, being G. kaustophilus ATCC 8005 the one that
proved to have the greatest biosynthetic capacity (Table 1).
This differential behavior can be explained by the different enzy-
matic selectivity, specificity or variability in the tertiary structure
of the enzyme to accept non-natural bases [23].
(Mf × 100)
Sphere mass loss(%) = 100 −
(3)
Mi
where Mi is the initial weight and Mf is the final weight of the
sphere.
Surface morphology and quantitative analysis of the chemical
composition of Ag-Ctrl and Ag-Bent beads were assessed using a
scanning electron microscope provided with energy dispersive X-
ray analysis (SEM-EDS) employing a Philips SEM 505 microscope.
3.2. Optimization of reaction parameters
2.6.3. Operational stability
Reusability of the stabilized biocatalyst was assayed through
successive ribavirin biosynthesis reactions. The catalyst reuse num-
ber was determined up to 50% of initial activity loss or matrix
integrity loss. Each reuse was performed for 6 h in optimized con-
ditions.
3.2.1. Biocatalyst load
Urd hydrolysis was tested using different amounts of G.
kaustophilus ATCC 8005 to prevent this step from becoming a lim-
iting factor for ribavirin biosynthesis.
Although Urd hydrolysis was detected using 1 × 109 CFU,
increasing the number of cells to 1 × 1010 CFU allowed to obtain
better bioconversion values at shorter reaction times. When higher
microorganisms loads (5 × 1010 CFU) were employed, a slight
increase in hydrolysis was observed (Fig. 1). However, due to oper-
ational difficulties similar to those previously discussed in other
biocatalytic systems [21], 1 × 1010 CFU was selected as the optimal
biocatalyst amount for successive assays.
2.7. Scale-up
The packed bed reactor was designed using 2.5-fold the opti-
mized biocatalyst load. Ribavirin biosynthesis was assayed in 10 mL
of media containing 2.5 mM Urd and 10 mM TCA, at 60 ◦C. Constant
flow (4 mL min−1) was achieved using a peristaltic pump (Apema
PC 26-20-F-D, Argentina).
3.2.2. Microbial growth phase
2.8. Analytical methods
Differential enzymatic expression is associated with nutritional
cell requirements at different growth stages. In view of this fact, rib-
avirin biosynthesis was assayed using the selected microorganism
at different growth phases [24].
When G. kaustophilus ATCC 8005 was evaluated at exponential
(3 h) or death growth phase (16 h), low rates of ribavirin biosyn-
thesis were detected. However, when these biocatalysts reached
stationary phase (6–12 h), a significant improvement in reaction
bioconversion values was detected, being this effect more marked
at early stationary phase (6 h) (Fig. 2).
Ribavirin biosynthesis was quantitatively monitored by HPLC
(Gilson) at 225 nm (Detector UV/Vis 156, Gilson) using a Phe-
nomenex Luna® C-18 column (5 m, 4.6 mm, 250 mm). An isocratic
mobile phase (100% water) was used with a flow of 1.2 mL min−1
,
using commercial ribavirin to determine retention time (Supple-
mentary data).
Product identification was performed by MS-HPLC under
the above mentioned conditions (Ribavirin; M+: 245.1; 2’-
deoxyribavirin: M+: 229.1) using a LCQ-DECAXP4 thermo finnigan
spectrometer with the electron spray ionization method (ESI) and
one ion trap detector.
This behavior may be explained by NP involvement in purine
and pyrimidine salvage pathways and their relation with nucleo-
side analogue biosynthesis. The activation of this recovery pathway