20
Letter / Journal of Molecular Catalysis B: Enzymatic 81 (2012) 19–24
Optimal conditions for DG oxidation with P2Ox in the presence
Symbols
of catalase depend on the enzyme source. For the recombinant P2Ox
used, literature information indicates 35 ◦C as being optimal tem-
perature for its activity, while catalase works better at 25 ◦C (at
the set temperature is close to the surrounding one, it is natural
to investigate the process characteristics at a lower temperature.
Consequently, the present paper is aiming at extending the study
of Maria et al. [1] by identifying the DG oxidation kinetic parame-
ters at the lower temperature of 25 ◦C for the optimal pH = 6.5, by
investigating the same range of CTP = 0–300 initial ratios. A com-
parative analysis of results at 25 ◦C and 30 ◦C allows determining
the economically favourable reaction conditions, by highlighting
the complex interferences among reactions through intermediate
species making the temperature and catalase addition influence the
process, sometimes in opposite directions.
cj
species j concentration, mM
saturation concentration of the dissolved oxygen,
mM
cD∗ O
D = d ln (V)/dt reactor dilution rate, s−1
kc, kd, Kj rate constants (units in s, U mL−1, mM)
koxl
m
n
a
overall gas–liquid mass transfer coefficient, s−1
number of observed species
Yano–Koya exponent
number of experimental points (recording times)
number of independent model parameters
reaction rate, mM s−1, U mL−1 s−1
model relative error standard deviation or variance
time (s), or statistical Student test of estimate sig-
nificance
N
p
r
sy, s2y
t
V
x
liquid volume, L
DG conversion
2. Experimental: enzymes, reagents, and batch experiments
Greeks
ꢀ2
ꢁ
Experiments have been conducted in
a thermostatically
statistical ꢀ2-test of model overall adequacy [21]
stoichiometric coefficient
controlled laboratory bioreactor using commercial grade com-
pounds from Sigma–Aldrich: recombinant pyranose 2-oxidase
(EC 1.1.3.10, product number P4234, activity of 10.4 U mg-
ꢂm
turnover−n1 umber of the main reaction, mM s−1
(U mL−1
)
protein−1
) from Coriolus sp. expressed in E. coli; catalase
ꢃ
measurement standard error deviation, mM,
(EC 1.11.1.6, product number C1345, activity of 2860 U mg-
protein−1) from bovine liver; glucose oxidase (GOD, EC 1.1.3.4,
product number G7016, activity of 154 U mg-protein−1) from
Aspergillus niger; -d-glucose; peroxidase (EC 1.11.1.7, product
number P6782, activity of 1000 U mg-protein−1) from horseradish;
2,2ꢀ-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS); 2-
keto-d-glucose; 2,3,5-triphenyltetrazolium chloride (TTC); 6 N
NaOH; acetic acid; ethanol; 0.1 M acetate buffer solution of pH = 5.0;
0.05 M and 0.01 M phosphate buffer solution of pH = 6.5.
U mL−1
Superscripts
*
ˆ
saturation
estimated
Abbreviations
ABTS
2.2ꢀ-azino-bis(3-ethylbenz-thiazoline-6-sulfonic
acid
The same spectrometric procedures described by Maria et al. [1]
have been used to determine the d-glucose and H2O2 concentra-
tion in samples, while the ABTS test of Leitner et al. [2] has been
followed to determine the P2Ox activity during the reaction. One
unit (U) of P2Ox activity is defined as the amount of enzyme nec-
essary for the oxidation of 2 mol of ABTS per minute under the
given conditions. Catalase activity is expressed in Sigma units and
tested according to the supplier instructions. One unit will decom-
pose 1 mol of H2O2 per minute at pH 7.0 and 25 ◦C. Enzyme assays
were performed in triplicate and mean values were considered. A
rapid and inexpressive method to determine kDG concentration in
samples, in the presence of DG, involves the use of TTC [22]. The
detection method is based on a different reduction rate by the two
sugars, meaning that DG reduces TTC to a red pigment (a triph-
enylformazan) about 100 times slower than an equivalent amount
of kDG. The analysis uses 0.010 mL sample in a 4 mL test tube, by
adding 0.020 mL 1% aqueous TTC and 0.08 mL 6 N solution of NaOH.
After 5 min, 3 mL acetic acid: ethanol (1:9) is added and the test tube
content is homogenized. The absorbance of each sample was ana-
lysed by spectrophotometry at 480 nm and the kDG content was
relatively quantified to a standard curve generated using kDG from
Sigma.
CTP
DG
DO
GOD
kDG
catalase/P2Ox initial ratio
d-glucose
dissolved oxygen
glucose oxidase
2-keto-d-glucose
NAD(P)H nicotinamide adenine dinucleotide (phosphate)
P2Ox
TTC
ꢁ•ꢁ2
pyranose 2-oxidase
2,3,5-triphenyltetrazolium chloride
Euclidean norm
is encouraging, indicating a 50% initial activity for catalase/P2Ox
(CTP) = 300 U U−1, and 70% initial activity for higher ratios than
1000 U U−1. However, the recommended CTP initial ratios are less
than 300 U U−1 for economical reasons [6,15].
A complete engineering analysis of the process can be bet-
ter done if an adequate kinetic model, of reasonable complexity,
is available. Recently, Maria et al. [1] proposed a three-reaction
schema of the DG oxidation on suspended P2Ox/catalase mixture
(recombinant P2Ox from Coriolus sp. expressed in E. coli), and esti-
mated the rate constants at 30 ◦C and pH = 6.5 in the range of
CTP = 0–300 initial ratios. A considerable decrease of the main reac-
tion rate is observed as the catalase amount increases (ca. 2 times
for a CTP = 100 up to 12 times for CTP = 300 initial ratio), while the
presence of catalase slows down the P2Ox inactivation. It results
that, for this P2Ox type, working at higher CTP ratios is not inter-
esting as long as the main reaction rate decreases sharply with
the catalase level in the reaction environment; a CTP = 100 ratios
appears to be the most favourable.
Experiments have been carried out in a 1 L laboratory bioreac-
tor, efficiently mixed, with fully controlled operating parameters
(temperature, pH, liquid level), and monitored DO. Separate exper-
iments, in the absence of reaction, allowed determining the overall
gas–liquid mass transfer coefficient koxla = 0.01–0.02 s−1 (1 L min−1
aeration rate, 300 rpm stirrer speed, reaction medium), by perform-
ing partial de-aeration (sparging with compressed N2) followed by
re-aeration of the liquid [16].
DG enzymatic oxidation has been conducted in batch mode
at 25 ◦C under continuous aeration, by using 100 mM glucose,