Production of fructosyltransferase by penicillium simplicissimum in submerged culture
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Date
2010-07
Authors
Mortan, Siti Hatijah (Binti)
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Abstract
Fructosyltransferase (FTase) is an enzyme responsible for the production of
fructooligosaccharides (FOS). FOS is an important ingredient in the cosmetic,
agrochemical, pharmaceutical and food industries. Recent increase in the demand of
FOS has led to new search of FTase producing microorganism and a low cost
substrate. Seventeen fungal isolates from different genera (Trametes, Pycnoporous,
Lentinus, Schizophyllum, Penicillium, Aspergillus and Trichoderma) were evaluated
for FTase production. Penicillium simplicissimum was found to attain the highest cell
growth at 3.8 g/L and FTase activities of 506 IU/ml for extracellular and 128 IU/ml
for intracellular, respectively. Sugarcane juice was found to have the highest sucrose
concentration at 1.249x 103 g/L among the seven sources oflocal sugar tested.
In shake flask culture, FTase production were carried out using "one-factorat-
a-time" method and a statistical design approach response surface methodology
(RSM) coupled with central composite design ( CCD). The optimum culturt
conditions was obtained at sugarcane juice concentration 20 g/L, fermentation time
36 h, pH 6, inoculum size 15% (v/v) and agitation speed 150 rpm with extra-cellular
FTase activity of 118.86 IU/mL, intracellular activity of 71.97 lU/mL and biomass
concentration of 14.16 g/L, respectively.
In bioreactor studies, the effect of initial sugarcane juice concentration,
agitation speed and rate of aeration for the optimization of FTase from Penicillium
simplicissimum were also investigated using a statistical tool, RSM. Results showed
that aeration rate of 2 vvm, agitation speed of 200 rpm and initial sugarcane juice
concentration of 30 g/L had a maximum extracellular FTase activity of 161.17
IU/mL. Under such condition, maximum specific growth rate (Jlmax) of 0.877 h- 1 and
biomass yield to sucrose consumed (Yxs) of 0.667 gig were obtained. The prediction
of FTase activity and P.simplicissimum growth using Artificial Neural Networks
(ANN) were also highlighted in this study. One step-ahead prediction process
through bootstrap resampling method have proved to be consistent with the
experimental data with R2 of 0.9903 and 0.9937 for FTase activity and biomass
concentration, respectively.
An unstructured kinetic models namely the Logistic, the Monod, the Logistic
incorporating Leudeking-Piret-like equation were proposed and validated. The
models were suitable to describe biomass growth, substrate utilization and FTase
production at different initial sugarcane juice concentration ranged from 1 0-50 g/L in
batch culture. The inhibition of substrate on the growth of the P.simplicissimum was
also studied. The data fit the competitive model with R2 of0.864.
The FTase enzyme was also characterized with respect to thermostability, pH
stability, optimum temperature and pH. The maximum activities were observed at
55°C and pH 5.5. The Lineweaver-Burk plots gave the Km and v;nax values of 6.51
g/mL and 6.39 IU/mL.min, respectively. Three enzyme kinetic models have been
proposed and validated to explain the behaviour of enzyme activity and reaction
rates. For deactivation of the FTase, first order enzyme deactivation model
satisfactorily fit the experimental data with R2 range from 0.971 to 0.979. Enzymatic
reaction rates model as a function of temperature and reaction time has been selected
as the model is able to fit the experimental data correctly in wide range of
temperature with R2 of 0. 982. In inhibition of the enzyme, a competitive inhibition
model fitted the experimental data better than non-competitive and un-competitive
with R2 of0.961.
Description
Keywords
Fructosyltransferase , Penicillium simplicissimum , Submerged culture