Thaumatin Recovery Via Bioengineering Route And In-Vitro Culture Of Thaumatococcus Daniellii
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Date
2008-01
Authors
Chan, Derek Juinn Chieh
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Abstract
Sweet tasting proteins have sparked new interest amongst researchers and
scientist alike due to the increased demand for natural sweetening ingredients. To
date. there are seven proteins identified to be able to elicit sweetness in humans. One
of them is thaumatin. a sweet tasting protein from the plant of Thaumatococcus daniellii.
Unfortunately. the limited supply and high processing cost has prevented its
widespread use. The current extraction method employs aluminium salt which is
present in the final product. Though considered negligible, it is not well accepted by
the consumers. Attempts to mass produce thaumatin using recombinant host is yet to
achieve an economically feasible level. Therefore this study was carried out to
establish a water based extraction protocol for thaumatin using the conventional
method as well as aqueous two phase system (ATPS) for potential industrial scale
application. The in-vitro culture study with the integration of membrane filtration system
was also carried as an alternative for thaumatin production in industrial scale. The
conventional extraction and purification method employed on both the water and salt
extracts of T. daniellii fruits showed varying results. A high purity of 92.4 % was
achieved for the water extract while the salt extract was only at 60.2%. The yield of
thaumatin was somewhat lower for water extract. 67.4 % compared to the salt extract
at 78.2 %. The developed extraction and purification protocols were found to be
suitable for water extracts but required further revisions in order to be applied to the
salt extracts. The performance of two ATPS composed of polyethylene glycol
(PEG)/potassium phosphate and PEG/sodium sulfate were evaluated with the aid of
fractional factorial design. The study showed that PEG/sodium sulfate system was
.superior to PEG/potassium phosphate system and consequently. PEG. sodium sulfate
and sodium chloride were identified as the significant factors against thaumatin
partitioning. Optimization carried out with the aid of response surface methodology
(RSM) with respect to thaumatin partitioning coefficient lead to a system with PEG at
the concentration of 5.92 % (wtlwt) PEG, 19.54 % {wtlwt} sodium sulfate with 0.95 M
sodium chloride at pH 7 and volume ratio of 0.12. The optimized condition led to a
relatively high yield (from 87 % to 94 %) of thaumatin when applied to both the crude
water and salt extracts. The results indicated that the A TPS developed showed
potential to serve as initial extraction and purification step as well as volume reduction
for subsequent purification procedure.
The in-vitro culture studies on the other hand showed that callus could be
obtained from root explants of T. daniellii cultured on Murashige and Skoog (MS)
medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-0). Detailed
studies would be required for possible in-vitro biosynthesis of thaumatin. The slow cell
growth of T. daniellii limited further studies on the production of thaumatin from its cell
suspension cultures. The integration of membrane filtration system in plant cell culture
was then carried out on Cyperus aromaticus, as model cell. Medium replenishment
through the membrane system leads to higher cell biomass and metabolite production
from the model cells which correlate to a value as high as 64 % and 112 %,
respectively, under the similar cultivation period as the control group. The reduction in
doubling time, extended exponential growth and increment in specific growth rate were
also demonstrated in culture undergoing medium replenishment. The medium
replenishment at 50 % (v/v) was identified for an economical and feasible application in
industrial scale for juvenile hormone (JH) III production from C. aromaticus cells.
Application of membrane filtration unit on the cell suspension culture of T. daniellii
using the, data obtained from the model cell however was shown not to differ
significantly. Establishment of a more suitable proliferation medium formulation would
be required before the incorporation of in-situ membrane filtration unit could be
venefited.
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Keywords
Thaumatin using recombinant host , achieve an economically feasible level.