Thaumatin Recovery Via Bioengineering Route And In-Vitro Culture Of Thaumatococcus Daniellii

Loading...
Thumbnail Image
Date
2008-01
Authors
Chan, Derek Juinn Chieh
Journal Title
Journal ISSN
Volume Title
Publisher
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.
Description
Keywords
Thaumatin using recombinant host , achieve an economically feasible level.
Citation