Method of purifying thuringiensin

Details Method of purifying thuringiensin Method of purifying thuringiensin

2000-06-02

2001-07-31

Prats, Francisco (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C424S093200, C424S115000, C424S467000, C435S041000, C435S072000, C435S087000, C435S089000, C435S122000, C435S135000, C435S136000, C435S137000, C435S252310, C435S252500, C514S047000, C514S252010, C514S255030, C514S264110, C536S026210, C536S028400, C536S028500, C536S028530, C536S028540

Reexamination Certificate

active

06268183

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of purifying thuringiensin, and more particularly to a method of purifying thuringiensin by using calcium silicate adsorption and dibasic sodium phosphate dissociation processes.
2. Description of the Related Arts
Traditional chemical insecticides are extensively applied in agriculture. However, their toxicity is also harmful to human and livestock, and the dosage used is increasing due to the increasing resistance of the insects to the chemical insecticides. Thus, the use of traditional chemical insecticides not only endangers agricultural workers, but also jeopardizes the health of consumers due to the residual pesticides on crops. Further, the impact of traditional chemical pesticides on the environment is of serious concern. Therefore, an insecticide obtained from nature and possessing high safety and low resistance is a key developing point in the field of insecticides.
Bacillus thuringiensis
is a naturally occurring, soil borne organism that has gained a great deal of attention for its ability to express compounds which control certain insect pests. Thuringiensin, one of eight toxins produced by
B. thuringiensis,
is a metabolic product and a heat-stable &bgr;-exotoxin, especially effective for fly control and often referred to as “fly factor”. Since the discovery of thuringiensin (McConnell, E. and Richards, A. G., (1959)
Can. J. Microbiol.
5:161-168), many aspects of its physical and biochemical properties, modes of action, and insecticidal/acaricidal selectivity have been described (Bond, R. P. M., et al. (1969)
Biochem. J.
114:477-488). Thuringiensin (C
22
H
32
N
5
O
19
.H
2
O) is a heat-stable compound with a molecular weight of 701 daltons. Its chemical structure is similar to that of nucleotides. The mechanism of insecticidal action is through inhibition of the production of DNA-dependent RNA polymerase by competition with ATP (Lecadet, M. M. and De Barjac, H., in Davidson, E. W. (Ed.): Pathogenesis of invertebrate microbial disease, pp.293-321, Totowa, N.J., Allanheld and Osmun, 1981). This toxic action generally applies to orders of insects such as Coleoptera, Diptera, Hymenoptera, Isoptera, Lepidoptera, Orthoptera, Neuroptera, Hemiptera and Acari in the families Tetranychidae and Phytoseiidae (Bond, R. P. M., et al. in Burges, H. D. and Hussey, N. W. (Eds.): Microbial control of insects and mites, pp.275-303, London Academic Press, 1971; Hall, I. M., et al. (1971)
J. Invertebr. Pathol.
18:359-362; Herbert, D. A. and Harper, J. D., (1986)
J. Economic Entomol.
79:592-595; Hoy, M. A. and Ouyang, Y. L., (1987)
J. Economic Entomol.
80:507-511). The toxicity of thuringiensin is much less than that of most chemical insecticides. Therefore, it shows great potential to become a very useful insecticide for controlling a wide range of insects.
Recent studies have shown that the production of thuringiensin could be improved by a net-draft-tube modified air-lift reactor (Tzeng, Y. M. and Young, Y. H., (1996)
World J. Microbiol. Biotechnol.
12:32-37). During the period of fermentation, penicillin-G may enhance the production (Tzeng, Y. M. and Young, Y. H., (1995)
Biotechnol. Prog.
11:231-234). These findings make thuringiensin more practical in terms of mass production. However, a lack of low-cost method for recovery of thuringiensin from fermentation broth has been the major rate-limiting step for industrial application. Traditional membrane ultrafiltration is not only expensive, but also time consuming and inefficient. Tzeng et al. (1999,
Biotechnol. Prog.
15:580-586) developed a micellar-enhanced ultrafiltration method to facilitate the efficiency of recovery by using a surfactant and cetylpyridinium chloride (CPC). However, some drawbacks in this method include limited adsorption rate, toxicity of CPC, and micellar complex accumulation within the filter, thereby deteriorating the efficiency of ultrafiltration.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a process of purifying thuringiensin, comprising the steps of: (a) preparing a sterile and acidic thuringiensin fermentation broth; (b) performing centrifugation of said thuringiensin fermentation broth; (c) adding an adsorbent to the supernatant obtained from step (b), and stirring to form a precipitate of said adsorbent and the thuringiensin; (d) isolating said precipitate by centrifugation; (e) adding a basic de-adsorption agent into said precipitate and vortexing to dissociate the thuringiensin; and (f) recovering and purifying the thuringiensin dissociated from step (e).
In the present invention, calcium silicate is added to the supernatant which is obtained from the thuringiensin fermentation broth by centrifugation, so that the thuringiensin dissolved in the solution can be precipitated by calcium silicate adsorption. After centrifugation, dibasic sodium phosphate is added to the precipitate and vortexed. The thuringiensin can be dissociated by the reaction of dibasic sodium phosphate with calcium silicate. After centrifugation, a preliminarily purified thuringiensin can be obtained.
The thuringiensin purified according to the method of the present invention has advantages of low-cost and quick production. The resulting thuringiensin can be formulated to a powder, thus allowing for easy release of the thuringiensin by adding water.
Another object of the present invention is to provide a high purity thuringiensin which is obtained by further purification using chromatography and electrodialysis, and which can be used as a standard for quantitative analysis.


REFERENCES:
patent: 5859235 (1999-01-01), Liu et al.
patent: 5976563 (1999-11-01), Liu et al.
Kim. Y. T. et al., The beta Exotoxins ofBacillus thuringiensis. I. Isolation and Characterization, 1970, J. Inv. Pathol., 15: 100-108.*
Jonson. D.E. et al., Limitations of HPLC for the Detection of beta Exotoxin in Culture Filtrates ofBacillus thuringiensis. 1983, Eur. J. Appl Microbiol., Biotechnol., 17:231-234.*
Yew-Min Tzeng et al.,Penicillin-G Enhanced Production of Thuringiensin by Bacillus thuringiensis SP. Darmstadiensis, Biotechnology Progress, vol. 11, No. 2, pp. 231-234 (1995).
Yew-Min Tzeng et al.,Recovery of Thuringiensin with Cetylpyridinium Chloride Using Micellar-Enhanced Ultrafiltration Process, Biotechnology Progress, vol. 15, No. 3, pp. 580-586 (1999).

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