Strain of streptomyces for the preparation of an alkaline...

Details Strain of streptomyces for the preparation of an alkaline... Strain of streptomyces for the preparation of an alkaline...

2000-03-17

2003-02-04

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S071300, C424S093430

Reexamination Certificate

active

06514748

ABSTRACT:

FIELD
This invention relates to a novel strain of Streptomyces sp. and a process for the preparation of an alkaline-protease inhibitor. More particularly, the invention relates to a process wherein the protease inhibitor exhibiting antifungal properties is produced using a strain of Streptomyces sp. isolated from the soil sample collected from Pune, Maharashtra, India.
BACKGROUND
Protease inhibitors are one of the most abundant classes of proteins in the world. They are found in numerous plants, animals and microorganisms (Kassel et al, Methods in Enzymol. 19, 839, 1970). Protease inhibitors have received increased attention due to the awareness that they control the action of proteases which are of vital importance in regulating many proteolytic processes involved in the mobilization of tissue proteins and in the processing of precursors of proteins. They also serve as an excellent model for studying protein-protein interactions. Based on their ability to inhibit the proteases of insect digestive tracts, protease inhibitors have been implicated to possess defensive role against insects and herbivores (Green and Ryan, Science, 175, 776, 1972). Genes coding for some of the protease inhibitors have been isolated and characterized (Johnson et al., Proc. Natl. Acad. Sci. 86, 9871, 1989, Ryan, Bio Essays, 10, 20, 1989). Direct evidence that the expression of different families of inhibitor genes provides resistance against insect pests has been presented (Hilder et al., Nature, 330, 160, 1987). Protease inhibitors have been the subject of research in many disciplines. Recently, their potential for developing therapeutic agents is being explored. Specific inhibition of the proteases that are crucial in the life cycle of causative microorganisms provides the basis for application of protease inhibitors as therapeutic agents against mortal diseases such as malaria, cancer and AIDS (Billings et al., Proc. Natl. Acad. Sci. 84, 4801, 1987, Seelmeier el al., Proc. Natl. Acad. Sci. 85, 6612, 1988).
Plants and animals are forced to survive and sustain life in a world full of pathogenic bacteria and fungi. Among crop plants, fungal diseases are one of the major biotic stresses that contribute substantially to the overall loss in yield. Fungicides play a vital role in controlling the agricultural economy. Agrochemical industry employs a wide variety of synthetic antifungal agents. However, they are associated with several drawbacks such as (i) the lack of specificity (ii) development of resistance upon prolonged application and (iii) the environmental hazards associated with the residual toxicity. Against this background, biodegradable antifungal agents provide high levels of safety to non-target species. Moreover, they are free from polluting residues and represent a reduced likelihood of developing resistant fungal strains.
Plants exhibit several defense mechanisms against the invading pathogen which can be broadly classified as localized (Staskawicz et. al., Science, 268, 661, 1995) and systemic responses (Boller et al., Planta, 157, 22, 1983). The systemic responses are responsible for accumulation of toxic phytoalexins and pathogenesis related proteins (PRPs). Recently, inhibitors of trypsin and chymotrypsin (Lorito et al., Mol. Plant Microbe Interact. 7, 525, 1994) and of cysteine protease (Joshi et al., Biochem. Biophys. Res. Commun. 246, 382, 1998) of plant origin have been shown to inhibit a few phytopathogenic fungi. Inhibitors of microbial alkaline protease, viz. subtilisin, have been isolated and characterized from microbial sources mainly Streptomyces sp. (Murao & Sato, Agri. Biol. Chem. 36, 160, 1973) and from plant sources (Bodhe, Biochem. Biophys. Acta, 1073, 11, 1990). They have been studied extensively with respect to their structure, mechanism of action and regulation of gene expression. However, none of the above processes provide for preparation of an alkaline protease inhibitor having antifungal properties.
Based on the fact that the alkaline proteases present in the midgut of insect pests play an important role in the digestion of food material, the applicants believe that the inhibitor produced as per the procedure of the present invention using Streptomyces sp. could be a potent inhibitor for proteases particularly alkaline protease, more particularly the proteases of lepidopterean insect pests and therefore, has potential application as a biocontrol agent.
OBJECTS
The main objective of the present invention is to provide a process for the preparation of an alkaline protease inhibitor employing a strain of a newly isolated Streptomyces sp. which was deposited at American Type Culture Collection (ATCC) on Dec. 2, 1999, and bears accession No. PTA 973.
Another object is to provide a biocontrol and anti-fungal agent employing the alkaline protease inhibitor developed according to the process of the invention.
SUMMARY
In accordance with the above and other objects, the invention provides a novel Streptomyces sp. having Accession Number PTA 973. The invention also provides a process for the preparation of an alkaline protease inhibitor, exhibiting anti-fungal properties against a wide spectrum of fungal pathogens and therefore, useful as a biocontrol agent and anti-fungal agent.
DETAILED DESCRIPTION
Accordingly, the invention provides a novel strain of Streptomyces sp. isolated by the Applicants from the soil sample collected from Pune, Maharashtra, India. The strain has been deposited at the American Type Culture Collection, USA, and bears Accession Number PTA 973.
Further, the present invention provides a process for the preparation of an alkaline protease inhibitor, said process comprising the steps of:
(i) growing Streptomyces sp. deposited at American Type Culture Collection, and bearing Accession Number PTA 973, in a fermentation medium comprising assimilable carbon and nitrogen sources at a temperature in the range of 28-30° C. for a period of at least 96 hrs.,
(ii) separating the solids by conventional methods to obtain cell free liquid, and
(iii) recovering the protease inhibitor by conventional precipitation method from the cell free liquid using salting out agent.
In an embodiment, the fermentation medium comprises carbon and nitrogen sources and micro-ingredients.
In another embodiment, the carbon source is selected from starch glycerol, glucose, sucrose, mannose, lactose and sorbitol.
In yet another embodiment, the nitrogen source is selected from casein, asparagine potassium nitrate, skimmed milk, soyabean meal, yeast extract, malt extract, peptone, casamino acids and urea.
In a further embodiment, the medium may be supplemented with salts of metals such as Na, K, Ca, Mg, Fe, etc. in the form of their sulfates, phosphates or carbonates.
In a feature, the fermentation medium further comprises
1)
Starch
 0.5-1%
2)
Casein
 0.05-0.1%
3)
potassium nitrate
 0.1-0.2%
4)
sodium chloride
 0.1-0.2%
5)
dipotassium hydrogen phosphate
 0.1-0.2%
6)
magnesium sulfate
0.002-0.005%
7)
calcium carbonate
0.001-0.002%
8)
ferrous sulfate
0.001%
In another embodiment, the salting out agent is ammonium sulfate.
Production of the inhibitor and its activity is also dependent upon the nature and composition of media components, inoculum size and parameters such as aeration, agitation, etc. Optimum growth and production of the protease inhibitor is obtained after 96-120 h of growth when a 5-15% (v/v) log phase inoculum is employed. Post fermentation processing of the broth for the isolation of the protease inhibitor includes centrifugation or filtration. The cell free supernatant is treated with ammonium sulfate to precipitate the proteinaceous inhibitor. The salted out inhibitor is recovered by centrifugation. The resulting product is subjected to preparative polyacrylamide gel electrophoresis (PAGE).
In another feature, the protease inhibitor produced in the fermentor medium is separated by conventional methods like filtration or centrifugation.
In yet another feature, the protease inhibitor in the cell-free culture filtrate is purified by employing various

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