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INULINASE PURIFICATION PDF

Keywords: Characterization; Inulinase; Production; Purification; Streptomyces. 1. Introduction. Inulin is a polyfructan in plant consisting of linear chains. Aspergillus niger exo-inulinase purification by three phase partitioning. Inulinase (2, 1-β-D-fructan fructanohydrolase, EC ) hydrolyses inulin into. 42(4), August , pp. Purification and characterization of Inulinase from marine bacterium,. Bacillus cereus MU S Meenakshi1, S Umayaparvathi .

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Abstract Streptomyces grisenus isolated from soil, was found to produce a very active inulinase enzyme.

The optimum growth and inulinase activity occurred in the presence of 0. Analysis of purified enzyme using gel electrophoresis jnulinase an apparent molecular weight of KDa. However pure enzyme was stable in the presence of CaCl 2. The purified inulinase enzyme was subjected to N-terminal sequence analysis. Microbial enzyme inulinase Puridication 3. Oligosaccharides are compounds with great potential of use purificatiin food industry particularly interesting are fructooligosaccharids FOSbecause of their favorable functional properties such as low calorie and noncariogenic sweeteners, improvement of the intestinal microbial flora, relief of constipation, decrease of total cholesterol and lipid in the serum and promotion of animal growth Vandamme and Derycke, Inulin can be considered as dietary fibre, a substitute for fat and low calorie sweetener Roberfroid, Prebiotics exert their beneficial effects through direct and selective stimulation of healthy bacterial species in the colon flora, Ingestion of the prebiotic iulinase leads to increased content of beneficial bacteria from genera Bifidobacterium and Lactobacillus in the colon inulimase by promoting gut health.

The fermentation products from these groups of bacteria give rise to local and systemic health effects. Increased bioavailability of minerals, lowering of serum lipids levels relevant for cardiovascular disease and stimulation immune response Coudray et al. Inulinase can be found in plants and microorganisms.

It is difficult to isolate purifidation inulinases in sufficient quantity. Therefore, microbial inulinases which can be induced by growing microorganisms, have a potential for industrial use in the production of fructose frminulin Edelman and Jefford, Many micro organisms, including filamentous fungi, yeast, bacteria and streptomyces sp.

Highly active isolate of Streptomyces grisenus isolated from soil sample and identified according to Cause et al. The medium used for the production of inulinase contained 8 L -1 according to Lim et al.

As a result of reaction, reducing sugar was determined by the 3. Inulunase activity units of the enzyme were determined from calibration curve of different concentrations of fructose solution inulin assay. Blanks were run simultaneously with the enzyme and substrate solutions. Protein content was determined ;urification the method of Lowry et al. Optimization of culture condition for inulinase production: The production broth medium was optimized for maximum inulinase production by addition of 0.

Purification of the extra cellular inulinase: At the end of incubation period, the cells were separated by centrifugation at rpm for 20 min under cooling and the clear supernatant crude extract was used for enzyme purification. The precipitate formed was obtained by centrifugation in an ultracentrifuge at Then, purifivation enzyme precipitate was dissolved in 0.

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Gel filtration using sephadex G The gel permeation chromatography on Sephadex G column provided large additional increase in the purity of the enzyme according to Ettalibi and Baratti Elution was carried out by 0. Assay of inulinase activity and protein content of each fraction were examined. Factors affecting purified inulinase enzyme: Inulinase activity was assayed in the reaction mixture inoculated with different enzyme concentration 0. N-Terminal amino acid sequencing: In order to determine the N-terminal amino acid purifocation, the protein was extracted from streptomyces grisenus cells by 8 M urea treatment and then separated in an SDS-PAGE gel as already described Laemmli, After separation, the proteins were transferred to a polyvinylidene difluoride membrane, and after staining with ponceau S solution, the inulinase protein band was cut from the membrane and subjected to inulinsse acid sequence analysis.

The bacterial culture used in this study was the strongest inulinalytic strain among 50 isolates: It was identified and confirmed according to description of ISP reported by Shirling and Gottliebaband and key of Bergey’s Manual of Williams et al.

The most potent isolate was identified as Streptomyces grisenus. Culture condition for inulinase production: As shown in Table 1 the maximum activity was found on the 2nd day of incubation period.

These results are similar to those obtained by yokota et al. The results in Table 2 revealed that the iinulinase activity was observed at pH 7. These results coincide with Allais purificatioh al. Purificstion effect of carbon source on inulinase production was shown in Table 4.

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Inulin was the best carbon source for inulinase production, while starch, maltose and lactose were inadequate. Similar results were obtained YoKota et al. These authors concluded that inulin induces inulinase production, where as cultivation using starch, maltose and lactose as carbon source s restrains the production of this enzyme by optimizing culture conditions for maximum inulinase production, purification of streptomyces grisenius crude inulinase in cell free extracts was the aim of further research in order to increase its specific enzyme activity.

Further purification by gel permeation sephadex G increased the specific activity to Representing the results of fraction 50 fraction of inuline lyase enzyme using the chromatograph resulted in one sharp peak and the molecular weight was obtained KDa.

Similar results were obtained by Burne et al. Inulinase was purified wit a recovery of 2. The results in Table 6 and 7 revealed the effect of some physical conditions on purified enzyme produced by streptomyces grisenus viz.

Present findings are in agreement with Lim et al. GNDuI produced high levels of extra cellular inlinase after 24 h at pH 7. Results represented in Table 8 Indicated that the inulinase activity increased with increasing the substrate concentration until 1 mu and the activity was constant between 1- muinulin. These results agreement with Abeer indicated that the activity of inulinase by streptomyces griseus increased with increasing the sub-state concentration until 1 mu. Data in Table 9 revealed the effect of metal ions on the activity of purified inulinase in the enzyme mixture.

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Similar result was obtained by Kim and Byum who stated that the immobilization of inulinase on various matrices such as, nylon and 2-aminoethyl – cellulose AE-celluloseshowed that immobilization on AE-cellulose with glutaraldehyde treatment gives good results in terms of activity and stability. These results agreement with Gupta et al. The analysis of the deduced inulinase amino acid sequence revealed the presence of a putative gram-positive signal peptide and a cleavage site following amino acid 39 was predicted with the program designed by Nielsen et al.

Studies on inulinases produced by some bacteria. Isolation and characlerization inuljnase bacterial strains with inulinase activity. Free and immobilized forms. Expression, purification and characterization of an exo-B-D-Fructosidase of Streptomyces mutans. Problems of Classification of Actinomycetes Antagonists.

Purification and characterizationof an endoinulinase from Xanthomonas oryzae No.

Effect of soluble or partly soluble dietary fibres supplementation on absorption and balance of calcium, magnesium, iron and zinc in healthy young men. The metabolism of fructose polymers in plants.

Beta fructofuranosidases of Helianthus tuberosus L. Purification, properties and comparison of invertase, exoinulinase and endoinulinase of Aspergillus ficuum. Acomparison of properties of inculinase of Fusarium oxysporum immobilised on various supports.

Hydrolysis of inulin from jerusalem artichake by inulinase immobilized on aminoethyl cellulose enzyme. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Purification and characterization puricication levanse from Streptomyces sp. Protein measurement with the folin phenol reagent. Use of dinitrosalicylic acid reagent for determination of reducing sugar.

Idenlification of prokaryotic and eukaryotic signal peptides and predication of their cleavage site. Recent developments in microbial inulinases. Its inulinae, properties and industrial applications. Utilization of chicory roots for microbial endoinulinase production. Dietary fiber, inulin and oligofructose: A review comparing their physiological effects.

Production, Purification, Characterization and Applications of Fungal Inulinases | BenthamScience

Methods for characterization of Streptomyces species. Co-operative description of type culture of St. Additional species description from first and second studies. Co-operative description of type culture of Streptomyces sp. Co-operative description of type strain of Streptomyces. Microbial inulinases formentation process, properties and applications. Optimisation of inulinase production by Kluyveromyces bulgaricus. Characleristics of an inulinase produced by bacillus subtilis Purifictaion, astrain isolated from the rhizosphere of Ueronia herbacea velly rusby.

Exocellular Inulinase of Bacillus subtilis Elsevier Applied Science, London, pp: Isolation of mutant of Kluyeromyces sp. Y resistant to catabolite repression. The Biology of ActinomycetesMordarski, M. Academic Press, London, pp: Purification and characteristics of two exoinulinases from Chrysosporium pannorum J. Production inulinawe inulotriose from inulin by Inulin-degrading enzyme from Streptomyces rochei E

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