Production, statistical evaluation and characterization of chitosanase from Fusarium oxysporum D18

(1) We screened and identified a chitosanase producing strain from the soil as Fusarium oxysporum D18 with an accession number OL343607.

(2) The enzyme activity of chitosanase was greatly improved by optimizing the fermentation medium of the fungus.

(3) The activity of chitosanase was optimized by statistical evaluation that was carried out by using Plackett-Burman and Box-Behnken designs.

(4) The production of chitosanase by Fusarium oxysporum D18 in solid state cultures using different solid substrates was studied for increasing activity.

(5) Partial purification of the enzyme was performed by using 75 % saturation of ammonium sulphate.

(6) Physical and chemical properties of the enzyme were performed leading to a highly thermostable chitosanase.

(7) An antitumour chitosanase activity was applied to the MCF-7.

Purpose The present research work focuses on the extraction of chitosanase enzyme from soil fungi.

Chitosan hydrolysis by chitosanase is one of the most effective methods to produce chitosan oligosaccharides which are new biomaterials that have many biological activities such as antitumour, antioxidant, antidiabetic and antimicrobial.

Method A strain producing chitosanase was screened and identified as Fusarium oxysporum D18 with an accession number OL343607.

Various physiological parameters (incubation type, carbon source, additive nitrogen source, statistical evaluation, solid state fermentation) were assessed to increase chitosanase production.

Results Fusarium oxysporum D18 produced a considerable value of chitosanase (1.220 U/ml).

After 7 days of incubation, the best carbon source was lactose, and the best nitrogen source was ammonium chloride.

Statistical evaluation was carried out by using Plackett–Burman and Box-Behnken designs.

The highest chitosanase production (1.994 U/ml) was induced by the medium composition g/l: KH_2PO_4 (1.5), MgSO_4 (0.269), lactose (18), NH_4Cl (1.26), pH (6.68), using a 5-day-old inoculum and chitosanase activity was 1.63 folds that of the original medium.

The production of chitosanase by Fusarium oxysporum D18 in solid state cultures using different solid substrates was studied and the best solid substrate for higher chitosanase activity (2.246 U/ml) was raw shrimp heads and shells and chitosanase activity was 1.13 folds that of the optimized liquid cultures.

An extracellular chitosanase was isolated and partially purified by using 75% saturation of ammonium sulphate.

The highest chitosanase activity (3.667 U/ml) with a specific activity of 0.390 U/mg protein was obtained at enzyme protein concentration of 9.391 mg/ml, substrate concentration of 1.2 % (w/v), V _max of the enzyme of approximately 0.430 U/mg protein, and K _ M of 0.26 % (w/v), at pH 5.6 and reaction temperature of 50 °C.

The activity of the purified and characterized chitosanase increased by 3 times than that the original isolate activity.

The enzyme was thermostable and retained about 55% of its original activity after heating at 70 °C for 15 min.

The enzyme preparations were activated by Ca^2+ ions and inactivated by Zn^+2, Cu^+2 ions, and EDTA.

Conclusion An antitumour activity of chitooligosaccharides produced by the chitosanase was applied to the MCF-7 (breast carcinoma cells) and they had a cytotoxicity inhibitory effect against them about IC_50 = 448 μg/ml.