Production Of Chitosanase From Mitsuaria Sp.141-2 And Preparation Of Low Molecular Weight Chitosan

Chitosan is the N-deacetylated derivative of chitin, it is useful for applications in food, pharmaceutics and cosmetics. However, their high molecular weights, high viscosity and poor solubility restrict the use. Low molecular weight chitosan (LMWC) reveals signification different characterization in application. The methods for preparing LMWC is chemical or oxidation, they have several defects: complex process, high costs, by-products formed and harsh condition.β-1,4-glycosidic bonds in chitosan can be split specifically and selectively. Hydrolysis process and the molecular weights of productions are controlled easily. Besides, enzymes operate under milder conditions; yield of production is high; pollution is slight. Therefore, it is attracting growing interest.A new isolated strain Mitsuaria sp.141-2 was employed in this study. The maximal chitosannase activity was optimized; the chitosanase was purified and its molecular weight was determined; LMWC was obtained successfully; antibacterial activity was proved with assay for antibacterial activity. The results in detail were as follows:1. Physiological characteristic of Mitsuaria sp.141-2 was studied. The results indicated that, the optimal growth was observed at 28℃, pH 6.8. Able to utilize a number of carbon compounds, such as glucose, glucosamine, maltose, glycerol, citrate. Ammonian can be utilized by this strain, nitrate can not be utilized, negative for cellulase and amylase.2. The optimal culture conditions for chitosanase production were determined as followed: chitosan(0.5%), tryptone(0.057%), MgSO₄.7H₂O(0.05%), KH₂PO₄(0.03), K₂HPO₄(0.07%). The maximum chitosanase activity was 3.6 U/mL using this broth. The chitosanase activity increased to 4.2 U/mL from 3.6 U/mL by adjusting pH of liquid to 5.7 using 1 mol/mL NaOH at 12 h of fermentation process.3. The extracellular chitosanase produced by strain Mitsuaria sp141-2 was purified using DEAE-Sepharose CL 6B ion-exchange chromatography. Its molecular weight was determined by means of SDS-PAGE, the result showed that the molecular weight of this enzyme was 3.1 ku.4. This paper studied the conditions under which low molecular weight chitosans . (LMWC) could be prepared by degradation of chitosan using chitosanase by single-factor experiments. It was found that the chitosanase from Strain Mitsuaria sp.141-2 showed optimum depolymerization under the conditions as followed: pH 5.0, temperature 65℃, and enzyme added 6.0 U/g chitosan, concentration of substrate 4%. Then the paper determined the concentration of reducing sugar in enzymatic solution and viscosity and analysed the oligosaccharide components, which showed that the chitosanase acted on chitosan in endorsplitting manner only. The determination of molecular weight of samples prepared by means of gel permeation chromatography and viscometric measurements indicated that under the above optimum condition, the molecular weight of chitosan dropped from 298.33 ku to 26.14 ku after 30 min of enzyme hydrolysis; to 10.59 ku after 1 h; to 6.477 ku after 2 h; to 3.822 ku after 3 h.5. The decrease of molecular weight led to transformation of crystal structure and the increase of water solubility, the action of chitosana with molecular weight on the growth of Escherichia coli k99 and G.tropicalis 621 were explored. The results indicated that, Chitosan-0, Chitosan-0.5, Chitosan-1 inhibit growth of E.coli k99and G.tropicalis 621, but both Chitosan-2 and Chitosan-3 did not.