Screening, Identification, Fermentation Of Chitosanase-produced Strain, And Its Application

Chitosanase (E.C.3.2.1.132) can catalyze the hydrolysis of chitosan specifically, the degradation product of which are chito-oligosaccharides (COSs). So the enzymatic preparation of COSs with this method has a high potential application. Chitosanase is mainly found in the cells of bacteria, fungi and other microorganisms, also in different tissues of monocotyledonous and dicotyledonous plants. At present, researchs on chitosanase are mainly based on micro-organisms, but its activity is relatively low, what greatly limits its application. So this paper aims at isolation, screening of chitosanase-produced strain, and selection, identification of high yield strain, and the best conditions for enzyme production, and enzymatic preparation of COSs and their inhibition for bacteria.(1) Isolation and screening of high-yielding chitosanase strain. The chitosanase-produced strain was preliminarily screened from soil samples with shrimp and crab shell accumulating by transparent ring method that based on colony diameter ratio (D/d) using powder chitosan as the sole carbon source. Then re-screening by fermentation of flasks had been done, three predominant strains were obtained. With the continue determination of enzyme activity curve, strain H2 was ultimately regarded as the best chitosanase-produced strain, of which, the D/d value and the corresponding enzyme activity were 4.5±0.5, 0.61±0.005U/mL respectively after 72h.(2) According to morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, referring to "Berger bacteria Manual " and "Streptomyces Identification Manual", the strain H2 was finally identified as Streptomyces roseolus DH.(3) Studying on the best fermentation conditions of high-prodution chitosanase strain. To improve the enzyme production, fermentation conditions were determined by single-factor test targeted on medium formula and culture condition. During optimization of fermentation conditions, three significant factors including temperature, initial pH and time of fermentation were established as a response surface methodology factors based on single-factor test. The best response value for the optimal fermentation conditions was established by Design Expert7.0 statistical software.Based on single factor experiments and optimization of fermentation conditions above, the best conditions for the production of chitosanase, which medium components were listed as follows(W/V): Yeast extract 0.5, K2HPO4 0.07, KH2PO4 0.03, NaC1 0.5, MgSO4·7H2O 0.05, colloidal chitosan 1.0, peptone 0.5; and the conditions of culture were also listed as follows: initial pH 7.2, temperature 30℃, shaking speed 150rpm, volume of medium 100mL/250mL, inoculum 2% and fermentation time 62.5h. The enzyme activity of strains DH increased from 6.10±0.12U/mL to 6.94±0.29U/mL, as 13% higher than before.(4) Preliminary study on the property of crude chitosanse in liquid and antibacterial activity of COSs. During the period of studying on effects of temperature, initial pH and enzymatic hydrolysis conditions were analyzed in detail and the optimal enzymatic reaction system for the hydrolysis: temperature 45℃, reaction time 30min, reaction pH 5.6. Degradation of colloidal chitosan was studied with broth of strain DH and COSs with a certain purity were successfully obtained, which was well water-soluble and similar to commercial ones. Then the deacetylation degree and polymerization degree of COSs made though the method above were determined as 94.41% and 4 respectively, which were important for their activity, especially in terms of antibacterial activity.The inhibition of pathogenic bacteria was preliminarily studied with the concentration of 0.5% COSs. The results showed that the growth of Escherichia coli, Staphyloccocus aureus, Bacillus subtilis, Vibrio parahemolyticus was inhibited, especially Staphyloccocus aureus, Bacillus subtilis. But the inhibition effect on Vibrio parahemolyticus was not obvious.