Screening Of Chitinase Producing Bacteria And Preparation Of Chitin Oligosaccharides By Enzymatic Hydrolysis

The native chitin has a very compact crystal structure. It is insoluble in water, dilute alkali anddilute acidand most organic solvents, therefore, native chitin is difficult to be hydrolyzed or derived by other chemicals. In this research, native chitin was treated by steam explosion, high pressure homogenization and Co-60 γirradiation, to destroy its crystal structure. The treated chitin was further hydrolyzed into chitin oligosaccharides using the chitinase produced by a strain of bacteria isolated from soil. This paper intended to solve the pollution problem in the production of chitin oligosaccharides by chemical method. The main results were as follows:(1) A strain of chitinase producing bacteria was isolated from soil, and was identified to be Paenibacillus polymyxa based on its morphological, physiological and biochemical characteristics and 16 S r DNA sequencing. The strain was named as A1.The fermentation conditions for chitinase production were determined as follows: the fermentation medium consisting of 1% chitin(120 mesh), 0.05% Mg SO4·7H2O, 1.2%CO(NH2)2, 0.5% Na Cl, 0.03% KH2PO4, 0.07% K2HPO4, 0.3% yeast extract powder,p H 4.5, starter culture age 12 h, inoculum size 1%, fermenting at 37 ℃ for 78 h.Under such conditions, the chitinase activity achieved 0.549 U/m L.(2) The chitinase produced by Paenibacillus polymyxa A1 was extracted by ammonium sulfate precipitation. Then the crude chitinase is purified by DEAE-52 ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The enzyme was purified 4.3-fold with an overall recovery of 18.4%.(3) The characterizations of purified chitinase were consequently studied. The molecular weight of the chitinase was estimated to be 30.0 k Da. The optimaltemperature of the chitinase was 45 ℃, and the enzyme was good thermostability when the temperature was lower than 45 ℃. The optimal p H of the chitinase was 4.5,and it was stable at range from p H 4.0 to 5.0. The enzyme activity is slightly activated by N-acetyl glucosamine, glucosamine and glucose, whereas SDS, VC and H2O2 can inhibit the enzyme activity. Metal ions have an influence on chitinase activity which was stimulated by Ca2+, Fe2+, Fe3+, Ni2+ and inhibited by Cu2+. Kinetic analysis of the chitinase was performed using colloidal chitin as the substrate in optimal conditions,and Km of the chitinase was 4.28 mg/m L. The chitinase can hydrolyze carboxymethyl chitin. In the initial stage of hydrolysis, the viscosity of carboxymethyl chitin solution decreased rapidly, which indicates that the chitinase is an endo-splitting enzyme.(4)The structural alterations of treated chitin by three methods, namely treatment by steam explosion method, high pressure homogenization method and irradiation method, were compared. The SEM micrographs revealed that the microstructure of the steam exploded chitin and high pressure homogeneous chitin came some changes,such as the emergence of many small holes or grooves, and structure is relatively loose, whereas the irradiated chitin was unchanged. The XRD results showed that the chitin treated by steam explosion three times exhibited the lower Cr I, with Cr I110 and Cr I020 values that were lower than those of the native chitin by approximately 23.2%and 24.7%, respectively. The Cr I110 and Cr I020 of the chitin treated by high pressure homogenization three times were reduced by approximately 12.4% and 28.7%,respectively, in comparison with those of the native chitin. These results indicated that the crystallinity of the steam exploded chitin and high pressure homogeneous chitin were reduced. The FTIR results showed that the steam explosion and high pressure homogenization method can increase the degree of deacetylation(DD) of chitin. The DDs of chitin treated with steam explosion three times and high pressure homogenization three times were increased from 20.3% to 33.5% and 26.0%,respectively.(5) Among the hydrolysis efficiency of the non-specific enzymes investigatedwith amorphous chitin as substrate, the papain showed the highest hydrolysis efficiency, but the papain was found to have no efficiency on raw chitin,steam exploded chitin and high pressure homogeneous chitin. Chitinase produced by Streptomyces griseus treatment of native chitin, steam exploded chitin and high pressure homogeneous chitin yielded 0.251 mg/m L, 0.145 mg/m L and 0.407 mg/m L of reducing sugars, respectively. When chitinase produced by Paenibacillus polymyxa A1 treatment of native chitin, steam exploded chitin and high pressure homogeneous chitin, the reducing sugars of the chitin treated by steam explosion three times and high pressure homogenization two times were improved by approximately 40% and300%, respectively. The results illustrated that chitin treated by steam explosion and high pressure homogenization can improve the reaction efficiency of chitin hydrolysis.(6)The analysis results from GPC, HPLC and mass spectrometry showed that the enzymatic hydrolysates from colloidal chitin using chitinase produced by Paenibacillus polymyxa A1 were N-acetyl glucosamine and N-acetyl chitobiose.