Mutagenesis Screening And Culture Optimization Of Strains Producing Chitin Deacetylase

Chitin deacetylase, the enzyme is a kind of enzymes that catalyzes the hydrolysis of acetamine groups of N-acetyl-D-glucosamine in chitin. And the enzyme that catalyzes the conversion of chitin to chitosan by the deacetylation of N-acetyl-D-glucosamine residues was first identified in the supernatant fraction of Mucor rouxii by Araki. Since then, chitin deacetylase has been purified and characterized from several other fungi. The main components of fungal chitin deacetylases are glycosidoprotein, with the molecular weight 75-150kd. Chitin deacetylase hydrolyzes the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, convering chitin to chitosan. Chitosan produced by microbial enzymes is better than produced by the method of alkali chemical heating. Chitin deacetylase enzyme producing chitosan from chitin has many advantages, for example, it doesn't pollute the environment, products the specific chitosan, and so on. Enzymes from biological source were very important to catalyze the hydrolysis of acetamine groups of N-acetyl-D-glucosamine in chitin. It is a vital study to obtain a bacterium with high ability producing chitin deacetylase. The research was started with the strain preserved in our laboratory, we screened for mutants with high chitin deacetylase activity and the main researches were as followings:Rhodococcus sp. strain Y-104 (chitin deacetylase enzyme activity 946.11U/mL) was preserved in our laboratory and a strain L79 (chitin deacetylase enzyme activity 1649.07U/mL) with high enzyme activity was screened by cataalyzes the hydrolysis of acetamine groups in nitro acetanilide and chitin. Then strain L79 was mutated by ultraviolet, diethyl sulfate and ultraviolet-LiCl. A mutant strain D9 was selected finally by preliminary screening and secondary screening with enzyme activity of 2855.36 U/mL, which had increased by73.15% compared with the initial. We analyzed cell morphology of mutant D9 and initial strain L-79.Conditions of mutant D9 to produce chitin deacetylation and some other natures could have changed. The culture mediums and the fermentation conditions of chitin deacetylation production by mutant strain D9 were optimized. The optimal culture medium for enzyme production were as follows: corn pulp 21.26g/L, sucrose 26.03g/L, KH2PO4 0.89g/L, sodium acetate 4.5g/L, peptone 2.5g/L.The optimal culture medium were determined by signal factor experiment, Plackett-Burman experiment and Box-Behnken experiment. We texted the optimal culture medium for enzyme production, and the results showed a high chitin deacetylation activity 6619.86U/mL, with the enzyme activity enhanced 3.01 times.The chitin deacetylase is stable under common temperature, and would be devitalized above 60℃; Its maximum activity was at 50℃,the enzyme activity was stable between pH7.0-9.0. The enzyme activity was strongly inhibited by Fe²⁺,Fe³⁺ and advanced by Mn²⁺.To sum up, chitin deacetylase produced by mutant strain D9 indicated stability of temperature and pH and potential to carry out research further.