Mutagenesis Breeding Of A Strain For Chitin Deacetylase Production And Study On Its Fermentation Condition

Chitosan,a deacetylation form of chitin,is more soluble,biodegradable and biocompatible than chitin.On the basis of excellent properties,such as antibacterial resistance,moisture retention and blood lipid regulation,chitosan holds its potential in food,biomedical fields and so on.Deacetylase(CDA),is the key enzyme for chitin-chitosan pathway.The catalysis approach of deacetylation is the trend for industry due to its mild reaction conditions,energy-saving controllability and eco-friendly.Penicillium janthinellum UV-OS(A marine filamentous fungi selected from the seaside soil of mangrove coast in Haikou of Hainan,China)was exposed to multiple-stage ultraviolet for mutation breeding.Fermentation profile of extracellular CDA production,optimization of fermentation conditions,properties of crude enzymology by the mutant were studied in the paper.The main contents and results are as follows:1.Multiple-stage UV-treatmentTo improve the CDA production of the penicillium janthinellum UV-OS,mutation breeding was carried out by ultraviolet treatment in this work.A selected mutant UV-3S exhibited the highest extracellular CDA activity(11.05±0.18 U/mL),2.13-fold increased in comparison with that of the wild strain UV-OS.2.Fermentation profile of CDA production by UV-OS and UV-3SExtracellular CDA avtivity,intracellular CDA avtivity,biomass production,residual glucose of Penicillium janthinellum UV-OS and mutant UV-3S were measured each 12 h(starting at 24 h during fermentation).From the time course of mycelia growth and CDA activity,it was concluded that the extracellular CDA was secreted together with the mycelia growth.Extracellular CDA enzyme activities by UV-OS and UV-3S were much higher than their intracellular CDA enzyme activities,and showed the highest CDA avtivities at 72 h.What different between UV-OS and UV-3S was that the activity of CDA of UV-3S significantly decreased in late fermentation.3.Changes of DD of cell wall chitinThe structure of fungal cell wall chitin was analyzed by scanning the fourier transform infrared spectroscopy(FT-IR).The chitins were obtained by fungal fermentation at 42 h,72 h,96 h.The DD of cell wall chitins by UV-3S were 74.04%,76.33%,79.62%,respectively.While the DD of cell wall chitins by UV-OS were 80.05%,84.01%,90.52%,respectively.4.Optimization of medium formulas and fermentation conditions of mutant UV-3SOptimum fermentation process was studied by single factor experiments and orthogonal test.The optimum medium formulas of strain UV-3S was as follows:maltose 1.3%,beef extract 2.2%,NaH2PO4 0.2%,CaCl2 0.07%and colloidal chitin 0.5%.The optimum fermentation conditions were NaCl 1.5%,initial pH 9.0,fermentation temperature 30 ?,liquid capacity 50 mL/250 mL,shaker oscillation rate 180 r/min.The highest CDA activity was 19.36 U/mL under these conditions,75%higher than that before optimization.5.Enzymatic properties of CD A by mutant UV-3STo stuy the thermo-stability of CDA,the enzyme were respectively pre-incubated at various temperatures 40?,50?,60?,70?,80 ? for 1 h before enzyme activity assay.To determination of the pH stability,the enzyme was pre-incubated at 4,5,6,7,8,9 buffersat for 1 h before enzyme activity assay.The highest CD A activities of UV-3S were detected at pH 7,and the relative residual CDA activities of mutant strain was above 80%when the pH in the range of 6-8.The appropriate temperature of CDA by UV-3S was estimated to be 50 ? and the relative residual CDA activities was above 80%in the range of 40-60?.CDA of UV-3S was activated by Ca2+,Na+,and inhibited by Fe3+,Cu2+.