Study On The Purification And Enzymatic Properties Of Chitin Deacetylase From Micromonospora Aurantiaca

Chitosan was formed after stripping acetyl groups from chitin,and the current crafts contains biological and chemical heat alkaline methods.Chitin deacetylase(CDA,E.C.3.2.1.4)plays a key role in biological method.Compared to the heat alkali method,it has many advantages,such as the production of high specificity of chitosan,good uniformity of the product,mild production conditions,cost savings,less environmental pollution,etc..In this paper,the CDA producing strains(Micromonospora aurantiaca)were screened from the soil of seaside man grove shrimp field.And the optimum fermentation conditions,purification methods and the enzymatic properties of CDA were studied.The main research contents and results are as follows:1.Screening of strains producing CDA enzyme:The soil samples collected from man grove shrimp farms on the beach were screened to select the strains producing CDA.2.Molecular biological identification of CDA producing strain:The strains of 1-1-1 were screened for morphological characteristics and the test of 16S rDNA.The results showed that the highest homology with Micromonospora aurantiaca was 98%.3.Optimization of fermentation conditions for producing CDA by Micromonospora aurantiaca:Based on the results of single factor experiments,the optimal fermentation conditions for producing CDA by Micromonospora aurantiaca were optimized by orthogonal test.The optimum conditions are:glucose 0.5%,yeast extract 1%,CaCl2 0.15%,colloidal chitin 0.03%,NaCl 1.0%;age 56 h,inoculum 4%,fermentation temperature 30 ?,initial pH 7.0,fermentation time 84 h,liquid volume 20%(v/v),180 r/min.After the optimized fermentation conditions,the enzyme activity of CDA was determined to be 23.53 U/mL.1.78 fold enhancement in CDA deacetylation degree.4.Isolation and purification of CDA:The fermentation broth was concentrated by ultrafiltration ? freeze drying ?DEAE-cellulose ion exchange ?dialysis bag for salt removal ?PEG 4000 enrichment ?freeze drying ?Sephadex G-100 gel column ?dialysis bag for salt removal ?PEG 4000 enrichment ? after lyophilization,CDA was detected by SDS-PAGE electrophoresis,and the molecular weight was 81.8KDa.The DEAE-cellulose ion exchange chromatography conditions identified as specification are:?1.6*30 cm;column volume:60cm3;sample volume:1.2 mL;flow rate:1.2 mL/min;50 mM pH 7.2 Tris-HCl;elution:Sephadex G-100 gel column chromatography conditions identified as specification:?1.6*50 cm;column volume:sample volume:100 cm3;1.2 mL;1.4 mL/min;flow rate of eluent:50 mM pH 7.2 Tris-HCl.5.Enzymatic properties of CDAThe effects of pH,temperature and metal ions on the enzyme activity of CDA produced by Micromonospora aurantiaca were studied.Detection by infrared spectroscopy and scanning electron microscopy of chitin purified CDA derived deacetylation effect and enzyme effect on chitin.The results of experiments show that the optimum CDA from the genus pH was 7;the optimum temperature is 40 ?;metal ions Ca2+,K+ promoted the activity of CDA,Zn2+showed significant inhibitory effect,Mg2+ and Cu2+ showed mild inhibition.Infrared spectra show that the purified CDA made chitin deacetylation degree increased from 39.03%to 78.40%,scanning electron microscopy showed that chitin deacetylation after the surface becomes porous enough.Groove,crystal disappeared,and further confirms the purification of CDA has obvious effect on removing acetyl.