Screening Of Chitin Deacetylase Producing Bacteria And Study On Its Production Technology

As the second largest biological resource in nature,chitin has a wide range of sources,but its strong hydrogen bonding structure greatly limits its application.Chitosan obtained by removing acetyl groups from chitin has improved biocompatibility and broad application prospects.Therefore,this study starts with screening strains with high production potential of chitin deacetylase,aiming to overcome the problems of high energy consumption and high pollution existing in current physical and chemical methods by enzymatic production of chitosan.In this study,the strains producing chitin deacetylase were screened from naturally fermented shrimps,identified them,and their identification and optimization of enzyme production conditions were carried out.The fermentation bioreactor was used to amplify the enzyme production,and the effect of inducers on the enzyme production of the strains was also explored.In addition,after obtaining the crude enzyme solution,the enzymatic properties were studied,providing new ideas for how to improve the enzyme production ability of the strains and a new direction for the green application of chitin.The main contents and conclusions of the study are as follows:（1）Six strains of chitin deacetylase-producing bacteria were isolated from naturally fermented shrimps through primary and rescreening,and one of the strains with strong enzyme production capacity,numbered X4,was selected as the test strain,and the enzyme activity was 3.909 U/m L.Through 16S r RNA molecular identification and morphological characteristics,it was found that the sequence similarity between this strain and known strains reached 97%,and it was identified as Lysinibacillus sp.X4（Lys-X4）.（2）The growth conditions of Lys-X4 were optimized,and the effects of carbon source,nitrogen source and inorganic salt on its growth were investigated.After that,the ratio of medium was optimized by orthogonal test,and the optimum growth medium was determined as follows:sucrose 0.8 g/100m L,beef extract 1.2 g/100m L,Mn SO₄ 0.1g/100m L,meantime,the number of cell can reach 2.06×10¹² cells/m L。（3）For one thing,the solution enzyme producing medium and enzyme producing conditions of Lys-X4 were optimized,for another,the effects of carbon source,nitrogen source,inorganic salt,chitin addition,p H and temperature and other conditions on the enzyme production of the strain were also investigated.Moreover,the proportion of medium and fermentation conditions were optimized by response surface methodology,the optimal enzyme production medium was determined as follows:corn meal 10 g/L,beef extract 10 g/L,sodium chloride 5 g/L,chitin addition 12 g/L,and the maximum enzyme activity was 17.228 U/m L at this time;and the optimum conditions for enzyme production are as follows:the initial p H was 7.06,the culture temperature was 35.96℃,the inoculation amount was 8.21%（V/V）,and the CDA enzyme activity was 17.237U/m L at this time.Withα-pentaacetyl glucose was induced to produce enzyme of Lys-X4,it turns out that the CDA activity was 6.17 U/m L when the addition ofα-pentaacetyl glucose was 5 g/L.Furthermore,the CDA enzyme activity was 10.83 U/m L while the ratio ofα-pentaacetyl glucose to chitin was 1:2.At this ratio,the maximum enzyme activity could reach 18.26 U/m L after 72 h fermentation.（4）The enzyme production of Lys-X4 was amplified in a 10L fermentation bioreactor.The results showed that when the stirring speed was 300 r/min,the CDA enzyme activity was 24.79 U/m L,and the deacetylation degree of chitin was 41.52%;along with dissolved oxygen wais controlled at about 75%,the enzyme activity of CDA was 31.08U/m L,and the deacetylation degree of chitin was 46.57%.After feeding the fermentation system in batches,the enzyme activity could reach 35.80 U/m L.At this time,after infrared characterization and structural characterization of the solid at the bottom of fermentation,it was found that the hydrogen bond structure of crystalline chitin was damaged to a certain extent,and partial acetyl group was removed,with its surface structure became loose.（5）The enzymatic properties of chitin deacetylase were studied,and the results showed that the optimum p H of the enzyme was 6.0,the optimum reaction temperature was 50℃,and the enzyme activity was relatively stable at p H 5～8;Mn²⁺,Fe²⁺and Fe³⁺had a strong enhancement effect on the enzyme,while Cu²⁺,Mg²⁺and Zn²⁺had an inhibition effect on the enzyme.Meanwhile,CDA produced by Lys-X4 could deacetylate colloidal chitin（71.75%）to chitosan（91.30%）,and improved the deacetylation degree of crystal chitin（52.62%）.