Study Of Chitin Deacetylase Production Of Mixed Fermentationof Rhodococcus HQcdagand Bacilluscereus CJPE209

Chitin/chitosan is a linear polymer.Both of them consist of N-acetylglucosamine and glucosamine as units in the sugar chain and are randomly connected alternately by?-1,4 glycosidic bond.When the degree of deacetylation is above 55%,they are chitosan.Chitosan has good biocompatibility.In addition to being used in industry,agriculture and fishery,it is also used in tissue engineering.Therefore,we started with the screening of high-producing chitin deacetylase strains,aiming to prepare chitosan by enzymatic method to overcome the shortcomings of high pollution and high energy consumption of the existing hot-alkali method,and to prepare high-quality chitosan products with uniform acetylation degree,small molecular weight change and fixed deacetylated position.In this study,the color circle method combined with the fermentation experiment was used to screen out the strains with the highest chitin deacetylase activity from the sludge of the crayfish farming plant;then,it was mixed with Bacillus cereus that can produce chitinase to improve the the degradation rate of colloidal chitin.And through single factor experiment,PB experiment,CCD experiment and response surface optimization experiment,the optimal conditions and medium composition for the two strains'cooperative fermentation were determined.Subsequently,the use of product component analysis and molecular biology experiments revealed the reason why the two strains promoted the production of chitin deacetylase through cooperative fermentation;finally,the extraction process of chitin deacetylase was explored and optimized.The specific results are as follows:1.Screening of chitin deacetylase producing strains:17 strains of chitin deacetylase producing strains were obtained from the soil sludge of shrimp ponds using p-nitroacetanilide as chromogenic agent and colloidal chitin as the only carbon and nitrogen source.The strain with the highest activity of chitin deacetylase was screened by pure culture,and the activity of chitin deacetylase was 19.35 U.Molecular biology and morphological identification showed that the strain was Gram-positive,belonging to Rhodococcus sp.,named HQcdag.2.Optimization of co-fermentation conditions:The authors found that the degradation rate of colloidal chitin in the culture medium was significantly increased when Bacillus cereus was mixed with Rhodococcus,and the deacetylase activity of the fermentation broth was greatly improved.Therefore,the mixed fermentation of the two strains was carried out in this paper,and the enzyme activity changes of chitin enzyme and chitin deacetylase were monitored by single bacteria fermentation as a comparison,and the single factors such as the mixing time of the two strains,the ratio of inoculation amount and fermentation conditions were optimized.Furthermore,PB experiment,CCD experiment and response surface optimization experiment were designed to determine the level of each component in the medium suitable for co-fermentation of the two strains,which could provide reference for industrial production of chitin deacetylase.The optimization results are as follows:The inoculation ratio of the two strains was 2:8(Bacillus cereus:Rhodococcus),the total inoculation amount was 3%(V/V),the fermentation period was 24 h,the fermentation temperature was 35?,the fermentation speed was 160 r/min,the initial p H was 7.0,and the loading liquid was 50m L/250 m L.The optimal medium group was as follows:peptone 0.25 g/L,K₂HPO₄0.6g/L,Mg SO₄1.5 g/L,yeast extract 3.0 g/L,Na Cl 8.5 g/L,KH₂PO₄0.8 g/L,colloidal chitin 8.0 g/L.The activity of chitin deacetylase production was 443.37 U/m L after optimization,which was 1.96 times of that before optimization.3.Revelation of the reason for the synergistic fermentation of the two strains to promote the production of chitin deacetylase:plate counting and enzyme activity monitoring experiments showed that the biomass of the two strains in the mixed fermentation was 2.85 times that of the single fermentation,and the optimal fermentation period for chitin deacetylase production was 24 h,which was 8 h shorter than that of the single fermentation of the two strains.Analyses of metabolites from Bacillus cereus fermentation showed that colloidal chitin was degraded to chitosan biose,chitosan triose,chitosan tetraose and chitosan hexaose.With two strains fermentation alone as a control group,the determination of two strains mixed fermentation and coli induced aureus fermented liquid fermentation of chitin and chitin deacetylase enzyme gene transcription,the results show that the two strains mixed fermentation,the enzyme of chitin and chitin deacetylase amount of gene expression are respectively 1.874 times and 63.22 times the size of its separate fermentation;the gene expression of chitin deacetylase in the bacteria fermentation group induced by bacilli fermentation broth was 22.202 times higher than that in the single fermentation group.This experiment showed that Bacillus cereus promoted the production of enzymes by promoting the growth and reproduction of Rhodococcus and degrading the substrate chitin,making it more readily available to Rhodococcus.4.Exploration and optimization of chitin deacetylase extraction process:six methods,including grinding method,freeze-thaw method,chemical reagent penetration method,enzymatic hydrolysis method,bead grinding method and ultrasonic method,were selected to optimize the chitin deacetylase extraction process.The results showed that bead milling had the best effect and the cell fragmentation rate was up to 48%.Three factors and three levels of orthogonal optimization experiments were carried out on the time,rotational speed and bead-liquid ratio of bead milling method.The optimal cell crushing scheme was obtained as follows:time(60 min),rotational speed(2500rpm)and bead-liquid ratio(2:1).The enzyme activity of cell fragmentation supernatant was 173.85 U/m L,and the cell fragmentation rate reached 59.5%.