Molecular Modification And Application Of Alginate Lyase

Alginate is one of the most abundant polysaccharides in marine resources.How to efficiently use alginate has received widespread attention.Alginate lyase can efficiently degrade alginate into fucoid oligosaccharides with various physiological activities,which has important application value in medicine,food,energy and other aspects.The current research on this enzyme is mainly focused on wild bacteria screening,heterologous expression of the enzyme,and characterization of the enzyme properties.It is necessary to use genetic engineering technology to further enhance the application potential of the enzyme.Through sequence comparison and analysis of aly-cob obtained in the previous stage,the key sites were selected for site-directed mutation,and a positive mutant strain R222L was successfully constructed and screened.The fermentation conditions were further optimized to increase enzymatic production and characterize enzymatic properties.The main conclusions are as follows:（1）Analyze the amino acid sequence of aly-cob obtained in the previous stage,and establish a mutation library based on the principle of homologous sequence consistency through BLAST alignment,homologous protein modeling,and molecular docking.And locate the key site Arg222,the forward mutant R222L was screened by site-directed saturation mutagenesis,and the enzymatic activity of the alginate lyase Aly-Cobm induced in E.coli Rosetta increased by17.01%compared with that before the mutation,reaching 6469.16 U·m L^-1.（2）In order to further improve the enzymatic activity of the mutant strain R222L,the fermentation medium was optimized at the shake flask level.The optimized fermentation medium components were:glycerol 5 g·L^-1,yeast powder 30 g·L^-1,cottonseed meal powder10 g·L^-1,Zn²⁺6μg L^-1,and the liquid volume is 50 m L per 250 m L.At the shake flask level,the highest enzymatic activity of using IPTG for low-temperature induction fermentation for24 h was 8702.71 U·m L^-1,and the cell concentration(OD₆₀₀)was as high as 9.11.The mutant strain was cultured in a fermenter.The highest enzymatic activity was 15159.92 U·m L-1 and the highest OD₆₀₀ reached 13.69 after 48 h of fermentation.The fermentation was further carried out by fed-batch fermentation,and the glycerol feed rate was 5.0 m L·h^-1.The highest enzymatic activity at 48 h was 47932.81 U·m L^-1,and the highest OD₆₀₀was 79.37.（3）Affinity chromatography was used to separate and purify the alginate lyase Aly-Cobm.Enzymatic characterization found that the optimal reaction conditions for Aly-Cobm were:temperature 45°C,p H 8.0;when the temperature is lower than 50°C and the p H is between 6.5and 9.5,the activity of Aly-Cobm is relatively stable;Na⁺,K⁺,Ca²⁺,Mg²⁺,Mn²⁺,Ba²⁺can promote Aly-Cobm,of which 1.5 mol·L^-1 Na⁺and 1.0 mol·L^-1 K⁺have the most obvious promotion effect on the enzyme;the surfactant Tween can also slightly promote the increase of enzymatic activity;the enzyme has substrate specificity and can simultaneously degrade three polysaccharides:alginate,poly M and poly G After 48 h of degradation treatment of the substrate,the main components of the product are disaccharides,trisaccharides and tetrasaccharides,indicating that Aly-Cobm is an endonuclease.