Alginate Degrading Gene Mining From Screened High Efficiency Strains And Its Heterologous Expression

China’s marine land area is vast,marine resources are very rich,brown algae resources are an important part of it.Sodium alginate is a natural polymer polysaccharide extracted from brown algae plant cells.It is widely used in the pharmaceutical industry,food industry,textile industry and agricultural production.In addition,alginate can be further degraded to obtain alginate oligosaccharides with smaller molecular weight.Alginate oligosaccharides have more excellent physiological activities than alginate,especially in the development of carbohydrate drugs.The preparation of brown algae oligosaccharides is of great significance for improving the economic value of cheap brown algae raw materials and realizing the effective utilization of brown algae resources.From alginate to alginate oligosaccharides,alginate lyase is one of the most critical tools.In order to promote the further development of brown algae industry and develop high value-added brown algae oligosaccharide products,it is necessary to continuously explore the excellent enzyme-producing strains and gene resources of enzyme.The main content of this study is to screen and obtain high-yield alginate lyase strains,excavate their genomic information,construct a recombinant expression system using molecular biology techniques,and achieve heterologous expression of alginate lyase in Escherichia coli.The enzymatic properties of the recombinant alginate lyase were further evaluated,and the antioxidant function of the enzymatic hydrolysate was preliminarily verified.The main findings are as follows:In this study,sediment samples collected from Dongshan Island and Xiatanwei Mangrove were screened,and 125 strains with alginate degradation potential were isolated,which were distributed in 4 genera and 11 species of Vibrio,Pseudoalteromonas,Acinetobacter and Achromobacter.Abundant alginate-degrading strain resources were obtained.Through the preliminary determination of enzyme activity by liquid culture and combined with the results of literature research,the strain DS32 with the highest activity isolated from the samples of Dongshan Island was selected for further study.In order to further explore the enzyme gene resources of strain DS32,the genome of the strain was sequenced.The sequencing results showed that the genome size of the strain was 3,782,364 bp,the GC content was 41.9%,and RAST found 3443 protein coding sequences,including 5 alginate lyase genes.The metabolic pathway of strain DS32 was predicted by KEGG database.Bioinformatics analysis of five alginate lyases showed that VRALG1～VRALG5 had no transmembrane region and were hydrophilic proteins.Combined with the enzyme production characteristics of the strain only under substrate induction conditions,it was speculated that VRALG1～VRALG5 were secretory proteins.The physical and chemical properties of the binding protein are predicted to have a half-life of more than 12 hours in E.coli,so the prokaryotic expression system should be given priority in the study of heterologous expression.The domain analysis of the genes showed that VRALG1～VRALG5 contained the conserved catalytic domain of the PL7family and did not contain the carbohydrate binding module.The three-dimensional structure model predicted by homology modeling was consistent with the typical jelly roll structure of PL7 family,and the predicted alginate lyase was PL7 family endo-cleavage lyase.Since the wild strain DS32 only produces enzymes under the condition of sodium alginate substrate induction,it is not suitable for industrial production.Therefore,the heterologous expression and enzymatic properties of the alginate lyase gene excavated in the strain were studied.The recombinant engineering bacteria of E.coli were constructed,and the enzymatic properties,substrate specificity and complete degradation products of the recombinant enzyme r VRALG1 were determined.The results showed that the optimum temperature of the recombinant enzyme r VRALG1 was 35°C,the enzyme activity reached more than 80%in the range of 0～50°C,the optimum p H was 6.5～7.5,and the enzyme activity was more than 90%after incubation for 1 h in the range of p H 6.0～9.0.Common metal ions and organic reagents such as Mg²⁺,K⁺,Cs⁺,Na⁺,imidazole,ethanol have little effect on the enzyme activity.The enzyme activity remains above 90%at a concentration of 5 mmol/L,which can better adapt to the complex environment of industrial production.In addition,unlike most of the currently characterized alginate lyases with polymannuronic acid preference,the recombinant enzyme r VRALG1 has high degradation activity on sodium alginate and polyguluronic acid,and the preference of polyguluronic acid is obvious.The degradation products of sodium alginate were mainly monosaccharide,disaccharide and trisaccharide mixture by TLC analysis,which provides a tool enzyme for the preparation of alginate oligosaccharides,especially guluronic acid oligosaccharides.In this experiment,the recombinant enzyme r VRALG1 was used to degrade high concentration substrate to prepare high concentration alginate oligosaccharides.The antioxidant function of the obtained oligosaccharides was preliminarily verified by ABTS total antioxidant capacity kit and FRAP total antioxidant capacity kit.The analysis results proved the antioxidant capacity of the enzymatic hydrolysate,which provided some theoretical support for the application of alginate oligosaccharides as an antioxidant.