| Alginate is a water-soluble and acidic polysaccharide derived from the cell wall and intercellular substance of brown algae.It widely distributes in brown algae,such as Laminaria,Sargassum and Macrocystis,etc.Alginate lyase can catalytically degrade alginate in a ?-eliminating manner,and its degradation product-alginate oligosaccharide,has been widely used in agriculture,medicine,cosmetics and other fields due to its wide biological activity.However,because of the low enzyme activity of the alginate lyase-producing strain it is not suitable for industrial production and restricts its wide application.In this paper,the alginate lyase-producing strains were screened from marine habitats,and the enzyme-producing abilities were optimized.The genetic information and enzyme-producing genes were analyzed by bioinformatics.The detailed research results are as follows:Seaweed samples were extensively collected from the coast of Hainan Island.Using two sample processing methods and using sodium alginate as the sole carbon source medium,15 alginate lyase-producing strains were screened.They distributed in 7 genera including Cobetia,Isoptericola,Mangrovicoccus,Microbulbifer,Paenibacillus,Paracoccus and Vibrio.Based on 16S rDNA sequence analysis,strain HB172198 has the highest similarity(97.63%)with Paenibacillus lemnae L7-75T.Phylogenetic analysis indicated that the novel strain formed a distinct subclade and clustered with the type strain of P.lemnae L7-75T.The results of the phenotypic,biochemical and physiological,16S rDNA and ANI analysis clearly indicated that strain HB172198T should be classified as representing a novel Paenibacillus species,for which the name Paenihacillus algicola sp.nov.is proposed.The medium and culture conditions of strain HB172198 were optimized by response surface methodology based on the alginate lyase enzyme activity.The optimal fermentation medium and conditions were:sodium alginate 7.50 g/L,tryptone 13.57 g/L,NaC129.75 g/L,MgS04·7H2O 0.08 g/L,original pH 7.0,30?.180 r/min for 36 h the enzyme activity reached 15.20 U/mL,which was 1.95 times of the enzyme activity under the initial culture conditions.The enzymatic properties of the crude enzyme solution after ammonium sulfate precipitation and ion dialysis were studied.The optimum reaction temperature of the enzyme was 50?,and the optimum pH was 7.0.It is stable in the environment of pH 5.0-9.0 and below 40?.Metal ions such as Ca2+,Mg2+and Fe2+ promoted the alginate lyase,but EDTA,Ba2+and Zn2+ have inhibitory effects.The zymolytic oligosaccharide component of sodium alginate was preliminarily identified by TLC method,and the degree of polymerization was between 1 and 6.The strain HB172198 was sequenced using PacBio RSII and Illumina X10 platform a complete circular genome was obtained,with 4,475,055 bp in length and the GC content 51.2%.Based on assembling by HGAP software,predicting by software glimmer 3.02,tRNA-scan RNAmmer,it was predicted that 4210 genes included 80 tRNA sequences and 27 rRNA sequences.The gene function was classified with COG and KEEG database,and it was showed that a total of 2950 proteins had clear biological functions,1842 proteins had KEGG homologous genes and 2946 proteins had COG classification.Three alginate lyase sequences were predicted based on the CAZY database,which belonged to PL5,PL7 and PL15 families,respectively.Conservative regions were compared by amino acid sequence alignment,and the possible alginate metabolic pathways of strain HB172198 were predicted based on bio informatics analysis as well.Mutagenic treatment of strain HB172198 was carried on using atmospheric and room temperature plasma(ARTP)technique,two strains with significantly increased and decreased enzyme activity were screened.The four strains were subjected to genome resequencing and their sequence differences were made clear. |
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