Resource Mining And Characteristics Of Alginate Lyases From Marine Bacteria

Alginate is a renewable carbohydrate resource.Alginate oligosaccharide(AOS),which was prepared by alginate degradation,has many biological activities such as anti-tumor,anti-oxidation,and lowering blood pressure.Since the industrial production and application of alginate are greatly limited by its high viscosity and low bioavailability,it is important to develop an efficient biological enzymatic hydrolysis method,which is the major process for alginate degradation.At present,there are two main directions for degrading alginate:first,to screen high-efficiency alginate degrading bacteria to degrade alginate directly,and explore the suitable condition for better alginate lyase production;second,to construct genetically engineered strains for better alginate degradation efficiency.In this study,we explored alginate lyase resources in both ways mentioned above.The details and results of the study are as follows.Screening and identification of alginate degrading bacteria and optimization of enzyme production conditions.A strain of high-efficiency alginate degrading bacteria FG2-1 was isolated from mangrove sediment samples by a series of alginate enrichment culture.Based on the similarity of the 16Sr RNA,the bacteria was preliminarily determined to belong to the genus Microbulbifer and named Microbulbifer sp.FG2-1.Taking carbon sources,nitrogen sources,sodium chloride,initial p H of the culture medium,culture temperature,and shaker speed as the experimental single factors,the optimal composition(g/L)of the fermentation medium of the strain was determined as Alginate 4,NH₄NO₃ 1,Na Cl 10,Mg SO₄·7H₂O 1,K₂HPO4 2.The optimal fermentation conditions was that the initial p H of the medium is 6.5,the culture temperature is 33?,and the speed of the shaker is 150 rpm.The enzyme activity can reach 11.5 U/m L after 24 hours of cultivation under the optimal conditions,which is 1.9times higher before optimization.Genome analysis and bioinformatics analysis of strain FG2-1.The genome of the strain FG2-1 was sequenced.The results showed that the genome size of FG2-1 was4,618,149 bp,the GC content was 47.0%,and there were 4248 protein coding sequences.Seven alginate lyases were annotated using RAST and CAZy database.KEGG database annotation indicated that the strain had a complete alginate metabolic pathway.Based on the bioinformatics analysis of the seven predicted alginate lyase genes,MAL1?MAL3 and MAL5 belonged to the PL7 family,MAL4 belonged to the PL6 family,and MAL6 and MAL7 belonged to the PL17 family.They are all hydrophilic proteins without any transmembrane region.Among them,MAL6 and MAL7 were predicted to be exo-alginate lyase of the PL17 family.MAL1?MAL3 and MAL5 have the conserved catalytic amino acid of the PL7 family and the typical three-dimensional structure of the PL7 family.MAL4 has the typical three-dimensional structure of the PL6 family alginate lyase,but the key amino acids at the catalytic site are poorly conserved,which indicates that it has the potential to degrade other polysaccharides.Heterologous expression and enzymatic properties of the alginate lyase gene val1 from Vibrio sp.YBCH1?8 isolated from the intestine of abalone.The recombinant expression strain E.coli BL21(DE3)/p ET22b(+)-val1 was successfully constructed from PCR products using the genome of Vibrio YBCH1?8 as the template,and the recombinant protein was induced by 1 mmol/L IPTG at 18?for 24 hours.The specific enzyme activity reached 6.6U/mg after protein purification.The enzymatic properties were studied in terms of temperature,p H,metal ions and surfactants,kinetic analysis,substrate specificity,and degradation products.The results showed that the optimal temperature of the recombinant enzyme r VAL1 was 30?,and the remaining enzyme activity could be maintained above80%after being held at 10-30?for 30 min;the optimal p H was 8.5,and the remaining enzyme activity remains above 50%after incubation at p H 4.5?10.0 for 30 minutes;1mmol/L Mn²⁺,Ca²⁺and Co²⁺had a significant promotion effect and Fe²⁺completely inhibited its activity,Fe³⁺and SDS also had a significant inhibitory effect on the enzyme activity;kinetic parameters V_max was 21.70 mg/(m L·min),K_cat was 3.28 s^-1,and Michaelis constant K_mwas 2.23 mg/m L;The substrate specificity of the recombinant enzyme to sodium alginate was significantly greater than that to Poly G or Poly M;TLC analysis of the recombinant enzyme degradation product was a monosaccharide.The results showed that the recombinant enzyme is an exo-alginate lyase with cold-adapted properties,and has potential application in the production of biofuels.The ocean is a vast enzyme reservoir,with lots of valuable alginate lyase yet to be explored.This research lays a foundation for the mining of marine alginate degrading bacteria and alginate lyase gene resources,and provides good enzyme resources for the production of AOS and the production of bioethanol from brown algae.