Characterization And Application Potential Analysis Of The Alginate Lyases From Vibrio

Alginate is the most abundant polysaccharide in brown algae,accounting for up to 40%of the dry weight of the algae biomass.Alginate consists of β-D-mannuronate(M)andα-L-guluronate(G)linked by 1,4 glycosidic bonds.Alginate lyases are polysaccharide lyases(PL),which are mainly involved in the alginate degradation and play important roles in the marine carbon cycle.Alginate lyases are mostly produced by marine bacteria.Vibrio speices are the ubiquitous aquatic bacteria and are also an important alginate-degrading group.A variety of novel alginate lyases have been found from Vibrio.Alginate oligosaccharides(AOS)derived from alginate have superior biological activities and exhibit broad application potentials in agriculture,food and medicine fields.Therefore,the study on the novel alginate lyases or the ones with application potential of Vibrio can not only enrich our understanding of the degradation of alginate by Vibrio,but also lay a foundation for the application of alginate lyases.In this thesis,we first found the gene alyC6’ encoding an alginate lyase with two PL7 catalytic domains from a Vibrio strain previously sequenced in our laboratory.The enzymatic properties of AlyC6’ were analyzed,and the synergistic effect between its two catalytic domains was revealed.The universality of the alginate lyases with multiple catalytic domains was also analyzed.In addition,we expressed and purified 11 alginate lyases from another Vibrio strain whose genome was previously sequenced,analyzed the enzymatic properties of the recombinant enzymes,and screened out the alginate lyase AlyC7,which had high activity and mainly produced alginate trisaccharides.The alginate lyase AlyC7 was expressed in Escherichia coli as a secreted enzyme and the culture conditions and additives in the medium were optimized.Finally,we prepared AOS with the extracellular recombinant alginate lyase,analyzed the composition and purity of AOS,and evaluated the promoting effect of the prepared AOS on plant root growth.1.Synergy of the two alginate lyase domains of a novel alginate lyase from Vibrio sp.NC2 in alginate degradationA novel alginate lyase AlyC6’ with two catalytic domains of the PL7 family was identified from marine bacterium Vibrio sp.NC2.The intact AlyC6’ and its two catalytic domains(CD1 and CD2)were heterologously expressed in Escherichia coli and purified.The CD1 and CD2 domains could degrade alginate independently in AlyC6’.The optimal reaction conditions of both CD1 and CD2 were 30℃,pH 8.0,and 1.0 M NaCl,which were consistent with those of AlyC6’.AlyC6’-CD1 showed high activities toward PM,PMG,and sodium alginate,but almost no activity toward PG;contrarily,AlyC6’-CD2 showed the highest activity toward PG,but almost no activity toward PM.AlyC6’ showed a similar substrate preference with AlyC6’-CDl but exhibited detectable,although not high,activity toward PG.The minimal substrate of AlyC6’-CD2 was tetramers,while AlyC6’ and AlyC6’-CD1 could degrade trimers into dimers.Compared with CD2,CD1 had higher catalytic efficiency,but lower substrate affinity.AlyC6’-CD1 significantly degraded the oligosaccharides produced by AlyC6’-CD2 into smaller products.The activity of the mixture containing AlyC6’-CD1 and AlyC6’-CD2 at a molar ratio of 1:1 was lower than that of AlyC6’.The results above showed that the two domains in AlyC6’ have a significant synergistic effect.Bioinformatic analysis showed that alginate lyases containing multiple catalytic domains are widespread in Alteromonadales and Flavobacteriales.2.Heterologous expression and characterization of the alginate lyases of Vibrio sp.C42We found 11 alginate lyase genes from Vibrio sp.C42.The alginate lyases were heterologously expressed and purified except for AlyC4.The results indicated that all the 10 recombinant alginate lyases had alginolytic activity.AlyC1,AlyC3,AlyC6,AlyC7,AlyC9,AlyC10 and AlyC11 were bifunctional alginate lyases that showed activities toward PM,PG,PMG and sodium alginate.Among them,AlyC7 mainly degraded sodium alginate into trisaccharides,which accounted for more than 80%of the products.Moreover,AlyC7 showed the highest specific activity among the bifunctional alginate lyases.These results suggest that AlyC7 has the potential to produce alginate trisaccharides.AlyC7 showed the highest enzyme activity at 30℃,pH 9.0 and 0.5M NaCl.3.Secretory expression of alginate lyase AlyC7 in Escherichia coli and optimization of culture conditionsIn order to express alginate lyase AlyC7 in Escherichia coli as a secreted enzyme,five signal peptides,OmpT,OmpA,PhoA,PelB and MalE,as well as the signal peptide of alyC7 gene,were selected to construct the recombinant vectors and the extracellular alginolytic activities of the recombinant strains were deterimined.The results showed that recombinant AlyC7 could be secreted by the recombinant E.coli strains with the signal peptides OmpA,PelB and MalE.The extracellular alginolytic activity of the recombinant E.coli strains was measured after adding 0.3%(w/v)sucrose,0.3%(w/v)sorbitol,0.03%(v/v)SDS,1%(w/v)glycine or 1%(v/v)Triton X-100 in the LB medium.The results indicated that PelB-AlyC7 showed the highest extracellular alginolytic activity after the addition of glycine.Moreover,the optimal conditions for the secretion and expression of AlyC7 were determined by single-factor optimization and orthogonal optimization.The highest activity in the culture supernatant was observed when the recombinant strain PelB-AlyC7 was cultured at 20℃ for 24 h with the addition of 500 mM glycine,and the extracellular alginolytic activity reached 1122.8 U/mL.4.Preparation of alginate oligosaccharides by recombinant AlyC7 and analysis of its application potential in promoting plant root growthIn order to prepare AOS from alginate using AlyC7 secreted by the recombinant strain PelB-AlyC7,we optimized the conditions for enzymatic hydrolysis.The optimal conditions were hydrolysis in 40 mM PBS(pH 8.0)with an E/S ratio of 25 U/mg at 30℃ for 45 min.The hydrolysis efficiency of sodium alginate reached 87.5%and the content of trisaccharides in the prepared AOS reached 84.89%.Moreover,the effect of the prepared AOS on the growth of seeds was analyzed.The results showed that 20 μg/mL AOS had significant promoting effect on the root growth of dicotyledon plants tomato and lettuce,but not on monocotyledon plants wheat and maize.In this thesis,the alginate lyases of Vibrio,an important alginate-degrading group,were explored,characterized and their application potential was analyzed.We first identified the synergism between the two catalytic domains of a novel alginate lyase,AlyC6’ from Vibrio.The alginate lyase AlyC7,which has the potential to produce alginate trisaccharides,was expressed in Escherichia coli as a secreted enzyme and the promoting effect of the AOS prepared with AlyC7 on plant root growth was evaluated.The results will enrich our understanding of alginate lyases and the degradation of alginate by Vibrio,and lay a foundation for the development and application of alginate lyases.