Strain Resource Excavation And Characterization Of Alginate Lyase

Alginate is a natural macromolecular polysaccharide originated from brown algae.It consists of?-D-mannuronic acid(M)and L-guluronic acid(G)linked by 1,4-glycosidic bonds.Alginate oligosaccharides(AOS)are low molecular weight oligomers of alginate with improved water solubility and viscosity.AOS could cross various biological barriers and cell membranes,thus expanding its application as physiological active substances,and present more outstanding biological activities,such as reducing blood sugar,antivirus,anti-oxidation and immune regulation,therefore,AOS have a broad application prospect in food,agriculture,biology,medicine and many other fields,and have become the focus of research and development of marine functional foods and drugs.Alginate lyase breaks 1,4-glycosidic bonds of alginate to produce AOS.Other than physical and chemical methods,enzymatic degradation requires only one step degradation,which has the advantages of simple operation,mild reaction conditions,high specificity,high yield,easy purification of products,etc.,and has a significant application prospect in AOS industrialization.However,alginate lyase preparation is rare and expensive,which restricts the large-scale production of AOS.The main purpose of this study was to screen and obtain strains with high alginate lyase yield,extracellularly express the alginate lyase in recombinant Escherichia coli via molecular biology technology,and further characterize the enzyme and study the multi-domain structure-function by truncation expression.On the other hand,Vibrio natriegens is the fastest-growing bacterium known to date,which presents doubling time almost half that of E.coli and is considered as a potential next-generation model organism,therefore,the feasibility of using the screened V.natriegens strain as new host bacterium was studied.The main contents and results of this study are listed:(1)An alginate lyase-producing strain V.natriegens SK42.001 was screened from sea mud,and was deposited in China Typical Cultures Preservation Center(CCTCC)with the preservation number No.M 2017011.Alginate lyase Aly01 produced by V.natriegens SK42.001 was an inducible enzyme,the fermentation activity was 5.20±0.14 U/m L,and the enzyme Aly01 mainly produced trisaccharides.The molecular weight of Aly01 was about 50k Da.When degrading alginate to different extent,with the increase of Aly01,the molecular weight and viscosity of the products decreased significantly,and the M content increased while the G content decreased.¹H NMR analysis further showed that Aly01 presented a preference for degrading poly G.(2)The gene encoding Aly01 was amplified from the genome of V.natriegens SK42.001by PCR,and the enzyme contains an N-terminal signal peptide consisting of 28 amino acids.Recombinant plasmid p ET28a-aly01 with the full length Aly01 gene was constructed and transformed into E.coli BL21,and the signal peptide of Aly01 could be recognized by E.coli and recombinant Aly01 was extracellularly produced when induced at 18^°C.Furthermore,the synergistic strategy of adding 120 mmol/L glycine and 10 mmol/L calcium ion in TB medium reduced the lag phase of protein secretion from 12 h to 3 h,meanwhile,the addition of calcium ion remedied the cell growth inhibition caused by glycine,and promoted the overall enzyme activity to 4.55 U/m L.In addition,the green fluorescent protein(GFP)was fused with the signal peptide of Aly01 to construct recombinant plasmid p ET28a-SPGFP,which was transformed into E.coli BL21.GFP was successfully secreted by recombinant E.coli,which further expanded the application of Aly01 signal peptide in the extracellular production of other heterologous proteins by E.coli.(3)From sequence analysis and the modelled three-dimensional structure,Aly01contains a carbohydrate binding module(CBM32)and a catalytic domain(CD),connected by an?-helix linker and a random coil linker.The activity of the CBM32-cleaved truncation CD167 was 3779.36±92.18 U/?mol,retaining only 60%of the original activity(6276.04±210.70 U/?mol).The K_m value and catalytic efficiency(K_cat/K_m)of CD167 were9.05±1.12 and 20.41±1.12 m M^-1 s^-1,respectively,while the K_m and K_cat/K_m of the original enzyme Aly01FL were 5.46±0.65 and 37.95±2.10 m M^-1 s^-1,indicating that CBM32 promoted the catalytic efficiency by improving the substrate binding ability of Aly01.Analyses of degradation products showed that Aly01FL mainly produced trisaccharides,while the products of CD167 was mixture of disaccharide and trisaccharide,indicating that CBM32truncation changed the certain product distribution.Molecular docking and dynamics revealed a conceivable degradation pattern of Aly01:CBM32 acts as a sensor to capture the substrate alginate,and regulates the degradation process by constantly pushing alginate into the catalytic domain.Both domains interacted with alginate like hands holding a stick,with trisaccharides being released as alginate gradually slides into the catalytic domain.(4)Genome sequencing and annotation suggested that V.natriegens SK42.001 had two circular chromosomes of 3,476,249 and 1,850,671 bp,respectively,encoding 4750 ORFs;the strain was found to be free of known Vibrio toxins according to the virulence factor database.The alginate metabolism of V.natriegens SK42.001 was proposed:extracellular Aly01 is secreted and degrades alginate to oligosaccharides,which further enter the cells and are degraded into monomers by oligo alginate lyase.The monomers naturally switch to DEH(4-deoxy-L-erythro-hexoseulose uronic acid),and further catalyzed by DEH reductase,KDG(2-keto-3-deoxy-D-gluconate)kinase and KDPG(2-keto-3-deoxy-6-phospho-gluconate)aldolase to G3P(D-glyceraldehdye-3-phosphate)and pyruvate,entering glycolytic pathway and then tricarboxylic acid cycle.The biomass and growth rate of V.natriegens SK42.001 in liquid were both about 1.5 times those of E.coli.On solid medium,V.natriegens SK42.001 single colonies were visible at about 5.5 h,approximately half that of E.coli(at about 10 h).Antibiotic tests showed that the strain is not resistant to chloramphenicol(Cm)and tetracycline.Using GFP as the reporter gene and Cm as the screening gene,the recombinant plasmid p AWP89-Cm-GFP was constructed and further introduced into V.natriegens SK42.001 by triparental conjugation.GFP was successfully expressed by the recombinant V.natriegens SK42.001,and green fluorescence was observed under the excitation of ultraviolet light,suggesting that V.natriegens SK42.001 has the potential to be further developed as next-generation model bacterium to express heterologous proteins.