| Alginate is a linear polysaccharide that consisted of ?-D-mannuronate(M)and its C5 epimer a-L-guluronate(G)by ?-1,4 glycosidic bonds.Alginate oligosaccharides showed important biological activities,such as anti-coagulation,anti-tumor,and immune protection.The biological activities are closely in accordance with the degrees of polymerization and M/G contents of oligosaccharides fractions.Therefore,how to efficiently prepare alginate oligosaccharides with specific molecular characteristics have been a research hot spot in the exploration field of marine biological resources with high value.Alginate lyases can degrade alginate by ?-elimination mechanism,producing oligosaccharide products that contain unsaturated sugar units(?)at the non-reducing end.When compared with the physical or chemical alginate-degrading technologies,enzymatic preparation of oligosaccharides showed the advantages of mild reaction conditions,definite product structures,and less environmental pollution.Many studies showed that the unsaturated oligosaccharides have stronger biological activities than saturated ones.Endolytic alginate lyases can completely degrade alginate into series of size-defined unsaturated oligosaccharide fractions;exolytic alginate lyases produce mainly one single oligosaccharide product,such as the unsaturated monosaccharide unit ?.Therefore,endolytic alginate lyases are more useful in the preparation of unsaturated oligosaccharide fractions.To date,more than 100 alginate lyases have been elucidated.Relevant researches have focused on the discovery of enzyme-producing strains,the enzymes,and gene resources;furthermore,the biochemical characteristics and three-dimentional structural properties of enzymes.However,the oligosaccharide-yielding properties(molar ratio between various sized-defined unsaturated oligosaccharide fractions,the structure properties,and their sucession rule of the final unsaturated oligosaccharides products),the substrate preferences,and particually the substrate-degrading modes(the endo/exo-lytic pattern,the smallest substrate,and the minimum product and its formation mode)are less eluciated,which has led to limited accurate application of reported alginate lyases as tool.Recently,we have elucidated that rAly5,G-specific alginate lyase from Flammeovirga sp.strain MY04,was efficient in degrading alginate to prepare large size-defined unsaturated oligosaccharide fractions ennriched with G units.Furthermore,deletion of the non-catalytic region of rAly5 has led to the yield increased of large sized-defined oligomer products.To further understand the application values of various other types of endolytic alginate lyases,we performed the following studies:1.Genome analysis of the polysaccharide-degrading marine bacteria Flammeovirga sp.strain MY04 revealed that the candidate alginate lyase Alyl contained a carbohydrate binding mode at the N-terminal and a putative catalytic module,Alg2,at the C-terminal.BLASTp searches showed the Alyl shared sequence identities of below 30%with the elucidated enzymes,and could be assigned into the PL7 superfamily.In this study,the whole protein(Alyl)and its truncated protein(Alyl-T185N)that contained only the catalytic module were individually expressed and purified.The recombinant enzyme rAlyl showed the optimal activity at 50? and pH6.0.It showed thermal stability and pH stability in a wide range of temperature(0-50?)and pH values(pH5?10).The activity of rAlyl could be increased by chemical such as Na+,K+,Li+,Co2+,Fe2+' Mg2+,Ni2+,glycerol,?-mercaptoethanol,DTT,and other chemical reagents.The activity of two enzymes were weakly inhibited by increasing NaCl concentration from 0 to 1 M.Primary substrate preference test and further oligosaccharide product structure identification indicated that rAlyl is bifunctional alginate lyase with G preference.The smallest substrates are tetrasaccharide-size fractions,and the minimal product types are M,G and the UDP2 fractions.The main novelties of rAlyl and truncated protein rAlyl-T185N are listed as follows:(1)To the best of our knowledge,we reported the novel oligosaccharide-yielding properties of a bifunctional alginate lyase with G preference.As is,rAlyl produced unsaturated disaccharide and trisaccharide fractions as the final main alginate degradation products.The unsaturated disaccharide fractions comprised of mainly ?G units,the unsaturated trisaccharides fractions contained ?M as well as ?G ends at the non-reducing ends,while the larger products fractions,e.g.,the unsaturated tetrasaccharie fractions contained only ?M ends.The truncated enzyme rAlyl-T185N showed oligosaccharide-yielding properties similar to those of rAly5,a G-specific endolytic alginate lyase from MY04.(2)The variable degradation modes of endolytic alginate lyases were systematically elucidated.The terminus types,molecular sizes,and M/G contents of oligosaccharide substrates are key factors that can cause mode changes of the enzymes and its regularity are described as follows.1)The effects of terminus types:the saturated units of M or G at the non-reducing ends of the substrates are the essential for the enzymes to act in monosaccharide-producing modes.In contrast,the unit ? is essential for the enzymes to cleave oligosaccharide products>UDP2 from substrates.The 2-AB labeling at the reducing ends significantly inhibited the enzyme activity,increased the size of the degradable substrate,and particually inhibited the ability of the enzymes to produce monosaccharides.2)The effects of molecular sizes:various small sized-defined unsaturated oligosaccharides and saturated ones can be produced from single or different saturated sugar chains with a parallel size enlargement of products to substrates.3)The effects of M/G contents:when the M or G oligosaccharides are degraded,the product types are the same and the proportions are different.This may be because the enzyme is bifunctional whereas with G preference.(3)The enzyme rAlyl-T185N showed similar biochemical characteristics and enzymatic degradation characteristics to rAlyl.The significant difference is that the NCR truncation of rAlyl enhanced the degradation proportion of small size-defind saturated M-enriched oligosaccharide substrates(e.g.,M4)and unsaturated substrates(e.g.,UDP4),and it showed no degradation pattern changes for saturated G-enriched oligosaccharides chains,thereby enhancing the M preference and enzymatic activity.2.In the alginate biosynthesis operon of Pseudomonas aeruginosa and Azotobacter vinelandii,there are two alginate lyase genes,algL,with similar arrangement,encoding M-specific alginate lyases.Previous studies have revealed the roles of AlgL in the alginate biosynthesis process,which randomly cleave alginate molecules to regulate the degree of polymerization of secreted polysaccharides.However,there are few data on the substrate-degrading pattern,oligosaccharide-yielding properties and structural properties have been reported.It cannot provide background information and detailed parameters for commercial application of the enzymes.Pae-AlgL from Pseudomonas aeruginosa and Avi-AlgL from Azotobacter vinelandii contained single alginate lyase modules belonging to the PL5 family.The amino acid sequence similarity of two enzymes is up to 66%.The biochemical characteristics and enzymatic properties of two enzymes are consistent with the previous reports,but they are slightly different from each other.The main novelties of Pae-rAlgL and Avi-rAlgL are listed as follows:(1)The genes were codon-optimized according to the codon bias of E.coli without changing the anmino acid sequences.While interestingly,the yield of water-soluble proteins were significantly improved.Biochemical characteristics analysis revealed that the activity of the recombinant enzymes was independent of NaCl,but 0 to 1 M NaCl could enhance enzyme activity.(2)The oligosaccharide-yielding properties of M-specific alginate lyases are revealed.That is,the final alginate degration products of two enzymes are series of oligosaccharides UDP2 to UDP7.For Pae-rAlgL,UDP2 fractions comprised of mainly ?M units,UDP3 and UDP4 fractions contained ?M as well as ?G ends at non-reducing ends,UDP5 to UDP7 fractions contained only ?G ends.Avi-rAlgL is similar to Pae-rAlgL,but only UDP3 fractions contained ?M as well as AG ends,and UDP4 to UDP7 fractions contained only ?G ends.On the whole,apparently with the degree of polymerization increasing,the ratio of ?M ends at the non-reducing end of oligosaccharides gradually decreases while the ratio of ?G ends gradually increases,finaly with complete AG ends.The larger unsaturated products of two enzymes could not be deeply digested,suggesting that they are G-enriched unsaturated oligosaccharides.These are the structure properties of the final oligosaccharide products that are contrary to those of the G-specific endolytic rAly5 but with similar regularity.(3)This study demonstrated that the oligosaccharide degradation by the two M-specific alginate lyases are similar to those by rAlyl,which used variable substrate-degrading modes.Furtermore,the terminal types,molecule sizes,and M/G contents were the key factors that cause mode changes of the enzymes.In conclusion,the bifunctional with G-preference endolytic alginate lyase rAlyl and its NCR-truncated protein can effectively and thoroughly degrade alginate,prepare oligosaccharides with small degrees of polymerization,and facilitate further biotransformation.The M-specific alginate lyases can be used to produce larger size-defined G-enriched unsaturated oligosaccharides products.Furthermore,the variable substrate degradation modes and their key factors for two types of endolytic alginate lyases were discovered and elucidated in this thesis. |
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