Studies On The Biochemical Characteristics,Enzymatic Properties,and Catalytic Mechanisms Of A Novel Mannuronate-Specific Endolytic Alginate Lyase

Alginate is a linear polysaccharide that is composed of ?-D-mannuronate(M)and its C5 epimer ?-L-guluronate(G),which randomly joined together in the backbone of polysaccharide.Alginate oligosaccharide has various important biological activities such as anti-coagulation,hypoglycemia,and anti-tumor.These biological activities are significantly affected by the composition and sequence of the sugar units,the degree of polymerization,and the molecular modification characteristics.Enzymatic degradation of polysaccharide substrates to prepare oligosaccharide products usually has the advantages of mild reaction conditions,strong specificity,low pollution,and strong controllability.It is a potential replacement technology for chemical and physical methods.Therefore,the exploration of tool-type alginate lyase is an important basis for the preparation of uniform size-defined oligosaccharide fractions.Alginate lyase is type of polysaccharide lyase that catalyzes the cleavage of glycosidic bonds of alginate via a?-elimination mechanism to produce oligosaccharide products containing unsaturated nonreducing(nr)ends.Unlike exolytic alginate lyases,endolytic alginate lyases can produce a series of size-defined unsaturated oligosaccharide product fractions when partialy but not completely digest the polysaccharide.Therefore,endolytic alginate lyases have greater accurate application values in the enzymatic preparation of oligosaccharide products.Recent studies have been foucused on the resource exploration,biochemical characteristics,and molecular modification of alginate lyase,whereas there are few systematic studies on the enzymes' substrate selectivity,oligosaccharide-yielding properties,and coresponding substrate degradation modes,although G-specific endolytic alginate lyase Aly5,G-prefered bifunctional endolytic alginate lyase Alyl and Aly2 derived from Flammeovir sp.MY04,M-prefered bifunctional endolytic alginate lyase Aly7B_Wf derived from Wenyingzhuangia fucanilytica CZ1127T,and M-specific endolytic alginate lyases and derived from Pseudomonas aeruginosa(Pae-AlgL)and Azotobacter vinelandii(Avi-AlgL)have been well elucidated.Overall,the in-depth study on the catalytic properties and corresponding mechanisms of M-specific endolytic alginate lyase have been rarely reported,which has led to the difficulties in direct rational evolution for enzymatic improvement.In this thesis,we analyzed a whole-genome sequence of the efficient polysaccharide-degrading strain of Persicobacter sp.JZB09,and identified a candidate alginate lyase gene ORF06925.By bioinformatics analysis,recombinant gene expression,protein purification,and molecular modification applications such as gene truncation and site-directed mutations,we compared the enzyme and its mutant proteins' differences on the substrate selectivity,oligosaccharide-yielding properties,and coresponding substrate degradation modes.The results are briefly as the following:1.Molecular Properties:Aly06925 consists of 399 amino acids and contains only one hypothetical functional module.Among the elucidated enzymes,the full-length Aly06925 protein shares the highest sequence similarity(9%)with AlgL,a PL-5 family member from Azotobacter vinelandii.Phylogenetic tree analysis indicated that although Aly06925 is closely related to PL-5 or PL-17 family members,it is branched separately and does not cluster with any family members currently identified.Therefore,Aly06925 should be classified as a member of a novel polysaccharide lyase family undefined yet.2.Biochemical Characteristics:The recombinant expression vector pET30a-Aly06925 was constructed using pET30a to yield the recombinant enzyme rAly06925.The optimal reaction temperature of rAly06925 is 35?,and the optimal pH varlue is 6.0.The enzyme shows certain temperature stability(0-20?)and pH stability(pH7-8).Ca2+(1 mM),DTT(1 mM),and NaCl(0.2 M)can all significantly promote the enzyme activity,which has increased to 120%,131%,and 160%,individually.The main novel properties of rAly06925 are listed as the following:1.The oligosaccharide-yielding properties of a novel M-specific endolytic alginate lyases are elucidated.When the recombinant enzyme rAly06925 completely degrades the alginate polysaccharide,the final main size-defined oligosaccharide product fractions contain unsaturated disaccharides(23.1%,w/w),trisaccharide(23.1%,w/w),tetrasaccharide(23.1%,w/w),and relative few unsaturated pentasaccharides(19.2%,w/w),unsaturated hexoses(11.5%,w/w).A series of size-defined oligosaccharide product fractions with purities greater than 99%are obtained by molecular gel column chromatography,and then subjected to 1H-NMR spectrum analysis.The results showed that all of these various size-defined oligosaccharide product fractions contain only ?M units,instead of ?G units,at the non-reducing ends.Notably,this type oligosaccharide-yielding property is significantly different from the structural characteristics and succession rules among the final main oligosaccharide products of the G-specific endo-lytic alginate lyase Aly5,G-prefered bifunctional endolytic alginate lyases Alyl and Aly2,M-specific endolytic alginate lyases Pae-AlgL and Avi-AlgL,which had been reported in our previous studies.That is,the final oligosaccharide products of Aly06925,i.e.,the purified various size-defined oligosaccharide product fractions,along with the chain length increasing from disaccharides to larger oligosaccharides,do not have the succession rule of changing from ?G to ?M ends,or changing from ?M to ?G ends,whereas being always a single ?M-type non-reducing end.Therefore,Aly06925 is a novel tool-type alginate lyase and can degrade alginate to produce a series of size-defined oligosaccharide product fractions,which contain only ?M unit at the non-reducing end,as the final oligosaccharide products.2.To reveal the corresponding mechanisms that determine Aly06925's oligosaccharide-yielding property described above,we performed the following studies in this thesis:(1)Substrate Preference:Aly06925 can significantly degrade a series of saturated M-enriched oligosaccharide chains,but it does not degrade any tested saturated G-enriched oligosaccharide chains,and therefore Aly06925's M-specificity is remarkable.(2)Substrate Degradation Mode:When the enzyme rAly06925 degrades the saturated oligosaccharide M5,it produces M2 and UM3 as the final main products.While being reacted with M4,rAly06925 produces M and UM3 as the final digests.When reacted with the artificially synthesized saturated oligosaccharide chain of 2AB-M5,the enzyme rAly06925 produces M2 and 2AB-UM3 as the final degradation products.It can degrade 2AB-M4 to produces M and 2AB-UM3 as the final main products.The enzyme can partially degrade unsaturated hexosaccharide fractions(UDP6)togenerate the UDP2,UDP3,and UDP4 product fractions,and it weakly degraded UDP5 fractions to yield UDP4 fractions and unsaturated monosaccharide unit(?).Therefore,the smallest oligosaccharide substrate types of the enzyme Aly06925 include the saturated M-enriched tetrasaccharide,the unsaturated pentasaccharide(UDP5)fractions,and the synthetic oligosaccharide substrate 2AB-M4.The minimal oligosaccharide product types includes the saturated monosaccharide M or unsaturated ? units.In addition,when the recombinant enzyme rAly06925 degrades the oligosaccharide substrate,the smaller oligosaccharide products are produced from the non-reducing end of the saturated oligosaccharide substrate.When the enzyme degrades various small size-defined saturated oligosaccharide substrates(e.g.,M4/M5,2-AB-M4/M5),the size of saturated final oligosaccharide products increases with parallel to the size increase of the substrate sugar chain.Notably,the monosaccharide product(M,?)is produced only when the smallest oligosaccharide substrates(M4 or UDP5)are degraded.These results also demonstrated that the M-specific endolytic alginate lyase Aly06925 possesses a variable substrate degradation mode.Based on the substrate preference and the substrate degradation mode described above,we hypothesize the key mechanism for rAly06925 to produce only ?M-ended unsaturated oligosaccharide product fractions as the following:The enzyme Aly06925 can specifically recognize and bind to M-enriched sugar sequences,i.e.,M-MMXn,G-MMXn,or ?-MMXXn,and cut efficiently at the '-' position,i.e.,the glycosidic bond(X,M or G;degree of polymerization n?1,nature number;the same below),thereby producing a series of unsaturated oligosaccharide product fractions with only?M unit at the non-reducing end.However,the enzyme Aly06925 does not recognize a G-enriched sequences such as G-GGXn,M-GGXn or ?-GGXn in alginate,and cannot cleave the glycosidic bond shown by '-'(X,M or G;degree of polymerization n?1,nature number;the same below),and thus making it impossible for the enzyme to produce various size-defined unsaturated oligosaccharide products with a non-reducing end of ?G unit.3.Primary sequence analysis revealed that the enzyme Aly06925 contains two potential catalytic motifs(N113-N114-H115-T116-D117-F118 and N238-N239-H240-G241-T242H243),semi-conserved with the highly conservative catalytic motif(NNHS YW)of the PL5 family members,whereas it is not convincing to predict the right catalytic motif that does play a key role in the catalytic degradation process through only bioinformatical analysis.Module composition analysis showed that the enzyme Aly06925 contained only one potential catalytic domain(V66-W345);and homology-based three-dimensional structure construction and analysis found that Aly06925 has a sleeve shape as a whole,mainly composed of ?-helix,and has a tunnel-like catalytic cavity,which is similar to the periplasmic alginate lyases AlgL of Pseudomonas aeruginosa and Azotobacter vinelandii,which belong to the PL5 family.While quite differently,Aly06925 contains an extra peptide(K45-N60)that constitutes an ?-helix with unknown function.Therefore,based on molecular truncation and site-directed mutation studies,this thesis analyzed and compared the enzymatic properties of the enzyme rAly06925 and its series of mutants.The results showed that N238-N239-H240-G241-T242-H243 within the recombinant enzyme rAly06925 is a catalytic motif,while N113-N114-H115-T116-D117-F118 is not a catalytic motif;Y87,Q170,H240,Y295 are the essential catalytic site residues;Deletion of the extra peptide K45-N60 of Aly06925 or the mutation of Y244 to A244 resulted in the loss of water solubility and enzyme activity of the resulting proteins.Therefore,the extra peptide K45-N60 and its Y244 residue are critical for maintaining the protein conformation or determining the hydrophilicity of the protein surface.In summary,Aly06925 is a novel M-specific endolytic alginate lyases encoded by the genome of the polysaccharide-degrading bacteria Persicobacter sp.strain JZB09.The enzyme can be classified into a new PL family to be defined.The oligosaccharide-yielding properties of Aly06925 are also co-determined by the restrict M-specificity and the variable substrate degradation modes.Therefore,when degrading alginate,Aly06925 could produce a series of size-defined unsaturated oligosaccharide fractions as the final products,with ?M unit as the unique non-reducing end.Studies on the enzymatic properties and catalytic mechanism in this study will be beneficial for the resource exploration and enzyme improvement of tool-type polysaccharide lyases.