Studies On The Agarase System Of The Agarolytic Marirne Bacterium, Flammeovirga Sp. MY04

Agarose is a neutral complex polysaccharide composed of3,6-anhydro-L-galactopyranose-α-1,3-D-galactose units that are joined by β1-4bonds. Agarases areglycoside hydrolases (GHs) that catalyze the cleavage of α1-3linkages or β1-4linkages of agarose, and are grouped into α and β types respectively. The α-agarases(E.C.3.2.1.158) degrade agarose into agaro-oligosaccharides (AOs), withβ1,4-3,6-anhydro-L-galactopyranose as the reducing end, while β-agarases (E.C.3.2.1.81) depolymerise agarose into neoagaro-oligosaccharides (NAOs), withα-1,3-D-galactose as the reducing end. Nearly50agarases have been characterized,and most of them are β-agarases.Agarases are useful in the preparation of algal protoplasts and the recovery of DNAfrom agarose gels. Recent studies have discovered some biological activities of NAOs,such as anti-oxidation, and prebiotic-and whitening-effects, which imply potentialapplications of agarases and oligosaccharides in food, pharmaceutical, and cosmeticindustries. However, few cheap and efficient agarases have been industrially produced.Thus, it is essential to find more bacteria with high agarase-yielding level for theexploration of more efficient agarases.Flammeovirga is a bacterial genus belonging to the family Flammeovirgaceaewithin the α-Proteobacteria. Five species have been reported in this genus: F. aprica, F.arenaria, F. yaeyamensis, F. kamogawensis, and F. pacifica. All the type strains areagarolytic strains, while only one β-agarase, AgaYT from F. yaeyamensis strain YT,has been reported so far. Flammeovirga sp. MY04is a polysaccharide-degradingmarine bacterium with agarose liquefying ability, suggesting that agarases areabundant in Flammeovirga sp. MY04.In this thesis, the agarase system of Flammeovirga sp. MY04has been exploredand studied, and the detailed results are,1．The extracellular agarase system of Flammeovirga sp. MY04The agarolytic marine isolate, MY04, is a member of the genus Flammeovirga. TheMY04strain is able to utilise multiple CPs as a sole carbon source and grows best onagarose, mannan, or xylan. This strain produces high concentrations of extracellularproteins (490±18.2mg/l liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose, for which theseproteins have an activity of310±9.6U/mg proteins. The extracellular agarase system(EAS), comprised in the crude extracellular enzymes, contains at least four agarosedepolymerases, with molecular masses of approximately30-70kDa. The EAS isstable at a wide range of pHs (6.0-11.0), temperatures (0-50℃), and sodium chlorideconcentrations (0-0.9M). Two major degradation products generated from agarose bythe EAS were identified to be neoagarotetraose and neoagarohexaose, suggesting thatthe major constituents of the MY04EAS are β-agarases.2. Gene cloning and sequence analyses of the agarase AgaG4from the genomicDNA library(1) The genomic library of Flammeovirga sp. MY04has been constructed using theEpifos fosmid library system. Five hundred nanograms of36-45kb blunt-repairedgenomic DNA has been ligated into the copy control vector pCC1FOS and transfectedinto E. coli EPI300-T1R. Totally, the genomic library of Flammeovirga sp. MY04contains more than500,000fosmids with insert sizes ranging from30kb to36kb,covering more than15.0Gbp of the genomic DNA.(2) More than3,000clones have been surveyed for activity screening of agarasegenes, but none of them showed any clear halos around bacterial colonies. Individualfosmids have been isolated from120random clones of the genomic library, and weresequenced using the T7primer of the pCC1FOS vector. After the trimming of vectorsequences, total118credible sequence tags with reading length longer than600bpwere obtained and analyzed. Partial agarase gene (agaG4) was initially identified atthe T7end of the fosmid pG4from clone R075. Special primers were designed,synthesized, and applied in PCR screening of agaG4from the genomic library. As aresult, the full-length gene of AgaG4was found in the fosmid pAI2from clone R012.Shotgun sequencing of pAI2revealed a31655bp DNA insert with eighteen deducedORFs, including the agarse gene agaG4and a potential gene cluster that may beinvolved in the degrading of mannan and cellulose. The5’-flanking region of agaG4lacked universal promoter elements such as the-35,-10, and RBS motifs, meaningthat the promoter can hardly be driven by the transcription systems of E. coli cells.GC contents of the ORF and GC3s are much lower than those of E. coli genes. These molecular characteristics, however, might lead to the unsuccessful screening of agaG4from the DNA library.(3) Several functional sites that were conservative among most GH16β-agaraseshave been found in the protein sequence of AgaG4. These conservative sites includedeight sugar-binding sites (N72, W74, W145, D151, F178, R180, I325, and V327), acatalytic motif of two sites (E154, and E159,), and three calcium binding sites (E22、N49、S365). AgaG4contains an N-termianl signal peptide, followed by a novel GH16module, a Ser-/Gly rich linker, a hypothetical chitin binding module (ChtBD3), and apor secretion system sorting domain. Although AgaG4shares high identity (98%)with the GH16β-agarase AgaYT from F. yaeyamensis YT, two novel modularproperties and a distinctive revolutionary position of both AgaG4and AgaYT havebeen discovered in this study. The catalytic modules of AgaG4and AgaYT arenon-typical GH16module, the inner peptide (N246-G302) of which share fewhomologous regions among the characterized GH16β-agarases except amongthemselves but with five different sites. The non-catalytic domains at the C-terminalconsists of a hypothetical chitin binding module (ChtBD3) and a por secretion systemsorting module, a novel modular organization type of agarase. Moreover, the agarasesAgaG4, together with the agarase AgaYT and the predicted agarase MS116fromMicroscilla sp. PRE1, was classified into a new β-agarase subclass of the GH16family.3. Heteologous expression of AgaG4and characterization of the novelmodular structures(1) The recombinant protein rAgaG4was overexpressed in E. coli using thepBAD/gIII expression system. Recombinant proteins were obtained from inclusionbodies using urea, purified by Ni-affinity chromatograph, and refolded throughdialysis. The agarase rAgaG4， with purity high than97%, showed optimalcharacteristics at50°C and pH7.5respectively, and was stable for a temperature rangeof0to50℃and a pH range of6-9. A novel non-13C-NMR spectroscopic methodusing2-amino benzamide labelling and MS/MS experiments was applied in thecharacterization of agarose-derived oligosaccharides by rAgaG4. The finaldegradation products of agarose by rAgaG4were confirmed as neoagarotetraose(NA4) and neoagarohexaose (NA6), with a mol ratio of1.53:1, and amounting to total99%proportion. Furthermore, rAgaG4did not digest NA4and NA6, while dividedneoagarooctaose (NA8) into NA4, and degraded neoagarodecaose (NA10) into NA4 and NA6. Moreover, NA4was produced from the non-reducing ends of NA10.Although AgaG4and AgaYT from F. yaeyamensis YT share98%identity of aminoacid sequences, they produce different final products in agarose degradation, thereforethey are different agarases. The biochemical characteristics of rAgaG4indicate that itis a thermostable endo-β-agarase of family GH16. The agarose-degrading propertiesshowed great promise of applying rAgaG4in the preparation of NA4and NA6specially.(2) The gene agaG4was truncated using PCR method, resulting one gene fragment(agaG4-GH16) that encoded the GH16module and the other fragment (agaG4-T57)that encoded partial AgaG4with the deletion of the unusual peptide. The genes wereover-expressed in E. coli strain TOP10using the pBAD/gIII expression system.Recombinant proteins were obtained from inclusion bodies using similar approachesas that of rAgaG4.The truncated agarase rAgaG4-GH16showed similar enzymecharacteristics to those of rAgaG4, but a lower starting refolding concentration and ahigher enzymatic activity, suggesting that the non-catalytic domain at the C-terminalof AgaG4had much to do with the protein solubility and enzyme activity. The othertruncated protein rAgaG4-T57showed optimum temperature at40°C, and degradedagarose into NA4, NA6and neoagarooctaose (NA8), with a final mol ratio of2.68:2.82:1. Furthermore, rAgaG4T57was unable to hydrolyze NA8, but degradedNA10into NA4and NA6. The results indicated that the special peptide in the GH16module of AgaG4plyays an essential role in binding and degrading of NA8.(3) The three-dimentional structural data was obtained by homologous constructiononline (http://swissmodel.expasy.org/), and observed using the soft Swiss PDBViewer3.7. The Y276residue, lying at the the entrance of the catalytic cleft andconsisting of a phenolic hydroxyl group, was determined as a candidate sugar-bindingsite in the special peptide of AgaG4. The plasmid pCTFG4, which can produce a fewsoluble fusion agarase of AgaG4in E. coli BL21(DE3), was mutated at the Y276siteusing PCR strategies, and formed a series of plasmid mutants. The plasmid pCTFG4,its mutants, and the negative plasmid pCold TF were individually transformed into thehost cells, and the recombinant fusion proteins were expressed by incubation usingIPTG. After the cells were centrifugated and sonicated, individual soluble fractionscontaing targeting proteins was prepared by futher centrifugation and reacted with the agarose solution retained at45℃as the crude enzyme。The degradation products wereanalyzed and compared usin TLC detection.As a result, when Y276(Tye276) was mutated into the Phe256site, the finaldegradation products of agarose by the crude enzyme were NA4and NA6, similar tothat by rAgaG4. While the Y276site was mutated into the sites of Asp276, Glu276,Asn276, Ser276or Thr276, the final degradation products of agarsoe changed intoNA4, NA6, and NA8, similar to that by rAgaG4-T57. Interestingly, when Y276wasmutated into the Gly276site, the degradation products of agarose by wereundetectable. The results indicated that the Y276site of the spectial peptide in theGH16module is a NA8-binding site of AgaG4and plays an important role in thedegradation of agarose. It suggested that the benzene ring is essential in the binding ofNA8.4. The agarase system encoded by the genome sequence MY04The genome of Flammeovirga sp. MY04is a circular chromosome of7,244,701bp. A total of5536protein-encoding genes were predicted. Fifteen agarasesharing identities high than30%with reported agarases have been found. The fourGH16agarases of MY04shared identities ranging from30%to42%with agarasesfrom Zobellia galactanivorans Dsij or Microbulbifer thermotolerans. Only oneagarase belong to the family of GH50, and the other ten agarases belong to the GH86family. The GH50and GH86agarases of MY04shared identities ranging from30%to45%with agarases from Saccharophagus degradans2-40. Moreover, the GH86agarases were clustered into two subclass, with relation to the GH86C and GH86E ofSaccharophagus degradans2-40respectively. However, three extracellular agarasesranging form30kDa to40kDa in the EAS of MY04were not found in the predictedgenes. It meant that biochemical and proteomic methods, more than genomic strategy,were neededed in the exploration of all agarases of Flammeovirga sp. MY04.Altogether, the agarases of Flammeovirga sp. MY04have been searched inthe extracellular agarase system, the genomic DNA library, and the chromosomegenome sequence. Moreover, the agarase AgaG4has been heterologouslyexpressed, biochemically characterized in detail. Furthermore, enzymatic functions and possible catalytic mechanism of the novel module structrures inAgaG4were analyzed and discovered.