| Alginate is a copolymer of α-L-guluronate and β-D-mannuronate, which are linked to form polyuronate of polymannuronate, polyguluronate, or alternating M&G blocks in the linear molecular. Alginate and alginate-derived oligosaccharides have been applied as raw materials in food, medical and agricultural fields. Recently, alginate bioconversion including alginate biosynthesis, enzymatic degradation and modification has attracted attention on their potential for expanding the uses of alginate.With the alginate lyase, the alginate could be gently depolymerized with a yff-elimination mechanism, yielding products with special bioactivities. The typical approach to purify the enzyme needed to isolate microorganisms for the desired alginate-degrading activity at first, with limited mediums containing alginate as the sole carbon and energy source, then induced enzymes were recovered and used in enzymatic method. With this method, a large fraction of the microbial diversity in an environment might be lost, for it was estimated that >99% of microorganisms observable in nature typically cannot be cultivated with standard techniques, at the same time alginate lyases with specific activity might be lost, too.The new approach of cloning genes from environmental genomic DNA library may be helpful, which was applied for cloning of Mannuronate (M)-specific lyase genes with a modified PCR screening approach in this thesis. As a result, the purifiedrecombinant enzyme showed an M-specificity in both quantitative and qualitative assays, which confirmed the cloned algL gene's function.A marine bacteria strain has also been isolated from seawater samples and identified to be some Pseudomonas strain with the 16S rRNA method, from which the whole alginate biosynthetic operon has been cloned and sequenced. Since the research has provided significant insights into the global and specific regulatory mechanisms which control the biosynthesis, modification and degradation of bacterial exopoly-saccharides. The structural genes cloned in this study will benefit the alginate bioconversion with reforming the constitutive biosynthetic pathway or the modifying alginate with recombinant enzymes.The detailed results are,(1) The DNA library was constructed with environmental genomic DNA isolated directly from seawater, and libraries stored and cultured in 96-well microtiter plates were screened by the rapid degenerate PCR approach with primers designed for M-specific-lyase genes. As a result, a new algL gene has been identified, the GeanBank accession number of which is AY163384. The novel gene was 1098bp and identified to be localized in the DNA fragment that shared high identity (86%) to partial alginate biosynthetic operon in Pseudomonas aeruginosa, and the deduced AlgL protein shared 66%, respectively. The homology-based three-dimension structure indicated that the predicted proteins should be an M-specific lyase.(2) The algL gene was cloned into pBAD gill/A plasmid and given a heterlogous expression in Kcoli Topi OF'. The his-tagged AlgL protein was purified with affinity-column from the periplasmic fragment, which was 40kDa about in molecular mass, analyzed with SDS-PAGE electrophoresis.(3) The recombinant AlgL protein was found to be an M-specific lyase by both quantitative and qualitative assay, with alginate, homologous poly(M) and poly(G) as substrate, respectively.(4) Specific primer pairs were designed for the resulting algL gene and used in PCR screening of an alginate-producing marine bacteria. The source strain was identifiedto be some Pseudomonas with the 16S rRNA method.(5) The whole gene of the alginate biosynthetic operon has been cloned with degenerate PCR step-walking strategy, which was 17.6kb about and corresponds for 12 functional proteins involved in the biosynthetic pathway. The GenBank accession numbers includes AY163384, AY216463 and AY279078.(6) A possible regulatory mechanism of alginate biosynthetic operon has been found, which may be a transcription induced by a two-comp...
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