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Biosynthesis Of Methanobactins And Characterization Of A New Alginate Lyase

Posted on:2017-05-23Degree:MasterType:Thesis
Country:ChinaCandidate:P WangFull Text:PDF
GTID:2480304841956939Subject:Organic Chemistry
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Methanobactins are a type of natural products produced by methanotrophs,which are capable of combining with copper ions.Methanotrophs are a kind of gram-negative bacteria,which use methane as carbon and energy source.Because of its special function of consuming the greenhouse gas methane,methanotrophs have become a focused issue in recent years.Methanobactins have a strong chelating activity for most transition metal ions,especially for copper ion,with the chelating constant as high as 1021 M-1,which is several orders of magnitude higher than the known chaperon copper proteins,that makes methanobactins good application prospect in industrial wastewater treatment with heavy metal ions and biomedical field.Recently,some methanobactins have been isolated from methanotrophs.These methanobactins from different bacteria all have special nitrogen heterocyclic structure,which are the metal ion chelating sites,indicated by crystal structures.The biosynthesis of these special heterocyclic structures has become the focus of attention,but there is still no report on the biosynthesis of methanobactins now.Recent research suggests that methanobactins may be a kind of ribosomally synthesized and posttranslationally modified peptides.On the basis of the biosynthetic gene clusters presumed by the previous studies,we carried out the study of the expression and purification of various related genes,including the precursor peptides and the modification proteins.To our delight,the expression and purification of precursor peptide MbnA of mbtin-OB3b and mbtin-SV97T were succeeded by constructing the mbnA to pACYCDuet-1 plasmid.After trying different expression conditions,we found that only MbnFSV97T and MbnC-SV97T were soluble among the putative modification enzymes,which were difficult for studying the correlation function of the biosynthetic gene cluster.The in vitro reaction of His6-MbnC-SV97T and/or His6-MbnF-SV97T with the standard peptides of MbnASV97T and MbnA-OB3b showed no modification by MALDI-TOF,which indicated that MbnC and MbnF may not be the first modification enzymes.So,related enzyme functions still need to be further studied.The alginate lyase is a kind of important polysaccharide lyase with the capable of degrading alginate through beta-elimination of the glycosidic bond,which is significant for analysis of alginate structure and seaweed scientific research.Alginate oligosaccharides,for their excellent biological functions,have been widely used in the field of medical and biological technology.Lacking of bioactivity of alginate oligosaccharides prepared by traditional chemical synthesis makes people turn to the biodegradation method.Nevertheless,different polymerization degree of alginate oligosaccharides show different bioactivities,which make customized alginate oligosaccharides to satisfy the need for specific purposes more important.Based on previous work of PhD.Benwei Zhu in our lab,a novel alginate lyase AlgNJU was identified from marine bacterium Cellulophaga sp.NJ-1.The algNJU was subcloned to pET21a and expressed with E.coli BL21(DE3).The purified enzyme had a molecular weight of about 36 kDa and exhibited activities towards both polyguluronate and polymannuronate indicating its broader substrate specificity.It showed maximal activity at 30? and pH 3.0-4.0.The alginate lyase was highly active at wide temperature range,from 10? to 50?;however the enzyme was not stable over time when incubated at 50? or even high.The alginate lyase maintained high activity after incubation at a broad range of pH overnight.Activity of the alginate lyase was influenced by some metal ions.ESI-MS analysis suggested that the alginate lyase mainly degraded substrates to di-,tri-and tetra-saccharides,which give a novel methods for preparation of alginate oligosaccharides.
Keywords/Search Tags:Methanotrophs, Methanobactins, genome mining, biosynthesis, alginate lyase
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