Research On Discovery And Reform Of Biological Enzyme For Modifying Alginate Thickener In Marine Coatings

Ocean exploration is an important part of the development of civilization.In this process,marine corrosion is also a problem that cannot be ignored.The coating protection as an effective method for ship anti-corrosion has been widely recognized,and the selection of coatings with good performance is the key to the implementation of this method.As an important class of coating additives,thickeners can make coating obtain excellent mechanical properties and good physical and chemical stability.Due to the good gel properties and environmental protection characteristics of alginate,it has broad development space in the direction of thickeners for marine coatings.Alginate is a linear polymer,consisting of?-D-mannuronic acid(M)and ?-L-guluronic acid(G),Because of the different distribution of M and G residues,there are often three types of fragment structures exist in the alginate long chain:MM-blocks,MG-blocks and GG-blocks.Among them,alginate with higher GG-fragment content has better gel strength,however,natural alginate tends to have a lower GG-fragment content.Therefore,mannuronan C-5 epimerase which can convert M to G could be used to increasing the content of GG-fragments to meet the demand of thickener for marine coatings.A strain with a high activity of alginate modification enzyme was isolated from the gastrointestinal tract of sea urchin using the sodium alginate as the sole carbon source.According to 16S rDNA phylogenetic analysis of the strain C-H,it belongs to the genus Pseudomonas.Further,through the bioinformatics analysis,a new mannuronan C-5 epimerase gene(pmc5a)was obtained from the genome of strain Pseudomonas sp.C-H,and its encoded enzyme PmC5A was subsequently heterologously expressed in E.coli with a theoretical molecular weight of 52.32 kDa.Accodring to the measurement of the enzymatic products by 1H NMR,PmC5A was demonstrated to have both mannuronan C-5 epimerase and alginate lyase activity.Due to the lyase activity of PmC5A,the long chain of alginate is degraded into shorter chains with lower degree of polymerization,which affects the formation of gel.Therefore,the work in this paper was to reduce the negative effects of the lyase activity of PmC5A through external environment and molecular modification.Studies have shown that the effect of lyase activity can be effectively reduced by controlling the temperature,because at 60?,the activity of epimerase is much greater than the activity of lyase.A series of variants of PmC5A were generated through site-directed mutagenesis,and the 348S-N was obtained which not only completely makes the lyase activity disappear,but also has an improved epimerase activity which ensures the gel stability of the product.This discovery not only provides important information for the understanding of the catalytic mechanism of the bifunctional epimerase,but also lays a solid foundation for further development and utilization of this enzyme in coating thickener.