The Research Of Sphingomonas Sp. ZHO Alginate Lyase And Alginic Acid Degradation Mechanism

Development of marine resources has become a hot topic, Because of the unique physical and chemical properties of the alginate, so that it is widely used in food, medical, chemical and other fields Degradation product of alginate, alginate oligosaccharide has a variety of physiological activity, has become the main direction of development of new drugs The past two years, alginate as abundant marine biomass, in the field of bio-energy has also been a lot of attention.Hydrochloric acid hydrolysis in high temperature and high pressure is the main technologies of degradation of alginate, but this technology have many shortcomings, such as high energy consumption, low yield and environmental pollution.Enzymatic procession of alginate by aliginate lyase has gradually become more popular than traditional technologies. the present often enrichment of alginate decomposition strains to produce alginate lyase, but this technology also have many shortcomings, such as low yields, complicated steps. So it is difficult to achieve a large number of industrial production.And the degradation mechanism of alginate remains unclear.Mass production through genetic engineering alginate lyase and alginate degradation mechanism study has important theoretical significance and application valueThis paper systematically studied the four alginate lyases of Sphingomonas sp. ZHO on several aspects:gene cloning and heterologous recombinant expression of alginate lyases, purification of alginate lyases, characteristics research of alginate lyases, molecular structure of alginate lyases, degradation mechanism of alginate.The main results are summarized as follows.1. Gene cloning and heterologous recombinant expression of the alginate lyase from Sphingomonas sp. ZHOFour alginate lyase genes were cloned from Sphingomonas sp. ZHO genome by PCR, which were named zhO-Ⅰ, zhO-Ⅱ, zhO-Ⅲ and zhO-Ⅳ.The recombinant plasmids containing zhO-Ⅰ, zhO-Ⅱ, zhO-Ⅲ and zhO-Ⅳ genes were designated pGEX-4T-1-ZHO-Ⅰ,pGEX-4-T-1-ZHO-Ⅱ, pGEX-4T-1-ZHO-Ⅲ, pGEX-4T-1-ZHO-Ⅳ, respectively. In E.coli BL21by IPTG induction of expression, the results showed that these four expression vector can be expressed in E.coli BL21with soluble protein. The recombinant alginate lyase were named ZHO-Ⅰ, ZHO-Ⅱ, ZHO-Ⅲ and ZHO-IV, respectively.2. Purification and characteristics research of the alginate lyase from Sphingomonas sp. ZHOAfter purification, the enzyme activity of ZHO-Ⅰ, ZHO-Ⅱ, ZHO-Ⅲ and ZHO-Ⅳ up to53.2U/mg,103.9U/mg,13.7U/mg and10.7U/mg, respectively.The enzyme activity of ZHO-II is much higher than the same type of alginate lyase.The optimum temperature of alginate lyase ZHO-Ⅰ, ZHO-Ⅱ, ZHO-Ⅲ and ZHO-Ⅳ were42℃,47℃,52℃and37℃respectively.The optimum pH of alginate lyase ZHO-Ⅰ, ZHO-Ⅱ, ZHO-Ⅲ and ZHO-IV were7.0,6.5,7.5and7.0, respectively.ZHO-Ⅰ purified from recombinant E. coli cells are specific for both poly(G) and poly(M),ZH0-Ⅱ and ZHO-Ⅲ purified from recombinant E. coli cells are specific for poly(G) and poly(M), respectively,ZHO-IV is a exolyase responsible for the degradation of unsaturated oligoalginates.3. Molecular structure of the alginate lyase from Sphingomonas sp. ZHOThe degradation products and family classification of ZHO-Ⅰ, ZHO-Ⅱ, ZHO-Ⅲ and ZHO-IV were analyzed by TLC and phylogenetic tree, respectively.The degradation products of the ZHO-Ⅰ and ZHO-Ⅲ were disaccharides and trisaccharides, ZHO-Ⅰ is belong to polysaccharide lyase family PL-5, also belongs to the family of PL-7, ZHO-Ⅲ belongs to the polysaccharide lyase family PL-5, the degradation products of ZHO-Ⅱ were disaccharide and tetrasaccharide, belonging to the polysaccharide lyase family PL-7, ZHO-Ⅳ is an exolyase, the degradation products as monosaccharides, belonging to the polysaccharide lyase family PL-15.Based on the homology modeling, from the molecular level to reaserch the structural and functional relationship of ZHO-Ⅱ, ZHO-Ⅲ and ZHO-Ⅳ by the tertiary structure.4. Degradation mechanism of the alginate in Sphingomonas sp. ZHOBy the research of synergy between alginate lyase ZHO-Ⅰ, ZHO-Ⅱ, ZHO-Ⅲ and ZHO-IV from Sphingomonas sp. ZHO, we found the synergy between endotype alginate lyases ZHO-Ⅰ, ZHO-Ⅱ and ZHO-Ⅲ with exolyase ZHO-IV was much more higher than the synergy only between endotype alginate lyases ZHO-Ⅰ, ZHO-Ⅱ, ZHO-Ⅲ, description exolyase ZHO-IV belong to the rate-limiting enzyme in the process of degradation of alginate. Thus initially revealed the mechanism of degradation of the alginate in Sphingomonas sp. ZHO.ZHO-IV exolytically degraded oligoalginates, which were produced from alginate through there action of ZHO-Ⅰ,ZHO-Ⅱ, ZHO-Ⅲ,into monosaccharides,indicating that the synergy actions of these four enzymes cause the completed polymerization of alginate in strain ZHO cells.