The Research Of Expression And Enzymatic Properties Of Alginate Lyase In Pichia Pastoris

The study on screening valuable substances from the ocean speices has become a significant topic in marine biotechnology.Alginate has broad application prospectand its degradation products has kinds of biological activities.It’s a trend that Enzymatic degradation of alginate represented by biodegradation take place of traditional chemical degradation. Therefore, alginate lyase production has far-reaching theoretical study and practical value.This study successfully cloned four kinds of alginate lyase gene zh0-Ⅰ,zh0-Ⅱ,zh0-Ⅲ and zh0-Ⅳ from Sphingomonas sp. ZHO which screened out by our laboratory.These gene was connected to the eukaryotic expression vector pPIC9K, constructed recombinant plasmid pPIC9K-zhO-Ⅰ.pPIC9K-zh0-Ⅱ,pPIC9K-zh0-Ⅲ and pPIC9K-zh0-Ⅳ. These expression vectors transformed into Pichia pastor is GS115 by electroporation and induced by methanol.The results illustrated four kinds expression vectors could expression in Pichia pastor is GS115.and interest protein secreted to the extracellular.The recombinant protein were named ZHO-Ⅰ.ZHO-Ⅱ,ZHO-Ⅲ and ZHO-IV, respectively. After optimization of fermentation conditions. The optimum temperature for enzyme production were 30℃、28℃、28℃、30℃; The optimum pH for enzyme production were 6.0、5.5、 5.5、5.5; The optimum methanol concentration for enzyme production were 1.5%、1%、1%、1%, respectively.The enzyme activity of ZHO-Ⅰ, ZHO-Ⅱ. ZHO-Ⅲ and ZHO-IV reach to 286.36U/mg.349.44U/mg、48.21U/mg�'�9U/mg, respectively,which belongs to a higher level in the reported research.The optimum temperature of alginate lyase ZHO-Ⅰ, ZHO-Ⅱ, ZHO-Ⅲ and ZHO-IV were 37℃.37℃,42℃and 37℃.respectively.The optimum pH of alginate lyase ZHO-Ⅰ, ZHO-Ⅱ, ZHO-Ⅲ and ZHO-Ⅳ were 6.5,6.5,7.0 and 7.0. respectively. ZHO-Ⅰ are specific for both poly(G) and poly(M),ZH0-Ⅱ and ZHO-Ⅲ are specific for poly(G) and poly(M), respectively,ZHO-Ⅳ is a exolyase responsible for the degradation of unsaturated oligoalginates.