Cloning, Expression And Characterization Of A New Alginate Lyase Aly-i7 From Antarctic Fungus

Antarctica is the resource treasure of whole humanity.It contains numerous mineral resources and abundant biological resources.The harsh living environment in the Antarctic region,with the low temperature,frostiness,high-salt conditions,large diurnal temperature variation,and strong ultraviolet radiation,makes large organism scarce.However,thanks to unique biochemical characterization and metabolic mechanisms,microorganisms can survive and multiply in this condition.Studying the microorganisms in Antarctica cannot only discover many undiscovered microbial populations and enrich microbial genbank,but also obtain varieties of unique bioactive substances.In this study,we sreened a kind of Antarctic fungus producing alginate lyase named Aspergillus sp.22-5,and cloned its alginate lyase gene by gene technology.The alginate lyase gene was in heterologous expression and the recombinant protein was in purification successfully.We studied the characterization of the recombinant lyase,and explored the utilization potentiality of degradation products characterization preliminarily,which will provide a scientific basis for preparation of alginate oligosaccharides by alginate lyase and its utilization in agriculture.In this paper we isolated 10 strains of fungus with alginate lyase activity from Antarctic sediment by using algin as the sole carbon source.The result showed there were 4 strains belonged to genus of Rhodotorula,3 strains belonged to genus of Penicillium,3 strains belonged to genus of Aspergillus.The strain of Aspergillus sp.22-5 with the highest activity was slected as the starting strain.The suspected alginate lyase gene（Aly-i7）was obtained by annotation of transcriptome analysis.Aly-i7 was inserted into pET-30（a+）by genetic engineering methods to transform E.coli BL21（DE3）achieving heterologous expression of Aly-i7successfully.The induction experiment showed that 0.8mmol/L IPTG could significantly enhance the expression of recombinant protein at 16℃ for 16 h.The SDS-PAGE showed that the molecular weight of Aly-i7 which was separated and purified by Ni-NTA resin was approximately 40kDa,the purification factor was 49.34,the recovery rate was 25%,the alginate lyase activity was 17.46U/mL,and the specific activity was up to 14.31 U/mg.The biochemical characterization of Aly-i7 was determined by dinitrosalicylic acid method,including substrate specificity,optimal temperature,pH,thermal and pH stability,metal ions and enzymolysis product.The result showed Aly-i7 had activities toward bothα-L-guluronic acid andβ-D-mannuronic acid,indicating that it is a bifunctional alginate lyase which prefersα-L-guluronic acid,and also it showed no activities to carrageenan,agar,soluble starch and fibrin.The optimal catalytic activity of Aly-i7 was showed at 50℃and pH7.0 and it can maintain half of initial activity at50℃,pH7.0 more than 230min.Aly-i7 was stimulated by metal ions Na⁺、K⁺and Ca²⁺,but it was inhibited significantly by Zn²⁺,Fe³⁺,Fe²⁺,Cu²⁺,Mn²⁺,EDTA,especially Cu²⁺,almost deactivation at 5mmol/L.Thin layer chromatography（TLC）showed the product of Aly-i7 was mainly disaccharide which was conducive to separation and purification.The control effect of alginate oligosaccharides prepared by Aly-i7 on Sphaerotheca fuliginea was tested by spraying different concentrations of oligosaccharides on cucumbers infected with Sphaerotheca fuliginea and by pot experiment.The results showed that the anti-effiency of 5%oligosaccharide was86.05%,which was higher than that of 1%Wuyiencin（63.71%）.The results also showed the spots on the cucumber leaves became smaller and fewer;the thickness of Sphaerotheca fuliginea became thinnner and the mortality became lower after spraying oligosaccharide on cucumbers infected with Sphaerotheca fuliginea,which showed potential application value of alginate oligosaccharide prepared by Aly-i7 in the field of plant disease control.