Heterologous Expression And Application Of Xylanase XynSPP2 Derived From Marine Microorganisms Marinifilaceae Bacterium

With the rapid development of economy,energy shortage and other issues are becoming more and more prominent,and search for economically renewable resources has become the most important for the sustainable development.Xylan is the main component of hemicellulose and is the second most available natural resource after cellulose.Due to its difficult to be decomposed and used,that results the waste of many natural resources.Xylanase plays a very important role in the process of xylan hydrolysis.It is the key enzyme of xylan hydrolysis into xylooligosaccharides,and has crucial value in commercial process and in reducing waste of resources.Xylanases from different environments and sources are different in structure and function,and in order to find more specific enzymes to meet the needs of industrial production,researchers tend to look for functional enzymes from extreme environments,such as glaciers,oceans,and polar regions.In this new study,a new dual-domain xylanase xyn SPP2 of the marine microorganism Marinifilaceae bacterium SPP2 source was studied,and its sequence was analyzed,it was found that it contained two structural domains,namely,GH10 catalytic domain and figprotein iii-type structural domain(Fn3).In this paper,the heterogenous expression in E.coli and Pichia pastoris was realized,and the effects of Fn3 domain on enzymes were analyzed.The composition of its enzymatic product was also analyzed in this study,which proved that it can be used to manufacture xylooligosaccharides.The major outcome of this study are:(1)M.bacterium SPP2 full-length xylanase XynSPP2-FL and Fn3 deletion XynSPP2-?Fn3 were cloned respectively,and heterologous expression was achieved in E.coli BL21,which has the optimum temperature of 50?,the most suitable p H is6.0,and the p H tolerance is wide.The molecular dynamics data of both are analyzed,and the Kcat values of XynSPP2-FL and XynSPP2-Fn3 are 178.19 and 117.27 s^-1,respectively.The catalytic efficiency of XynSPP2-FL is approximately three times that of XynSPP2-Fn3,and xyn SPP2-FL's specific activity in E.coli BL21 is 226.56U/mg.The results show that the addition of Fn3 domain can improve enzyme activity and catalytic efficiency effectively.(2)In order to adapt to industrial applications,XynSPP2 was transferred to Pichia pastoris,and the expression of exocytosis was realized successfully.Its most suitable reaction temperature in yeast expression system is 40?,the influence of elemental metals on it is not obvious,positive butanol will inhibit its activity completely,and its activity is reduced about half in organic solvents such as 5%of methanol and ethanol.XynSPP2 has obvious substrate specificity,in the test substrate only shows activity on beech wood xylan,its activity can be increased by about 10%in the 0.5 M Na Cl environment,with strong salt resistance.The hydrolysis reaction is carried out with beech wood xylan as the substrate,and the main components of the hydrolysis products are xyloose,xylotriose,and xylotetraose,indicating that it can be used for the preparation of xylooligosaccharides.(3)Through the optimization of single-factor conditions,the optimum prerequisite for the self-made corn cob xylan were obtained,and the solid liquid ratio is 1:10,Na OH concentration is 10%,the boiling time is 100 min,through this method obtains 3.20 g of xylan,the yield is 6.2%.The optimal conditions for XynSPP2hydrolyzing corn cob xylan are 40?,the enzyme concentration is 35 U/mg,the substrate concentration is 10%,and the hydrolysate components are xyloose to xylotetraose.