Cloning And Characterization Of An Endo-?-1,6-galactanase From Penicillium Oxalicum

Galactan is widely distributed in all organization of advanced plants.Galactan hemicelluloses and lignin make up the cell walls and xylem of plants.Many studies have shown that oligogalactan has excellent bioactivity.So it is of great significance to obtain galactanases with high efficiency and specificity.It could be divided into?-1,3,?-1,4 and?-1,6 three kinds of galactanase according to structures of their substrates.In this study,a gene encoding endo-?-1,6-galactanase was cloned and characterized.The main results are as follows:?1?In this study,we cloned anendo-?-1,6-galactanase gene?pogal6?belonging to glycoside hydrolase family 30 from Penicillium oxalicum,which contains 487 amino acids residues.The gene of pogal6 was ligated to the bacterial expression vector pET-32a?+?and the fungal expression vector pPIC9K,respectively.And recombinant proteins PoGal6-R and PoGal6-G were successfully expressed in both Rosetta-gami B?DE3?pLysSand GS115.?2?Enzymological characterization of recombinant PoGal6:The activity of PoGal6-R was measured using pNP?gal as a substrate.The results showed that optimal pH of the purified protein was 6.0,and enzyme activity is still more than 60%whenPoGal6 was incubated at pH range from 3 to 7.Optimum reaction temperature of PoGal6 is 50°C,and it still maintained more than 80%activity within 60 min at 55°C;the tolerance results of metal ion shown that Hg²⁺could inhibit the activity of PoGal6-R completely,and Ca²⁺could increase the activity of PoGal6-R to 143%.The enzymatic activity of PoGal6 to degradegalactan was used RB5-dGA as substrate and detect the ability of degradation.The results exhibited that the enzyme activity of PoGal6-R was 35.2 U/mg,and the enzyme activity of PoGal6-G was 2.52U/mg when we used recombinat PoGal6 to degrade RB5-dGA.?3?The ability of recombinant enzymes to degrade galactan in plant polysaccharides was further investigated.The results of ion chromatographic analysis and methylation analysis showed that the recombinant protein PoGal6-R exhibited high activity with compared to the currently reported endo-?-1,6-galactanase.It could specifically hydrolyze?-1,6 linkage galactan,and had different degrees of hydrolysis ability for different molecular weight galactans.In this study,an endo-?-1,6-galactanase?PoGal6?was obtained from Penicillium oxalicum for the first time,and its enzymatic properties and functions were investigated in detail.Our study provided the theory evidence for the structural analysis and preparation of active galacto-oligosaccharides and polysaccharides.