Recombinant Expression Of β-galactosidases From Paenibacillus Polymyxa And Their Pectin Degradation Activity

β-galactosidase is able to hydrolyseβ-1,3,β-1,4 andβ-1,6-galactosidic bonds and can be applied in the fields of enzymatic modification of polysaccharides,structural analysis and preparation of oligogalactans and oligosaccharides.RG-Ⅰ pectins have complexβ-galactan side chains and the use ofβ-galactosidase to hydrolyse these side chains is an important part of elucidating the structure of RG-Ⅰ pectins.Therefore,the discovery ofβ-galactosidases that hydrolyse RG-Ⅰ pectins is important for the analysis of pectin structures and the preparation of the corresponding oligosaccharides.Paenibacillus polymyxa is a good source of newβ-galactosidases as it contains many glycosidases capable of degrading carbohydrates.Paenibacillus polymyxa was able to completely degrade potato RG-Ⅰ pectin,indicating that the bacterium can produce a variety of glycosidases includingβ-galactosidase.Based on the annotation results of the Paenibacillus polymyxa genome in NCBI,six recombinant strains containing theβ-galactosidase gene were successfully constructed and high purity target proteins were obtained.Six recombinant enzymes acted in an exocytic manner,with four of them possessing mainlyβ-galactosidase activity and the other two possessing arabinopyranosidase andβ-mannosidase activities,respectively.Pp Gal42A,Pp Gal42B and Pp Gal2A were selected for in-depth study.The optimum reaction conditions and kinetic parameters of the recombinant enzymes were investigated using p NPβGal as the substrate.The optimum p H of the threeβ-galactosidases was6.0-8.0 and the optimum reaction temperature was 30-40 ~oC,all of which showed a moderate temperature neutrality.All the divalent heavy metal ions had a severe inhibitory effect on the activity of the three recombinant enzymes.Pp Gal42A showed the highest affinity for the p NPβGal substrate with a K_m value of 1.14±0.19 m M.The selectivity of the recombinant enzymes was investigated in detail using p NP-glycoside,β-galacto disaccharide andβ-galactan as substrates.Pp Gal42A was broadly selective with an exocyticβ-1,3/1,4/1,6-galactosidase activity,Pp Gal42B was an exocyticβ-1,3/1,4-galactosidase and Pp Gal2A was a selective and specific exocyticβ-1,6-galactosidase.Among them,PpGal42B has a better degradation activity of potato RG-Ⅰ pectin.The hydrolysis of polygalacturonic acid and RG-Ⅰ structural domains in RG-Ⅰ pectin contributes to the enzymatic activity of Pp Gal42B.In summary,this thesis identified newβ-galactosidases,Pp Gal42A,Pp Gal42B and Pp Gal2A,from Paenibacillus polymyxa,which enriched theβ-galactosidase resources.The study of the properties and selectivity of the three recombinantβ-galactosidases and their enzymatic activity on RG-Ⅰ pectin provides new techniques for the structural analysis of sugar compounds and the preparation of active sugars.