Gene Cloning,construction Of Engineering Bacteria,expression And Application Of Inulinases

Inulin is a linear chain polysaccharide,which consists of?-2,1-linked D-fructofuranose bonds,and terminates with a glucose residue.Inulinase can catalyze the hydrolyzation of inulin.Two typical inulinases are endoinulinase and exoinulinase.Endoinulinase can hydrolyse the internal?-2,1-fructofuranosidic linkages to yield inulooligosaccharides,while exoinulinase splits off terminal fructose units successively from the non-reducing end of the inulin molecule to yield fructose and glucose.In the study,two inulinase were cloned from a inulin-degrading strain Paenibacillus sp.Lfos16,which were named inu-1 and inu-2.The ORF of inu-1 was 2274bp,which encoded757 amino acids,and GC%content of it was 51.67%.It belonged to the glycoside hydrolase32 family,which had 98%homology with glycoside hydrolase derived from Paenibacillus polymyxa DSM365.The ORF of inu-2 was 2265bp,which encoded 754 amino acids,and GC%content of it was 49.49%.It belonged to the glycoside hydrolase 32 family,which had99%homology with glycoside hydrolase derived from Paenibacillus polymyxa F2.inu-1 and inu-2 were cloned into expression vector pET-28a?+?and heterologously expressed in E.coli BL21?DE3?.The recombinant enzymes Inu-1 and Inu-2 were purified using affinity chromatography,and the biochemical characterization of Inu-1 and Inu-2 was investigated.The results showed that the molecular weight of recombinant Inu-1 was estimated to be 88kDa by SDS-PAGE.The specific enzyme activity of the crude enzyme solution was 46.07U/mg.The main product of the hydrolyzed inulin was F3,and the yield of it was 78.5%in the final hydrolyzed products of inulin,indicating that the recombinant Inu-1was an endoinulinase.After purification,the specific enzyme activity of Inu-1 was 185.16U/mg and the purification fold reached 4.02.The optimum temperature of the Inu-1 was 35oC,and the activity was stable when the temperature was lower than 45oC.The optimal pH of the Inu-1 was 7,and the activity was stable when the pH was 7-8.The activity was increased by Ca²⁺and Mn²⁺,while Ag⁺,Cu²⁺,Zn²⁺,Hg²⁺,Fe²⁺and Fe³⁺had a significant inhibitory effect on Inu-1.The V_maxax of Inu-1 was 0.14mg/?mL·min?and K_m was 19.91mg/mL.The molecular weight of recombinant Inu-2 was estimated to be 87kDa by SDS-PAGE.The specific enzyme activity of the recombinant Inu-2 crude enzyme solution was 87.43U/mg.The I/S of it was0.499,and the main product of hydrolyzed inulin was fructose,indicating that the Inu-2 was an exoinulinase.After purification,the specific enzyme activity of Inu-2 was 348.30U/mg,the purification fold reached 3.98.The optimum temperature of Inu-2 was 40oC and the activity was stable when the temperature was lower than 30oC.The optimum pH was 6,and the activity was stable when the pH was between 6.6-7.6.Mg²⁺,Ca²⁺,Fe²⁺had a certain inhibitory effect on Inu-2,and Ag⁺,Cu²⁺,Zn²⁺,Hg²⁺,Fe²⁺,Fe³⁺had a significant inhibitory effect on recombinant Inu-2;V_maxax of Inu-2 was 0.18mg/?mL·min?,and K_m was 19.28 mg/mL.