Cloning And Expressing Of Pullulanase Gene From Bacillus Thuringiensis

Pullulanase(EC 3.2.1,41)are also called true pullulanase,limit dextrinases,debranching enzymes,amylopectin 6-glucanohydrolases or ?-dextrin 6-glucanohydrolases,which can typically hydrolyze in an endo-way the 1,6-?-D-glucosidic linkages in pullulan and in branched oligosaccharides to maltotriose and linear oligosaccharides,respectively.As an industrially important enzyme,therefore,pullulanase is generally employed together with other amylolytic enzymes(?-amylase,glucoamylase)to efficiently break down recalcitrant biomass into fermentable sugars for generating biofuels and other chemical commodities.So far,pullulanase has been discovered and identified from various microorganisms,such as Thermotoga neapolitana,Clostridium thermosulfurogenes,and species of the genus Bacillus.In this paper,our target is to build highly efficient gene-expression of pullulanase engineering strains,the main results of this research were as follows:1.We cloned gene and obtained the gene structure of pullulan enzyme by the conventional polymerase chain reaction(PCR),with genomic DNA from Bacillus thuringiensis BtR05 as the template.The recombinant prokaryotic expression vector pET(K)-Trx-pul was constructed successfully through the pullulan gene inserted into the prokaryotic expressing vector pET(K)-Trx.we achieved soluble expression of pullulan genes in E.coli BL21(DE3)with the molecular chaperone help.2.The fermentation conditions of recombinant strains were optimized.The optimum condtions: induction after each strain OD600 value of 0.6,The optimal cultivation temperature was 28 ?,cultivated temperature was 25?,after cultivation for 48 hours the activity of the recombinant pullulanase was up to about 32 U/mL.3.Enzyme characterization test showed that the optimum temperature is 45?,the recombinant pullulanase showed higher activity when the temperature was 35?-45?,and the half-life of the enzyme was 20 hour at 60?.The optimum pH for recombinant protein was 6.0,and recombinant was highly active in the pH range 4.0-9.0.Hydrolysis ability to pulluanase,soluble starch synthase,dextrin and amylopectin of recombinant pullulanase is reduced in turn.4.The recombinant pullulanase proceeded auto-induction training using by the induction culture medium.After the cultivation for 24 h at 25?,the intracellular enzyme activity of pullulanase reached 27.7 U/mL.We optimized auto-induction medium,the results indicated that the optimum culture medium is: Tryptone 2%,Yeast extract 0.25%,glycerin 1.0%,glucose 0.025%,lactose 0.4%.Under the optimum conditions show that the activity of the recombinant pullulanase was up to about 50.7 U/mL.