Cloning And Co-expression Of Pullulanase Gene And Vitreoscilla Hemoglobin Gene In Escherichia Coli BL21

Pullulanase is a kind of debranching enzyme,which can efficiently and specifically cut the ?-1,6-glycosidic bond in the chain starch,thereby cutting off the entire branch of amylopectin to form a straight starch.This feature determines the use of pullulanase protein in starch-based industrial production activities,whether used alone or in combination with other amylases,reducing the use of starchy raw materials,reducing production costs,and reducing waste tails.The convenience of protecting the environment has high application value.The high value of the enzyme product that does not match the application value of the pullulanase is that the pullulanase is the only enzyme in the amylase that cannot be produced by itself in China.The high-yield pullulanase engineering bacteria for industrial production has always been a hot topic.Due to the large molecular weight of pullulanase,expression in E.coli is subject to considerable limitations.The expression of vitreoscilla hemoglobin is regulated by the oxygen content in the environment,which can help oxygen enter the cell,accelerate the transmission of oxygen in the cell,and play a positive role in the accumulation of microbial metabolic products.The laboratory preserved a strain that can produce pullulanase protein.In this experiment,the complete coding sequence pul A of the pullulanase protein was obtained by combining protein profiling with high-throughput sequencing.It was successfully expressed in E.coli BL21(DE3)by molecular cloning technology,and the vitreoscilla hemoglobin-encoding gene vgb and pul A gene were expressed in the host bacteria together with the symbiotic plasmid.By comparing the enzyme activity,the expression of vgb gene in the host can promote the expression and accumulation of host metabolites,and the enzyme activity of pul A is increased by 3-4 times compared with the recombinant bacteria expressed alone.The pullulanase-encoding gene pul A belongs to the type I pullulanase,and there is currently no identical sequence in NCBI.The main difference is in the sequence from the firstbase to the 114 base,from 115 bases to The last base is identical to the NCBI search number MH411123.1.The recombinase peptide chain is composed of about 927 amino acids and has a size of about 93.64 KDa;it has good acid and high temperature resistance,p H=4.0~5.0,and has a relative enzyme activity of 65.78%;p H ranges from 5.0 to 5.5.At the same time,it still has a relative activity of 73.63%;the relative active activity of the enzyme exceeds 90% in the temperature range of 50 ? to 60 ?,and the relative enzyme activity exceeds 80% in the temperature range of 45 ? to 50 ?.The above studies show that the protein encoded by the pul A gene obtained in this experiment has good acid and high temperature resistance properties,and has similar enzymatic properties compared with the industrially produced pullulin product.The bacteria successfully expressed pullulanase and vitreoscilla hemoglobin,and the activity of the pullulanase enzyme shake flask fermentation activity was up to 67.25 U/m L,compared with the pullulanase activity expressed alone,pul A and vgb genes.When co-expressed,the activity of pullulanase was increased by more than three times to 191.47 U/m L.Therefore,the recombinant pullulanase has superior protease properties,and the recombinant enzyme has high enzyme production ability.At the same time,the vitreoscilla hemoglobin gene is expressed in the recombinant bacteria to increase the activity of the pullulanase enzyme.These are the development of the pullulanase engineering bacteria.Provides a new technical approach.