Isolation Of Strain Producing Pullulanase, Gene Cloning And Expression, Molecular Modification Of Type I Pullulanase And High Cell Density Cultivation Of Recombinant Escherichia Coli

Pullulanase (EC3.2.1.1/41) specifically hydrolyzes the α-1,6-linkages in branched oligosaccharidesof amylopectin, and leads to the formation of amylose. Pullulanase together with other amylases is veryuseful in starch industry with good market prospect. It has been a good enzyme in the amylose industry.Pullulanase has an expansive prospect as feed additive. The reasons are pullulanase can remove theanti-nutrition factors which are formed by amylopectin in the feed materials and promote effectivelyanimal digestion and absorption of nutrients in the feed, improve feed utilization, and improve thedisease resistance of animals together with the animal endogenous and exogenous amylase. In thisthesis, we study the isolation of bacteria producing pullulanase, cloning and expression of pullulanaseand high cell density cultivation which are propitious to feed. The research includes the items asfollows.1A strain H4producing pullulanase was obtained by screening from the soil close to a starchprocessing plants using spread-plate method. Characteristics of morphological, physiological,biochemical and16S rDNA sequence were applied to identify the strain. The strain was closely relatedto Exiguobacterium sp. A series of experiments were conducted about the strain H4for the optimizationof fermentative medium and growth conditions. Under the optimum condition,yield of pullulanasecould reach to6.35U/mL, which was110.2%higher than the control (3.02U/mL).2Primers were designed used CODEHOP approach based on the conserved nucleic acid sequencesof pullulanase genes from Exiguobacterium sp. A gene fragment was amplified with these degenerateprimers. Tail-PCR strategy was used to get full gene from this fragment. Thus the whole gene sequencehad been gotten and registered in Genbank, whose accession number was JQ686229. Pullulanase fromExiguobacterium sp. H4is type I pullulanase, and it belongs to glycoside hydrolases13familes throughbioinformic analysis.3Six expressing vectors were constructed in order to express original and modified genes ofpullulanase from Exiguobacterium sp. H4. The vectors were transformed to Escherichia coli BL21(DE3)plysS. Six recombinant pullulanases were expressed in the E.coli respectively. The modifiedpullulanase deleting100amino acids from the amino terminus of original pullulanase got highestactivity of pullulanase, and the enzymic characteristics of it were studied. Its optimal activity pH was6.0, and optimal temperature was50℃. Metallic ions and chemical reagents had very little influence onthe modified pullulanase, and Co²⁺showed an enhancing effect. The modified pullulanase specificallyhydrolyzed the α-1,6-linkages in pullulan as well as soluble starch and dextrin, and its degradationproducts were maltritose, maltose and glucose. The modified pullulanase could tolerate the action ofpepsin and trypsin.4After analysis of the data in the shake flask culture, the strategy of producing of pullulanase byhigh cell density cultivation of recombinant E.coli was investigated. A pre-determined feeding(μset=0.12) strategy was chosen to maintain carbon-limited growth using a defined medium. Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor in order toprevent the accumulation of acetic acid. Expression of pullulanase was induced when cell concentrationOD600was70.0. Applying the feeding strategy, final cell concentration of53.3mg/mL dry cell weight (gL^-1DCW) and activity of pullulanase in a volumetric concentration of67.0U/mL were obtained.This was the first report for producing pullulanase in Exiguobacterium. It was also obtained thenovel gene encoding pullulanase from Exiguobacterium, and provided recombinant E.coli expressingoriginal and modified pullulanase. The fed-batch process for high cell density cultivation based on thevalue of E.coli yield on glucose (YX/S, g g^-1) was firstly reported in China. Feeding was carried out toincrease the cell mass concentration exponentially in the bioreactor, and it was instructive in industrialproduction of pullulanse.