| Pullulanase (EC3.2.1.41) is a debranching enzyme, which specifcally cleaves α-1,6glycosidic bonds in pullulan, starch and other related amylaceous polysaccharides. Hence,pullulanase can be employed to break down starch to produce glucose, fructose, maltosesyrups, and amylose, in conjunction with or without α-amylase, β-amylase, glucoamylase. Inaddition, pullulanase could also be used in the industry of pharmaceuticals, feed, brewing etc.to enhance the utilization rate of starch. However, low yield and productivity have been thebottleneck for the large-scale production and widespread application of pullulanase.In order to obtain pullulanase-producing microbes of high efficiency, this study beganwith the screening of pullulanase producers from the soil samples taken from severalprovinces in China. Subsequently, an engineered Escherichia coli was constructed, whichcould produce extracellular pullulanase with high efficiency. For the sake of improved yield, acombined strategy involving auto-induction, temperature regulation, and osmolyte additionwas developed. As a result, its application greatly enhanced the yield of extracellularpullulanase.(1) A pullulanase-producing strain was obtained. In order to overcome the problem oftraditional screening method, such as heavy workload and complex operation, a plate assay,based on red-pullulan, was employed to reduce the scope of screening. Subsequent abilityconfirmation of the isolates was carried out with the traditional screening method.Consequently, more than60isolates were obtained, which presented the ability to de-color thered-pullulan. In order to achieve an excellent strain, the isolates with haloes2times thecolonies were subjected to the second round of screening. Fortunately, an isolate with thehighest capability was obtained, which was identified as Klebsiella sp. SHN-1(GenBankAccession Number: HM037179). Under the optimized fermentation condition, the Klebsiellasp. SHN-1could produce about10.98U mL-1extracellular pullulanase.(2) An engineered E. coli BL21(DE3) with the ability to produce extracellularpullulanase was constructed. Although extracellular production is important for the large-scaleproduction of pullulanase, most constructed engineered strains produced pullulanaseintracellularly. Considering the close relationship between E. coli and Klebsiella sp. SHN-1, aprimer pair was designed based on the published pullulanase sequence to clone thepullulanase encoding gene. As a result, a DNA fragment of3000bp was obtained, whichshared99%homogeneity with that from K. variicola At-2and K. pneumoniae342.Subsequently, the sequence was subjected to GenBank (Accession Number: JX087429).When the DNA fragment was cloned into E. coli BL21(DE3) with the plasmid of pET-28a(+)as expression vector, recombinant pullulanase was detected in the supernatant of culture brothin the presence of IPTG.(3) An expression vector without signal peptide sequence was constructed. Numerousstudies suggested that the secretion of pullulanase from Klebsiella in E. coli required theexistence of secretion-related genes, including the signal peptide sequence. In order to explorethe reasons of pullulanase secretion in E. coli, a signal peptide-deleted plasmid was constructed. However, the engineered E. coli BL21(DE) carrying the expression vector couldstill express the gene encoding pullulanase and release the product into surroundingenvironment. The distribution of recombinant pullulanase in subcellular fractions, such as cellmembrane, cytoplasm, petriplasmic space, cell-associated fraction was also investigated. Theresult showed that up to65.49%recombinant pullulanase was secreted, indicating the releaseof recombinant pullulanase was not close to the existence of the signal peptide sequence.Analysis on the sequence of mature pullulanase suggested two inner regions might beresponsible for the secretion of pullulanase. Subsequently, the fermentation conditions of thisstrain in LB medium were optimized to enhance the production of extracellular pullulanaseand10U mL-1extracellular pullulanase was obtained. The recombinant pullulanase was alsopurified by one-step affinity chromatography. Investigation on the purified pullulanaseshowed its optimum reaction pH and temperature were5.0and55oC, respectively. When itwas employed to hydrolyze1%pullulan, maltotriose as the main end products was obtained,demonstrating the property of type I pullulanase.(4) The yield of extracellular pullulanase was enhanced by using a combined strategyinvolving the visible screening method, auto-induction, temperature control strategy. In orderto ensure the stability of the engineered strain, colonies used to carry out the production ofpullulanase were evaluated with the visible screening method. Subsequently, auto-inductionmethod was employed to simplify the production of pullulanase and increase the cell densityof culture. Results showed that the yield of extracellular pullulanase was improved to50U mL-1. Additionally, the ability of the engineered strain was further improved to68.23U mL-1by applying various natural osmolytes. Finally, pullulanase production was performedin a7L fermentor with batch and fed-batch strategy. As a result, the yield of extracellularpullulanase was elevated to97U mL-1.The yield of extracellular pullulanase was greatly improved by performing systematicresearch work. An isolate producing extracellular pullulanase was obtained with color platemethod and pullulan precipitation method. During the construction of an engineered E. coliproducing extracellular pullulanase, the signal peptide-independent secretion of pullulanase inE. coli was discovered. This observation provided the study of effective proteins secretionwith reference and theoretical basis. According to the property of recombinant proteinexpression and secretion, auto-induction-two stage temperature control strategy wasdeveloped. By combination with osmolytes, the yield of extracellular pullulanase was greatlyincreased. Therefore, the study presented theoretical guideline for the large-scale productionof pullulanase in the future. |
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