Isolation Of Strain Producing Pullulanase,Cloning And Expression Of The Pullulanase Gene And Its Enzymatic Characterization

Pullulanase?EC 3.2.1.41?is a starch debranching enzyme which can specifically hydrolyze the?-l,6 glycosidic bond in pullulan,amylopectin,and limit dextrinase,converting them into amylose.Therefore it plays a key role in the starch processing industry,and exhibited great application requirements in the food processing,beer brewing,chemical industry and medical industry,etc.In this study,the pullulanase-producing strain was isolated and further identified from sewage water samples of a starch factory,and the enzyme activity was increased through heterologous expression of the pullulanase gene,showing great significance in the development and application of pullulanase in China.The main research contents are as follows:?1?The strain LK18 was isolated using spread-plate method and further enzyme activity determination.It exhibited high pullulanase activity?1.53±0.16 U/mL?and was identified as Paenibacillus puldeungensis according to its morphological observation,physiological and biochemical tests,16S rDNA sequence and phylogenetic tree analysis.?2?The pullulanase protein sequence of the strains which showed high homology with Paenibacillus puldeungensis LK18 was selected and aligned to define the conservative regions,and the degenerate primers were designed through CODEHOP methods to amplify the partial pullulanase gene.Other primers to amplify the full-length pullulanase were further designed according to the alignment result of the partial sequence.The sequencing result showed that the full-length pullulanase gene?1968 bp?encoded 655 amino acids.Pullulanase from Paenibacillus puldeungensis LK18 is type I pullulanase,and it belongs to the glycoside hydrolase 13 family according to bioinformatics analysis.?3?The recombinant expression vector pET-28a?+?-pulA was constructed and expressed in E.coli BL21?DE3?,after which the induction conditions were further explored.The results showed that the recombinant enzyme exhibited the optimized expression when induced at 22°C with 0.5 mmol/L IPTG for 12 h,under which the enzyme activity reached248.52 U/mL.?4?The specific enzyme activity of the recombinant pullulanase purified by nickel affinity chromatography was 508.8 U/mg,with the molecular weight of 76.95 kDa.The enzymatic properties showed that the optimal reaction temperature of the recombinant enzyme was 45°C,and the optimum pH was 6.0.The enzyme was stable in the range of35°C⁴0°C or pH 6.0⁸.0.The enzyme activity was activated by K⁺and Mg²⁺in concentration of 10 mmol/L,but was inhibited by Zn²⁺,Ni²⁺,Fe²⁺,Cu²⁺to different extents.Other metal ions had no obvious effects on this enzyme.When the pullulan was used as the substrate,the Km and Vmax values of the enzyme were 3.69 mg/mL and 384.62umol/?min·mL?,respectively.