Study On Heterologous Expression Of The Acid Pullulanase

Pullulanase is a kind of debranching enzyme which specific degradeα-1,6-glycosidic. Pullulanase was used in the brewing industry, starch industry, amino acid industry and other processing bio-industry. It is important enzyme of the starch industry. However, because of technical limitations, we can not produce the pullulanase until now.According to the sequence of pullulanase gene from Bacillus deramificans (NCBI accession number: AX203843), the gene encoding mature peptide of pullulanase was synthesized and designated pulA. The pulA was cloned into the vector pUC18 to get recombinant plasmid pUC-pulA. The pulA was amplified by the method of PCR and cloned into the expression vector pET-28a to yield recombinant plasmid pET-28a-pulA. The recombinant plasmid pET-28a-pulA was transformed into the E.coli BL21 (DE3) and the gene of pullulanase was correctly expressed.The pulA fragment subcloned from pET-28a-pulA into expression vector pHY-WZX to yield hybrid plasmid pHY-WZX-pulA. Recombinant plasmid pHY-WZX-pulA was introduced into the B.subtilis WB600. Active pullulanase was expressed by recombinant B.subtilis and secreted into medium. Recombinant plasmid pHY-WZX-pulA was also transformated into the B.licheniformis B60608.Culture condition of recombinant B.licheniformis B60608 was optimized for production of pullulanase. The activity of pullulanase increased from 0.115 ASPU/mL to 5.11ASPU/mL under the fermentation of the shake flask. The activity of pullulanase arrived at 12.32 ASPU/mL on fermenter; the initial strain could not produce pullulanase. The optimized medium consists of 7% of glycerol, 0.4% of (NH4)2SO4, 2.5% of cotton seed protein, 11.75 mmol/L of phosphate. The recombinant strains grow well at temperature of 42℃and pH of 7.0. The optimized fermentation conditions were: fermentation temperature of 30℃; pH of 6.5; seed age 16 h; inoculation 10%.The properties of pullulanase were characterized which showed that the enzyme has an activity optimum at 50℃and at pH 3.5. It was active below 60℃and high stability at pH values ranging from 5.0 to 7.0. Most of metal ions such as Mg²⁺, Mn²⁺, Li⁺, Na⁺ , Zn²⁺, Fe³⁺, Cu²⁺ and Co²⁺enhance activity of enzyme, but K+ has no significant effect. Surfactant EDTA and Tween 20 also enhance activity of enzyme. The activity of the enzyme was strongly inhibited by SDS.