The Breeding And Heterologous Expression Of Pullulanase High-yield Strain

Pullulanase(EC 3.2.1.41)plays an important role in the pharmaceutical,feed,food and other fields,while,the industrial production of pullulanase was limited by many factors,such as the low yield of pullulanase,poor stability of pullulanase,high toxicity of Escherichia coli expression system and so on.In this work,the atmospheric and room temperature plasma(ARTP)was used to mutate the wild pullulanase producing strain,and four high pullulanase producing strains were obtained.Furthermore,the pullulanase was successfully expressed in Bacillus subtilis WB600.The research contents were as follows:(1)Identification of pullulanase producing strain and ARTP mutation.A pullulanase producing strain was obtained from the soil of the starch factory,which enzyme activity was 10.83 U/m L.The SDS-PAGE analysis showed that there was a clear band near 75 k Da,which was consistent with the theoretical size of pullulanase(78 k Da).The strain was identified as Bacillus by PCR amplification of 16 S r DNA fragments,sequenced analysis and phylogenetic tree.The L5 strain was mutagenized by ARTP,and the results showed that the optimal mutagenesis time was 60 s.Four strains with high production of pullulanase were obtained from the mutagenesis,among which the strain of G4 had the highest activity of producing pullulanase(22.01U/m L),which was about 2 times higher than that of the original strain.The stability analysis of pullulanase fermentation of the above four strains showed that their enzyme activity fluctuated within 5%.(2)Optimization of fermentation medium and study on enzymatic properties of Strain G4.Based on the single factor experiment,an orthogonal experiment with 7factors and 3 levels was designed to optimize the composition of fermentation medium.The optimal combination was as follows: dextrin 1%,soluble starch 1.5%,peptone 0.5%,soybean powder 1.5%,Fe2+ 0.1%,Ca2+ 0.05%,Zn2+ 0.15%.Under the optimal fermentation medium conditions,the enzyme production activity of strain G4 was 27.22 U/m L(increased by 23.7%).The optimum reaction conditions for the producing pullulanase were 50? and p H6.5.The enzyme activity could be kept above 80% at 30?60? for 2 h and above 60% at p H5 ? 9 for 4 h,indicating that the enzyme had good stability.The enzyme kinetic test further verified that the enzyme has a strong affinity for pullulanose.(3)Heterologous expression of pullulanase in B.subtilis WB600.The pullulanase expression system was constructed by bifunctional Bacillus subtilis inducible high-efficiency expression and secretion vector p SBPTQ,and the highest activity of pullulanase was 14.12 U/m L by sucrose induction.Then,the effects of sucrose concentration,pre-induction culture time,induction culture temperature and induction expression time on the expression level were optimized.It has been verified that the sucrose concentration of 3.2%(w·v-1),the pre-induction culture time of 3.5 h,the induction temperature of 20 ? and the induction time of 52 h were the optimal induced expression conditions of the recombinant strain C9,and pullulanase activity was 17.11 U/m L under these conditions.Further SDS-PAGE analysis showed that there was a characteristic band of the same size as pullulanase(78 k Da)above 75 kDa.