Improve Heterologous Expression Of Pullulanase Using M Rna Stability Sequence From Bacillus Thuringiensis

Pullulanase(EC3.2.1.41)specifically hydrolyzes ?-1,6-glycosidic linkages of amylopectin and cleaves the the smallest unit of starch branch.Together with amylase and glucoamylase,it can greatly improve the speed of hydrolysis rate and saccharification of starch after liquefying,which significantly improves the utilization of raw materials in fermentation and food industry.However,neither the homologous expression or heterologous expression,the expression level of pullulanase is low.Bacillus thuringiensis insecticidal protein can be abundantly expressed,the mechanism is that it connectes to a conservative SD(Shine-Dalgarno)sequence in the 5 ' upstream and a stem loop structure sequence in the 3' end,which makes the m RNA very stable.Thus,the present study,we constructed this pullulanase stable expression vector and expressed in E.coli,the expression of pullulanase significantly increased.The main results of this study were as follows.(1)We cloned pullulanase gene(pul A)from Klebsiella sp.HN9 and synthetized the stem loop structure sequence(3t)of insecticidal protein gene from Bacillus thuringiensis.(2)Using E.coli-B.subtilis shuttle plasmid pHT43 skeleton,we placed the pul A gene connected with the SD sequence or stem-loop structure in the downstream of an inducible promoter Pgrac to generate pul A heterologous expression vector p HT43-pul A,p HT43-SD-pul A,p HT43-pul A-3T and p HT43-SD-pul A-3T respectively.Among the four expression vectors,the connection type which linked to SD sequence or stem-loop structure sequence were pul A gene(SD sequence or stem loop structure is not connected,as a control),5'-SD-pul A-3',5'-pul A-3t-3' and 5'-SD-pul A-3t-3' respectively.(3)The recombinant plasmids were transformed into E.coli BL21(DE3)and induced by IPTG to express.The intracellular enzyme activitives of plasmids p HT43-pul A,p HT43-SD-pul A,p HT43-pul A-3T and p HT43-SD-pul A-3T were 0.0344 U / m L,12.233 U / m L,0.0378 U / m L and 24.111 U / m L respectively.(4)We analysed the expression of recombinant plasmids in E.coli using semi-quantitative RT-PCR technology.The expression of p HT43-SD-pul A,p HT43-pul A-3T and p HT43-SD-pul A-3T were increased 2.4 times,1.3 times and 3.4 times than p HT43-pul A respectively.(5)The recombinant plasmids p HT43-pul A,p HT43-SD-pul A,p HT43-pul A-3T and p HT43-SD-pul A-3T were digested with Sca?and connected to Kana-resistant gene expression cassette sequence to generate p KAC-pul A,p KAC-SD-pul A,p KAC-pul A-3T and p KAC-SD-pul A-3T respectively using Easy Geno Assembly Cloing kit.These results show that it is necessary that the expression of the structural gene connected to the SD sequence in the upstream.Meanwhile,the stem loop structure connected to the downstream of structural sequence can further enhance the expression of target gene.