Exploration Of The Methods For Expression Of Pullulanase In Bacillus Amyloliquefaciens

Pullulanase is a debranching enzyme for the hydrolysis ofα-1,6-linkages in starch. It is wildly used in the starch processing industry. The molecular weight of the pullulanases produced by Bacillus sp. or its relatives is always approximately 100 kDa; hence, its secretory expression is difficult. In the current study, the secretory expression of pullulanase is investigated using Bacillus amyloliquefaciens as expression host.Bacillus amyloliquefaciens BF7658 was used for the commercial production of mesophilicα-amylase in China. Considering that B. amyloliquefaciens BF7658 secretes a large amount ofα-amylase, the promoter ofα-amylase could be used to drive the gene expression in an expression vector. The DNA fragment Pamy, which contains the promoter and the signal peptide-encoding region ofα-amylase was amplified using PCR, with the genomic DNA of B. amyloliquefaciens 7658 used as a template. A B. amyloliquefaciens espression vector was constructed with Pamy as promoter, the pMB1 ori from pBR322 as the original replication region in E. coli, with the ori-pama and the tetracycline-resistance (tet) gene from pHY300PLK as the replication original region in Bacillus and the selection marker, respectively. The constructed vector was designated as pCHA03.In B. amyloliquefaciens BF7658, the production ofα-amylase always ends because of spore germination; therefore, disruption of the gene responsible for sporulation may delay spore germination,which would increase the time for enzyme production. In B. amyloliquefaciens, spoIIAC is the key gene responsible for initiation of sporulation. A vector used for the deletion of spoIIAC, designated as pHY-psp-pem, was constructed with erythromycin resistance as the selection marker. B. amyloliquefaciens BF7658 was transformed with pHY-psp-pem, a mutant strain with spoIIAC, which was disrupted, selected, and designated as B. amyloliquefaciens sj08. Together with pCHA03, a new expression system was formed.The BdP gene that encodes pullulanase was inserted into the MCS of secretary expression vector pUC980 and pCHA03, resulting in recombinant plasmids pUC980-BdP and pCHA03-BdP. The two recombinant plasmids were transformed into B. subtilis 1A717 and B. amyloliquefaciens BF7658, respectively. The pullulanase activities produced by B. subtilis 1A717/pUC980-BdP and B. subtilis 1A717/pCHA03-BdP were rather low. The pullulanase activities produced by B. amyloliquefaciens BF7658/pUC980-BdP and B. amyloliquefaciens BF7658/pCHA03-BdP were at 1.1 ASPU/mL, and 3.1 ASPU/mL, respectively. The results indicate that the pullulanase activity expressed in B. amyloliquefaciens were higher than that in B. subtilis. The gene expression, driven by Pamy is more efficient than that by the P43 promoter of pUC980. Plasmid pCHA03-BdP was transformed into B. amyloliquefaciens sj08. The fermentation experiment in a 15 L fermentor showed that compared with B. amyloliquefaciens BF7658/pCHA03-BdP, pullulanase production with B. amyloliquefaciens sj08/pCHA03-BdP was prolonged to 8 h, and the pullulanase activity increased by 23%. Culture condition of B. amyloliquefaciens sj08/pCHA03-BdP in flask scale was optimized for pullulanase production. The maximum enzyme activity under the optimized conditions reached 6.9 ASPU/mL. The recombinant pullulanase produced by B. amyloliquefaciens sj08/pCHA03-BdP was purified to homogeneity and the enzyme properties were investigated. The recover rate of the enzyme was 20.1% and fold of purification was 49.9. The molecular weight of the enzyme was about 100 kDa. It showed optimum enzymatic activity at pH5.0 and has an optimal temperature of about 60℃; more than 50% of its initial enzyme activity was still detectable after incubation at 60℃for 3 h.