| The pullulanase from Bacillus naganoensis shows excellent enzymatic properties, which meet the requirements of the saccharification process for high temperatue and acid-resisting. Pullulanase has high application value in the starch processing industry, on account of improving the utilization rate of starch and accelerating saccharification process. Bacillus subtilis is a recognized safety strain(GRAS) and capable of secreting biologically active proteins directly to the medium. Hence, In this study, the gene encoding B. naganoensis pullulanase was extracellularly expressed from recombinant B. subtilis. By optimizing the promoter and the host strain, analyzing the stability of the constitutive expression systems, constructing inducible expression system and optimizing induction conditions, extracellular activity of pullulanase was enhanced. The construction and optimization of the recombinant B. subtilis not only offered valuable strains for getting rid of the dependence on import of pullulanase in China, but would also provide advantageous options for efficient production of recombinant enzymes from B. subtilis. The main contents are as follows:(1) Using B. naganoensis genomic DNA as the template, the pullulanase gene pul was amplified by PCR. The PCR product was inserted into the shuttle vector pMA0911 and recombinant plasmid pMA0911-PHpaII-pul was transformed into the expression host B. subtilis WB800 by electroporation to obtain the recombinant B. subtilis WB800/pMA0911-PHpaII-pul. SDS-PAGE analysis of the supernatant of the culture broth indicated a distinctive protein band at around 110 k Da. After incubation for 60 h, the extracellular activity of expressed pullulanase was 3.9 U?mL-1. It indicated biologically active pullulanase was successfully expressed in B. subtilis and was secreted extracellularly.(2) By introducing the strong promoter P43 instead of PHpaII, the extracellular activity of expressed pullulanase from the constructed B. subtilis WB800/pMA0911-P43-pul was improved to 8.7 U?mL-1, which was 2.23 folds increase of that from the original strain. The constructed plasmids were transformed into B. subtilis WB600 to obtain B. subtilis WB600/pMA0911-PHpa II-pul and B. subtilis WB600/pMA0911-P43-pul with the extracellular pullulanase activity 21.3 U?mL-1 and 24.5 U?mL-1, which were 5.46 and 6.28 folds increase of that from the original strain, respectively.(3) By exploring the stability of recombinant plasmids from the constructed constitutive strains during fermentation process, it was found that the loss of recombinant plasmid did not obviously occurred to B. subtilis WB800/p MA0911-P43-pul and B. subtilis WB800/ pMA0911-PHpaII-pul while B. subtilis WB600/pMA0911-PHpaII-pul and B. subtilis WB600/ pMA0911-P43-pul suffered the loss of recombinant plasmids, which proved that the higher expression burden generated the loss of recombinant plasmids. The supplement of the corresponding antibiotic to fresh transformants in fermentation medium as a selection pressure was proposed as an effective approach for overcoming this limitation. In addition, from the respect of structural stability of plasmid, deletion or mutation of the gene pul would not happen to B. subtilis WB600/pMA0911-P43-pul with the highest extracellular pullulanase activity for at least 50-generation subculture.(4) Sucrose-induced promoter Psac B was introduced to replace original promoter PHpa II and inducible recombinant B. subtilis WB800/p MA0911-Psac B-pul was constructed. The extracellular activity of expressed pullulanase was 2.5 U?m L-1 after induction. By optimizing host strain and induction conditions, the maximum of extracellular pullulanase activity of 10.6 U?m L-1 was achieved from B. subtilis WB600/p MA0911-Psac B-pul, under the optimized expression conditions that induction was initiated at 20 °C with 3%(w?v-1) sucrose and OD600 of growing cells reached 0.3. Compared with previous recombinant B. subtilis WB800/ pMA0911-PsacB-pul, extracellular activity of pullulanase was enhanced 4.24 folds. |
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