Integrative Expression Of Bacillus Naganoensis Pullulanase From Bacillus Suhtilis

Pullulanase from Bacillus naganoensis exhibits great enzymatic properties especially the high temperature and acid resistance,which properly suits the starch saccharification process and enhances industrial production.Bacillus subtilis is generally regarded as safe for food production with a well-developed protein transcriptional and secretion system.Hence,in this present study,two pul expression cassettes including a constitutive promoter PHpa? and an inducible promoter VsacB,respectively,were inserted into the genome of Bacillus subtilis.Thereafter,fermentation conditions of promising recombinant strains were investigated to obtain the optimum enzyme activity.The main contents were as follows:(1)Taking the conducted shuttle plasmid pMA0911-PHp?-pul as the template,the pul expression cassette PHpa?-pul was amplified by PCR.The resulting product was inserted into the genomes of B.subtilis WB600 and B.subtilis WB800.After fermentation for 72 h,the extracellular pullulanase activity of B.subtilis WB800-PHpa?-pul(hereinafter referred to as 8H)and B.subtitlis WB600-PHpa?-pul(hereinafter referred to as 6H)was 30.32 U·mL-1 and 18.83 U·mL-1,respectively.As the extracellular pullulanase activity of 8H was higher than that of 6H,the following studies on determining the expression conditions for pullulanase expression were conducted using 8H.The maximum enzyme activity achieved was 60.85 U·mL-1 under the optimum fermentation conditions,which were as follows:inoculum size 2%(v·v-1),incubation temperature 30?,shaking speed 250 r min-1,and initial pH 7.0.Compared with what was achieved under the primary conditions,extracellular pullulanase activity of 8H was enhanced around 2 folds;(2)Taking the conducted shuttle plasmid pMA0911-PsacB-pul as the template,the pul expression cassette VsacB-pul was amplified by PCR.The resulting product was inserted into the genomes of B.subtilis WB600 and B.subtilis WB800.After fermentation for 72 h,the extracellular pullulanase activity of B.subtilis WB600-PsacB-pul(hereinafter referred to as 6S)and B.subtilis WB800-PracB-pul(hereinafter referred to as 8S)was 5.34 U·mL-1 and 13.19 U·mL-1,respectively.As the extracellular pullulanase activity of 8S was higher than that of 6S,the following studies on determining the induction conditions for pullulanase expression were conducted using 8S.The maximum enzyme activity achieved was 48.59 U mL"1 under the optimum induction conditions,which were as follows:sucrose concentration 2%(w·v-1),induction temperature 20 ?,and induced occasion OD600 0.4.Compared with what was achieved under the primary conditions,extracellular pullulanase activity of 8S was enhanced around 3.7 folds.