Abstract
A strain CGT-1 producing β-cyclodextrin glucanotranferase (β-CGTase) was identified as Paenibacillus campinasensis by morphological analysis and 16s rDNA analysis. The gene coding for β-CGTase was cloned, sequenced, and expressed in Escherichia coli BL21(DE3). Recombinant β-CGTase was purified and its purity evaluated by SDS-PAGE, showing it encodes a mature protein with a molecular mass of 74 kDa. The β-CGTase was most active at pH 7.0 and 65 °C, respectively. More than 80% activity was retained after incubation at 55 °C for 5 h. The stability of the enzyme was in a pH range from 5.5 to 10.0. The K m and V max for the enzyme activity on CGTase were 3.75 mg/mL and 290.75 μmol/min, respectively. The recombinant plasmid pET28a-DacD-cgt-his, pET28a-OmpA-cgt-his, pET28a-OmpT-cgt-his, and pET28a-CGTase-cgt-his were constructed by cloning the signal peptide genes DacD, OmpA, OmpT, and signal peptide derived from cgtase gene into pET28a-cgt-his, respectively. The production of the recombinant β-CGTase with pET28a-DacD-cgt-his reached 60.89 U/mL after 72 h of culture, which produced an approximately 1.98, 2.93, 4.15 to 9.74-fold higher activity than those containing OmpA, CGTase, OmpT, and the control without signal peptide, respectively. The culture conditions for extracellular production of the recombinant β-CGTase in E. coli BL21(DE3) were optimized. The CGTase activity reached the highest level (37.67 U/mL) under the induction of 0.03 mM IPTG at OD600 of 0.8 at 30 °C after 48 h of culture. Optimization of the extracellular secretion of the β-CGTase from Paenibacillus campinasensis in recombinant E. coli laid the foundation for further industrial production and application of β-CGTase.