The Cyclodextrin Glycosyltransferase of Paenibacillus pabuli US132 Strain: Molecular Characterization and Overproduction of the Recombinant Enzyme

Abstract

The gene encoding the cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to the β-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37°C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19°C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed Vmax and Km values of 253 ± 36 μmol of β-cyclodextrin/mg/min and 0.36 ± 0.18 g/L, respectively.

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

“…The monitoring of the CGTase activity in the cell crude extract, periplasmic fraction and the supernatant, showed a clearly detectable activity of 2 U/mL (95%) in the extracellular juice of DH5α/pAD23 strain against of only 0.08 U/mL (1.5%) and 0.2 U/mL of culture (3.5%) in the cytoplasmic and periplasmic frac- tions, respectively. In contrast, in the case of the CGTase US132 with its natural signal peptide, the CGTase activity was mainly periplasmic evaluated to be 1 U/mL (98%), while no detected activity was found in the supernatant and only 2% in the cytoplasmic fraction, as already reported by Jemli et al (2008). Thus, enzyme localization showed that the enzyme was efficiently expressed and secreted into the supernatant in catalytically active conformation with specific activity of about 400 U/mg.…”

“…After centrifugation (7,500×g, 10 min), the supernatant was used as crude enzyme solution for assaying enzyme activity. The periplasmic and cytoplasmic proteins were prepared as described by Jemli et al (2008).…”

“…Despite the same enzymatic properties of CGTases expressed in E. coli, the enzyme ended up in the periplasm and sometimes a formation of insoluble inclusion bodies was reported (Lee et al 1994;Jemli et al 2008). These phenomena were due to an additional transport barrier, the outer membrane, of Escherichia coli (Pugsley 1993).…”

Mutations affecting the activity of the cyclodextrin glucanotransferase of Paenibacillus pabuli US132: insights into the low hydrolytic activity of cyclodextrin glucanotransferases

“…The plasmid pSJ8 carrying the wild-type US132 cgtase gene was previously described (Jemli et al 2008). The pCR2.1 vector (Invitrogen) was used for the cloning of mutated US132 cgtase genes.…”

“…In previous works, the conventional CGTase from Paenibacillus pabuli US132 (US132 CGTase) was characterized, cloned and overexpressed (Jemli et al 2008;Zouari Ayadi et al 2011). In addition, the enzyme has been reported as a potential good candidate for application as an anti-staling agent particularly because of its action on bread firmness (Jemli et al 2007).…”