Optimization Of Extracellular Secretion Of Peanibacillus Macerans α-cyclodextrin Glycosyltransferase In Recombinant Escherichia Coli

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is capable of converting starch or starch derivates into cyclodextrins through an intramolecular transglycosylation reaction. With application of cyclodextrins expanded in the industries related to food, pharmaceuticals, etc, CGTase has become a research focus nowadays.In our eraly study, Paenibacillus maceransα-cyclodextrin glycosyltransferase was successfully expressed into culture medium in recombinant Escherichia coli. In order to enhance the extracellular secretion efficiency of P. maceransα-CGTase in recombinant E. coli, several strategies including signal peptide optimization, co-expression of bacteriocin release protein(BRP) and addition of media additives were employed in this study. The main results are listed as follows:(1) Several signal peptides which were mostly used in mediating secretion of recombinant proteins in E. coli were obtained by routine PCR and overlap PCR. Effects of four signal peptides including OmpA, PelB, OmpT and Endoxylanase were fused to the N-terminal of the matureα-CGTase and expressed in E. coli BL21 (DE3). Theα-CGTase secretion efficiency mediated by different signal peptide was investigated. The results showed that OmpA was the optimal signal peptide among all the studied signal peptides under the same culture conditions. After 72 h of cultivation, the extracellularα-CGTase activity could reach 32 U/mL, which was 2.4-fold and 4.3-fold that of the PelB and OmpT signal peptide, respectively. However, the secretion efficiency of Endoxylanase was the lowest, the detectableα-CGTase activity was quite low and a large amount ofα-CGTase existed in the form of inclusion bodies.(2) Six primers were used in the overlapping PCR to obtain the gene encoded Lpp-BRP protein. The target gene was cloned into the expression vector pSTV 28 and co-transformed withα-CGTase expression vector into the host cell E. coli BL21 (DE3). Effects of BRP co-expression on extracellular secretion of recombinantα-CGTase were investigated. The results showed that BRP co-expression had almost no promotion effect on the extracellular secretion of recombinantα-CGTase in the existing expression system.(3) Several kinds of media additives such as glycine,SDS, Tween-80, Triton X-100 and so on were added into the culture to test their effect on extracellular secretion of recombinantα-CGTase. The results showed that glycine and Triton X-100 had a significant promotion effect on extracellular secretion of recombinantα-CGTase. 0.7% glycine was added into the culture after 15 h of cultivation, the extracellularα-CGTase activity could reach 30 U/mL after 48 h of cultivation; 0.2% Triton X-100 was added into the culture at the beginning of the cultivation,α-CGTase activity in the supernatant could reach 28.9 U/mL after 48 h of cultivation, which was 9.7-fold and 9.3-fold higher than that of the control group without any additives, respectively.(4) On the basis of the result of the singe factor experiment, a two-factor and three-level fractional factorial design was employed to optimize the concentration of glycine and TritonX-100. The optimal condition to achieve maximal extracellular production ofα-CGTase was supplementation the culture with 0.5% glycine and 0.5% Triton X-100 together. The extracellularα-CGTase activity reached 48 U/mL after 48 h cultivation, this enzyme productivity was 20-fold that of the control group without any additives and 95 times higher than that by cultured P. macerans JFB05-01.(5) Theα-CGTase from the culture containing additives (0.5% glycine and 0.5% Triton) and without any additives were purified through a combination of ion-exchange and hydrophobic interaction chromatography. Specific activity of theα-CGTase from these two sources were 195.1 U/mg and 199.8 U/mg, respectively. Therefore, addition of glycine and Triton X-100 had almost no effect on folding of the recombinantα-CGTase.(6) Inner and outer membrane permeability of E. coli cells cultivated under different conditions was monitored using ONPG and NPN as molecular probes, respectively. The results showed that addition of glycine and Triton X-100 greatly increased cell membrane permeability. Under the optimal condition, inner and outer cell membrane permeability was 9-fold and 3-fold that of the control group, respectively. The increased cell membrane permeability may play an important role in the extracellular secretion of recombinantα-CGTase and the specific mechanism remained to be further study.