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Optimization of Extracellular Secretion of Peanibacillus Macerans α-cyclodextrin Glycosyltransferase in Recombinant Escherichia Coli

Author: LiBin
Tutor: WuJing
School: Jiangnan University
Course: Fermentation Engineering
Keywords: α-cyclodextrin glycosyltransferase Softening Bacillus E. coli Extracellular secretion Bacteriocin release protein
CLC: TQ925
Type: Master's thesis
Year: 2010
Downloads: 63
Quote: 2
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Abstract


Cyclodextrin glycosyltransferase (CGT enzyme for short, EC 2.4.1.19) can be transferred through intramolecular glycosylation reaction conversion of starch and related matrix synthesis cyclodextrin. With the increasingly wide application of cyclodextrins in food, medicine and other fields, CGT enzyme has become a hot research today. Our previous research work, success the Paenibacillus maceransα-cyclodextrin glycosyltransferase (α-CGT enzyme) in E. coli extracellular secretion. In order to further improve the efficient expression of recombinant α-CGT enzyme in E. coli, the present study from the optimization of the signal peptide, the bacteriocin release protein co-expression and the medium is added to the culture medium additives to promote softening Bacillus P. maceransα-CGT enzyme in Escherichia coli Escherichia coli BL21 (DE3) in the extracellular secretion. Main results are as follows: (1) obtained by the conventional PCR and overlap PCR commonly used in E. coli secretion of recombinant protein expression of the exogenous signal peptide gene fused to the signal peptide of α-CGTase of the mature protein N-terminal, and in the E . coli BL21 (DE3) expressed. The the OmpA, PelB, OmpT and Endoxylanase four signal peptide recombinant alpha-CGT enzyme in E. coli exocytosis. Under the same fermentation conditions, the OmpA signal peptide mediated recombination α-CGT enzyme secretion effect is preferably fermented for 72 hours, up to 32 U / mL. Extracellular enzyme activities were PelB of the OmpT mediated efficiency of 2.4 times and 4.3 times. Endoxylanase mediated recombination of the signal peptide of α-CGTase secretion effect is relatively poor and can only be detected in the extracellular α-CGT enzyme activity to low, a large number of α-CGTase exist in the form of insoluble inclusion bodies. (2) by overlapping PCR obtained after transformation of the protein the gene encoding Lpp-BRP BRP cloned into the vector pSTV 28, together with recombinant α-CGT enzyme expression plasmid was transformed into E. coli BL21 (DE3). Investigated BRP protein co-expression of the secretion of the extracellular domain of the target protein. The results are displayed in the existing expression systems, BRP, co-expression of the extracellular secretion of the recombinant α-CGTase almost no role in promoting. (3) the effects of different culture media additives, such as glycine, SDS, Tween-80 and Triton X-100 and so the expression of recombinant α-CGT enzyme secretion. The experimental results showed that glycine and Triton X-100 recombinant α-CGT enzyme extracellular secretion has a significant role in promoting. Extracellular activity reached at 15 h of fermentation, adding 0.7% glycine, 48 h of fermentation activity of the supernatant up to 30 U / mL; initially adding 0.2% Triton X-100 in the fermentation, the 48 h of fermentation 28.9 U / mL, increased 9.7-fold and 9.3-fold, respectively, compared with the control group. (4) on the basis of single factor experiments, the two factors and three levels fractional factorial design experiments optimized added concentration of glycine and Triton X-100 overlay effect. When the medium supplemented with 0.5% glycine and 0.5% Triton X-100, and to promote the role of the most obvious; fermented for 48 h, extracellular enzyme activity of 48 U / mL, does not contain any additives control group 20 times the intensity of production, under the optimum conditions for enzyme production compared with wild mushroom P. macerans JFB05-01 production of high strength 95 times. (5) through the anion exchange and hydrophobic chromatography step purification of the fermentation supernatant of the α-CGTase from containing additives (0.5% glycine and 0.5% Triton X-100) and does not add any additives, the two sources was measured the specific activity of the α-CGTase, respectively, was 195.1 U / mg, and 199.8 U / mg, and no significant difference. Thus, the glycine and Triton X-100 added to the folding of the recombinant α-CGTase almost no effect. (6) ONPG and NPN were used as molecular probes, analysis of the E. coli cell outer membrane permeability under different culture conditions. Under optimum culture conditions, the cell outer membrane permeability were 9-fold and 3-fold of the control group. Membrane permeability increase may strengthen α-CGT extracellular enzyme secretion process plays an important role in the specific mechanism remains to be further studied.

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