Effect Of Medium Additives On Extracellular Production Of Recombinant α-Cyclodextrin Glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is an extracellular enzyme capable of converting starch or starch derivates into cyclodextrins through an intramolecular transglycosylation reaction. With cyclodextrin applications expanded in the industries related to food, pharmaceuticals, etc, CGTase has become the focus of scientific research nowadays. To overcome the low productivity of wild strains, the overexpression of CGTase in Escherichia coli has been expected. However, previous reports showed that the CGTases expressed in E. coli were usually accumulated in the cytosol as biologically inactive inclusion bodies and/or the periplasm as soluble forms, which limited its industrial utilization. Thus, it is highly desirable to target the CGTase into the culture medium of E. coli. E. coli cells harboring plasmid cgt/pET-20b(+), constructed and conserved in our lab, has the ability to produceα-CGTase. In this study, extracellular recombinantα?-CGTase production by this strain when increase some chemical additives into the fermentation medium was systematically investigated for the purpose of highα-CGTase yield and productivity. The mechanism of it was also explored. The main contents of this dissertation follow:(1) In shaking flasks experiment, the effects of addition of surfactants on the production of extracellular recombinantα-CGTase were investigated. The result indicated that surfactants inhibited the growth of E.coli. The more surfactant were added, the inhibition was stronger. The inhibition of CTAB towards the growth of E.coli was the strongest, while Tween-80 was the weakest. The production of extracellular recombinantα-CGTase is able to be improved by surfactants. When adding 0.02% SDS at 20 hours, the final yield of enzyme could reach to 5.31 U/mL , which was 5.25 times higher than that of control.(2) In shaking flasks experiment, the effects of addition of ions on the production of extracellular recombinantα-CGTase were tested. The result indicated that the production of extracellular recombinantα-CGTase was able to be improved only by Ca2+. When its adding concentration was 2.5 mM, the final yield of enzyme could reach to 1.12 U/mL , which was 1.3 times higher than that of control. The different addition time during culture stage could not further improve the yield. Ca2+ improved the production of extracellular recombinantα-CGTase has related with cell membrane penetrability, also has related with its slight promoter action to the growth of E.coli.(3) In shaking flasks experiment, the effects of addition of penetrants on the production of extracellular recombinantα-CGTase were tested. The result indicated that sorbitol and sucorse had better promoter action to the growth of E.coli, other penetrants had more or less inhibitory action to the growth of E.coli. Only L-proline and Na+ could promote the production of extracellular recombinantα-CGTase. When the adding concentration of L-proline and Na+ are 75 mM and 500 mM respectively at the beginning of culture,α-CGTase activity of culture supernatant reached 1.72 U/mL for L-proline and 3.6 U/mL for Na+ and were respectively 2.02 and 4.2 higher than that without addition of penetrants.(4) Using SDS, Na+, glycine and Ca2+ as four factors, a L9(34) orthogonal experiment was designed for the target ofα-CGTase activity. By the implement of an orthogonal array experiment design, the importance of factors on enzyme production was obtained as such, glycine > SDS > Na+ > Ca2+. The optimal adding concentration of glycine , SDS , Na+ and Ca2+ were 0.03%, 400 mM, 0.3% and 10 mM respectively. Compared to the result obtained before optimization, the maximal yield ofα-CGTase reached 12.89 U/mL, which was increased by 15-folds.(5)The change of permeability of the E. coli membrane after adding different chemical additives were studied. The hydrophobic fluorescent probe NPN was used as an indicator of outer membrane integrity, and the permeabilization of the inner membrane was monitored using theβ-galactosidase substrate ONPG as a probe. Compared to the result obtained before optimization, the permeability of E. coli membrane was increased, which allowed more recombinantα-CGTase releasing into the culture medium.(6) The analysis of phospholipid composition of cell membrane demonstrated that under the control condition, the amount of phosphatidylethanmine (PE) and phosphatidylglycerol (PG) of the E.coli membranes were 75.8% and 18.2% respectively. However, the amount changed to 71% for PE and 20.3% for PG under the optimization condition. The increaseed amount of PG may stimulat the binding of chaperonins SecA to membrants, which stimulated the translocation of precursor protein across the membrane. Thue the yield of extracellular recombinantα-CGTase enhanced.(7) Compared to the control, the amount of unsaturate fatty acids (C16:1, C18:1, C19:1) under the optimization condition increased from 1.3%, 13.41% and 2.28% to 2.34%, 20.85% and 23.27 % respectively. The amount of C14:1 was descended from 0.29% to 0.11%. At the same time, the amount of C17:0 was descended from 27.45% to 26.11%. Medium additives changed the ratio of saturated acids and unsaturated acids, and the fluidity of cell membrane was increased, which allowed more recombinantα-CGTase releasing into the culture medium.