Stability Of Recombinant Enzyme Preparation Of α-cgtase

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.In our early study, Paenibacillus maceransα-cyclodextrin glycosyltransferase was successfully expressed into culture medium in recombinant Escherichia coli. In order to enhance the stability of P. maceransα-CGTase in recombinant E. coli, several strategies including site-directed mutagenesis, chemical modification and addition of chemical additives were employed in this study. The relation between thermal stability and protein structure was also analyzed by the Circular dichroism spectra. The main results are listed as follows:(1) Theα-CGTase from P. macerans, E. coli and Bacillus subtilis were purified respectively through a combination of ion-exchange and hydrophobic interaction chromatography. Specific activity of the CGTase from these three sources were 233.8 U/mg, 224.6 U/mg and 211.5 U/mg, and the difference of the thermal stability between them was analyzed, after water bathed at 50℃for 30 min, the residual activity ofα-CGTase from P. macerans, E. coli and B. subtilis were 28%,25% and 30%. It can be seen theα-CGTase from the three strains had a similar thermal stability. None of them reached the requirements of the industry application. According to the production ofα-CGTase, we chose theα-CGTase from E. coli as the object of the flowing research.(2) The storage stability ofα-CGTase was studied at the optimum pH and protein concentration, the CGTase from E. coli with pH 6,6.5,7 and protein concentration 1-6mg/mL were prepared. The result showed when protein concentration was 4mg/mL, the stability ofα-CGTase was better than any other concentration after water bathed at 50℃for 15 min, and the residual activity is above 60%. The highest residual activity is 75%, it was found at pH 6.5. Theα-CGTase with the optimum protein concentration and pH was preserved at 4℃,25℃and 37℃, the result showed when preserved at 4℃and 25℃, the T1/2 ofα-CGTase is 108 days and 50 days respectively, when preserved at 37℃, the activity ofα-CGTase was decreased rapidly, the T1/2 is 2.9 days, and the activity was completely lost after a week.(3) Through site-directed mutagenesis to enhance the thermal stability ofα-CGTase. Through the simulation ofα-CGTase spatial structure and the study of homologous enzyme and thermal stability, we identified the mutant site where to introduce the salt bridge. The result showed it formed a salt bridge after one amino acid mutation when analyzed by PyMOL, however the thermal stability of mutant's hadn't been improved much more. After three amino acids mutation, the thermal stability ofα-CGTase was improved noticeably. And the residual activity of mutant a-CGTase was two fold than control after water bathed at 50℃for 30 min. The mutant a-CGTase with the optimum protein concentration and pH was preserved at 4℃,25℃and 37℃, and the T1/2 is 120 days,80 days and 7 days.(4) Through chemical modification to improve the thermal stability ofα-CGTase. Theα-CGTase was cross-linked into a stable macromoleculs by glutaraldehyde. The stability of a-CGTase was improved compared with contol after water bathed at 50℃for 30 min. However the enzyme activity had a large loss during the cross-linking process. Then we cross-linked the preparation of a-CGTase and solved the problem of enzyme inactivation during the cross-link process. But the stability of cross-linked enzyme still did not meet the requirements of industrial application.(5) Through adding specific chemical additives to enhance the thermostability and storage stability of a-CGTase, The effect of additives on the stability of a-CGTase was tested at 50℃. The preparation of a-CGTase was added with chemical additives at an optimized concentration and then they were preserved at 40℃to determine the storage stability. The results indicated that the thermostability of a-CGTase could be enhanced dramatically by the addition of selected additives (Gelatin, Glycerin, CaCl2 and PEG400). A 100% of the enzyme activity was remained after 45 days at 40℃with different additives superimposed at the optimum concentration. Subsequent analysis of the relationship between thermostability and protein structure was also carried out through Circular dichroism (CD) spectra. The CD spectra value of a-CGTase was measured in the far UV (200 nm-250 nm) and near UV (250 nm-320 nm) ranges respectively to detect the change of the secondary and tertiary structure under high temperature. The CD spectra of a-CGTase showed that glycerin can protect the secondary and the tertiary structure of the a-CGTase under high temperature. We studied the protective mechanism of polyol to the enzyme through adding PEG with different molecule weight and glyserol. The result showed that it was the hydroxyl groups in glyceral protected the a-CGTase from degradation.