Product Specificity And Preliminary X-ray Analysis Of γ-cyclodextrin Glycosyltransferase From Alkalophilic Bacillus Clarkii 7364

Cyclodextrin Glycosyltransferase (CGTase, EC2.4.1.19) is an extracellular enzyme capable of converting starch or starch derivatives into cyclodextrins through cyclization. Due to their capability of forming inclusion complexes with various hydrophobic molecules, cyclodextrin have been widely used in many industries such as food, pharmaceuticals and so on. However, all wild type CGTases produce the mixture ofα,β,γ-cyclodextrins, and the isolation of pure cyclodextrin from the mixture requires a series of steps which lead to the increased cost of cyclodextrins.The molecular mechanism of product specificity from B. clarkii 7364γ-CGTase was investigated in this research. Theγ-CGTase gene from B. clarkii 7364 was expressed in E. coli BL21 (DE3), and the relationship between the lack of 6 amino acid at subsite -7 and theγ-CGTase specificity was described. Additionally, the three-dimensional structure of B.clarkiiγ-CGTase was resolved for further study the relationship betweenγ-CGTase and product specificity. The main results were as followed:(1) The B.clarkii 7364γ-cgt gene was cloned into the downstream of OmpA peptide signal sequence in vector pET-20b (+). The expression plasmids pET-20b (+)/γ-cgt was transformed into E.coli BL21 (DE3). The recombinant E. coli cells were cultured in TB medium at 25°C, 200rpm. After 72 h of culture, the extracellular activity ofγ-CGTase achieved 4 U/ml.(2) The recombinantα-CGTase with a C-terminal His-tag could be purified to homogeneity through a nickel affinity chromatography, but the recombinant enzyme could not combine in the chromatography. Thus, the recombinant enzyme was purified by hydrophobic interaction chromatography and ion-exchange chromatography and the high purified protein was obtained.(3) Using overlapping PCR, 6 amino acids were inserted into subsite -7 ofγ-CGTase. The mutantγ-CGTase gene was ligated with pET-20b (+) and expressed in E. coli BL21 (DE3). The recombinant mutant and wild typeγ-CGTase were used to convert soluble starch into cyclodextrins (CDs). HPLC analysis results showed that, compared to wild CGTase, theγ-CDs produced by mutant enzyme decreased from 76.0% to 12.5%, while the ratio ofα- andβ-CDs increased from 8.7% and 15.2% to 37.5% and 50%.(4) Using sessile drop vapor diffusion synthesis, crystals were cultured and screened at 20°C at different conditions. After a week, theγ-CGTase crystal had come out in many conditions. After optimized the initial screen condition, two crystals was obtained for X-ray diffraction. In the Shanghai Synchrotron Radiation Facility, one set data of 1.65 ? was obtained and processed using HKL2000. Then using molecular replacement technique, the crystal structure ofγ-CGTase was resolved.(5)γ-CGTase crystal structure was aligned with other CGTase structures from PDB database, it was shown thatγ-CGTase has small ring in subsite -7, whileβ-CGTase has larger ring structure. The small ring provided more space for glucose combination and was thus advantageous for formingγ-CD. But when the 6 amino acids were inserted into the subsite -7 of wildγ-CGTase, the space to bind with glucose reduced and consequently resulted in less γ-CD production.