| Compared with α-CD and β-CD, γ-CD can be used for embedding the larger guest molecular with higher molecular weight, because of a larger cavity and higher solubility. So there will be a wide range of application prospects of γ-CD in the future. γ-CD was produced by cyclodextrin glycosyltransferase(CGTase, EC2.4.1.19) catalyzing with starch. In this research, the enzymatic properties of CGTase from Bacillus sp.G-825-6 were studied. The reaction conditions for producing γ-CD were discussed. Moreover, site-directed mutagenesis of CGTase was made and the enzymatic properties of mutagenesis was investigated. This study will provide reference for industrial production of γ-CD.Study on the purification of CGTase and the enzymatic properties. CGTase was purified by starch adsorption method. The purification result was verified by SDS-PAGE. Results showed that the purification ratio reached to 9.88 and recovery rate was 34.20% after crude CGTase purification. The single band on the electrophoresis map indicated electrophoresis pure.grade target enzyme was received. The results of enzymatic properties of the purified CGTase showed that the optimum temperature and pH value of the CGTase was 50℃ and 8.5, respectively; it is relative thermal stable at 40~50℃ and pH stable within the pH 7~10; the relative residual enzyme activity was more than 80% within the same pH; the enzyme activity could be activated by Ca2+ and Mg2+ and inhibited by Ni2+, Cr2+ and Cu2+, especially by Ni2+.Study on the effect of substrates on the γ-CD yield. The effects of substrate with different molecular weight and different chain length on the γ-CD yield were explored. When corn starch, soluble starch, maltodextrin(DE=15) was used as substrate, γ-CD yield reached to 7.91%, 6.21%, 5.11%, separately. Which showed that the less molecular weight of the starch leads to decreasing of γ-CD yield. If the starch substrate was debranched by isoamylase or the chain length of starch was lengthen by 4αGTase, then γ-CD yield was increased to10.69%, 10.43% respectively, indicating that an appropriate molecular weight and chain length of substrate are very important for the production of γ-CD.Study on the reaction conditions on the γ-CD yield. Corn starch was pretreated by 4αGTase, the effect of 4αGTase dosage, CGTase dosage, CGTase reaction time and substrate concentration on γ-CD yield was investigated. The optimal conditions for the γ-CD production was deserved as 5% of substrate concentration, 4 U/g starch of 4αGTase, 8 U/g starch of CGTase and 30 h reaction time, and the γ-CD yield is 12.83% under this condition.Structure analysis of CGTase. The multi amino acid sequence of CGTases from different strains was aligned, and the three dimensional structure of γ-CGTase from Bacillus sp.G-825-6 was compared with B.circulans 251 β-CGTase by Pymol. Nine subsites in substrate binding groove was analyzed. Results showed that the sequence similarity of Bacillus sp.G-825-6 γ-CGTase with B. firmus/lentus 290-3 and Bacillus clarkia 7364 was 95% and 70%, separately. Y211 was highly conserved in all strains, the three dimensional structure of Bacillus sp.G-825-6 γ-CGTase was similar as B.circulans 251β-CGTase. Amino acid in the substrate binding groove nearby-7 subsite was important for the product specificity. And Y211 was located nearby-7 subsites.Mutagenesis of CGTase was constructed and the enzymatic property of mutant was studied. Tyrosine 211 of CGTase was site-direct mutated into leucine by PCR method and the sequence was verified. The enzymatic properties study on mutant showed the similar properties with wild type. Moreover, the mutant was used for γ-CD production, the ratio of γ-CD in total cyclodextrins was increased from 68.40% to 96.40%, which indicated high product specificity of mutant Y211L. |
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