| a-cyclodextrin glycosyltransferase, which belongs to the a-amylase family, can catalyze starch into cyclodextrins by intramolecular transglycosylation reaction. Cyclodextrin is composed of several glucose residues, which has hydrophilic outside and inside hydrophobic. Because its special space structure, cyclodextrins can form inclusion complexes with many compounds. As a result, it has a widespread applica-tions in food, cosmetic, medicine, textile industry, environmental protection and so on. Consequently,α-CGTase has been paid more and more attention for its applications.Two problems were met during the a-cyclodextrin glycosyltransferase research: One is the low or insoluble expression of the gene in Escherichia coli; The other lies in that a-CGTase has a low product specificity, which also increased the cost for downstream processing. In present study, the gene encoding a-CGTase from Bacillus sp.602-1was cloned and recombinant plasmid with expression vectors were constructed. engineering bacteria were obtained. We optimized the conditions that can increase the active recombinant enzyme releasing. By directed evolution method and library screenning. a mutant strain that had higher product specificity were obtained. The main results are listed as following:1. The gene of α-CGTase from Bacillus sp.602-1was inserted into expression vectors pET22(b+). PQE30. respectively. and then recombinant plasmids pET22(b+)/cgt, PQE30/egt were constructed, and then transformed into the host E.coliBL21(DE3), E.coliM15. Finally we got the engineering bacteria E.coliBL21(DE3)(pFT22(b+)/cgt) and E.coliM15(PQE30/cgt).2. The strain of E.coliBL21(DE3)(pET22(b+)/cgt) were cultured in shaking flasks to produce the recombinant α-CGTase. The optimum expression conditions in the process of fermentation were studied. As a result. the best conditions for the extracellular production of recombinant α-CGTase were determined as follows:IB medium.0.01mmol/L IPTG.1150mmol/L Glvcine and0.25mol/L mannitol. and16℃the α-CGTase activity in the culture medium was11702U/mL In auto-induction medium.1.2g/L glucose and lg/L. lactose were the best concentration for the extracellular expression, and the activity reached10092U/mL.3. The optimal conditions for the expression of α-CGTase from E.coliM15(PQE30/cgt) using the IPTG induction were as follows:TB medium,0.01mmol/L IPTG,16℃, with the activity of5209U/mL culture. In addition, glycine and mannitol were found to be able to help secretion of the α-CGTase into the culture. The activity in an optimized auto-inducing medium, that of TB medium containing1.2g/L glucose and3.0g/L lactose, achieved8635U/mL.4. The recombinant expression plasmid pET22(b+)/cgt was transformed into expression bacteria E.coliOrigamiB(DE3). By SDS-PAGE analysis, we found there were less inclusion body formed than that in E.coliBL21(DE3)(pET22(b+)/cgt),less secretion occurred. Thus, in this paper, E.coliBL21(DE3) was the best expression host we chose.5. The recombinant α-CGTase catalyzed starch into cyclodextrins and the products ratios were analyzed by HPLC. The propotion of α-,β,γ-CD were74%,21.7%,4.3%. respectively. And as a result, an α:β ratio was3.4:1. In relative to native α-CGTase, which converted starch into CDs with the peak ratio of α:β was2.4. Thus the recombinant α-CGTase showed better industrial perspective in the α-CD production.6. The recombinant α-CGTase converted raw starch into CDs. Firstly. we liquefyed6%corn starch by isoamylase. and then400U/g α-CGTase was added into this system. After24h incubation, the ratio of α:β was2.3:1.7. We constructed the mutations library by Error PCR. which was about70.000strains. By preliminary screening, we chose153strains for further analysis. Fortunately, we successfully screened a mutant strain which had a higher product specificity, we named95. After incubation, the ratio of α:β was7.3. while the recombinant α-CGTase was3.3. We analyzed its sequence that it had two amino acides changed:Y16711, A536V. respectively.8. The mutation of167subsite was changed from histidine into tyrosine by reverse mutation. after conversion. the ratio of α:β falled to2.8:On the other hand. we changed valine into alaninc with α:β ratio of6.7:1. Consequently. we inferred that Y167H was important for higher product specificity, and A536V did not distinctly influence its product specificity. In addition, we found that the mutation of167lies in-6subsite, thus we inferred this subsites may be important for product specificity.9. In order to search the best amino acid at167subsite, we constructed saturation mutgenesis library. From it, we screened15amino acids, which contains nonpolar, polarity, acidity and alkalinity. By HPLC analysis, at present Histidine was the optimum amino acid.10. The mutation a-CGTase was cultivated in fermentation mudium, and then purified with a nickel affinity chromatography. The specific activity of the purified enzyme reached54230.7U/mg protein, and7.3fold purification was obtained with a yield of25.2%. We also analyzed the main properties of mutation a-CGTase:the optimum temperature and pH was50℃and7.4, respectively, and the activity reached11705U/mL. However, the cyclization activity of a-CGTase was strongly inhibited by Cu2+. Fe2+or Zn2+. K+and Na+did not make an obvious effect on enzyme activity. In addition. Ba2+and Ca2+did not obviously activate its cyclization activity. |
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