| 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, cosmetic, agriculture, etc, CGTase has become the focus of scientific research nowadays.To overcome the low CGTase productivity of wild strains, the overexpression of cgt gene in Escherichia coli has been expected. However, previous reports showed that the CGTases expressed in E. coli were usually accumulated in the cytosol as inactive inclusion bodies and/or the periplasm as soluble forms, which limited its industrial utilization. Thus, it is highly desirable to extracellular production of the recombinant CGTase in E. coli. In addition, a major disadvantage of cyclodextrin production by CGTase is that all known wild-type CGTases produce a mixture ofα-,β-, andγ-cyclodextrins, which were not favorable to its downstream processing. In the present study, the gene encodingα-CGTase from Paenibacillus macerans JFB05-01 was expressed in E. coli and the recombinant enzymes were targeted into the culture medium of E. coli through some culture strategies. The product specificity of CGTase was also analyzed and improved. The main results are listed as follows: 1. The cgt gene encodingα-CGTase from P. macerans JFB05-01 was cloned into the downstream of pelB signal sequence in a vector pET-20b(+) and pET-22b(+), respectively. Thus, the expression plasmids pET-20b(+)/cgt and pET-22b(+)/cgt were constructed and transformed into the host E. coli BL21(DE3).The culture conditions for extracellular production of the recombinantα-CGTase in E. coli BL21(DE3)(pET-20b(+)/cgt) were optimized in shaking flasks. The E. coli cells were cultured in TB medium at 25°C. After induction with 0.01 mM isopropylβ-D-thiogalactoside (IPTG) for 90 h, the activity ofα-CGTase in the culture medium achieved 22.5 U/ml, which was about 43-fold higher than that of the parent strain, P. macerans JFB05-01. In E. coli BL21(DE3)(pET-22b(+)/cgt), higher IPTG concentration (0.2 mM) was needed to induce the expression ofα-CGTase and, after 90h of induction, the activity ofα-CGTase achieved only 17.5 U/ml. Thus, E. coli BL21(DE3)(pET-20b(+)/cgt) was more suitable for the extracellular production of the recombinantα-CGTase. To the best of our knowledge, this is the first report on the extracellular production ofα-CGTase in E. coli.2. The optimum temperature for extracellular production of the recombinantα-CGTase in E. coli was 25°C. However, at 25°C, only very few recombinantα-CGTase was secreted into the culture medium within first 50 h, while most were accumulated in the periplasmic space.The extracellular secretion of the recombinant enzyme was suppressed at higher temperatures. The possible reason was that the precursor protein with a signal peptide aggregated as inclusion bodies in the inner side of inner membrane due to too high synthetic rate, which might subsequently block protein translocation across the inner membrane. Extracellular production of the recombinantα-CGTase could be stimulated by raising the induction temperature at the later stage of culture. The maximum extracellular production was achieved when the temperature was shifted to 30°C after 32 h of induction at 25°C. After 90 h, the activity was 32.5 U/ml, which is 1.45-fold higher than that at 25°C constant.3. The addition of glycine enhanced the extracellular secretion of the recombinantα-CGTase and markedly shortened the culture time. Especially in the culture with 150 mM glycine, theα-CGTase activity in the culture medium reached 23.5 U/ml at 40 h, which was 11-fold higher than that of the control culture at the same time. The productivity of the recombinant enzyme also reached the maximum value of approximately 0.60 U/ml/h at 36 h, which was 2.5-fold higher than that of the control culture at 80 h. The potential mechanism is considered to be the significantly increased membrane permeabilities of E. coli cells.However, glycine inhibited the growth of E. coli cells, which prevented further improvement in overall enzyme extracellular productivity. Ca2+ could remedy cell growth inhibition induced by glycine as demonstrated by significantly increased cell number and viability, reduced cell autolysis, and repaired cell morphology. In the culture with 150 mM glycine and 20 mM Ca2+, theα-CGTase activity in the culture medium reached 35.5 U/ml at 40 h of culture, which was 1.5-fold higher than that in the culture with glycine alone. The productivity reached the maximum value of approximately 0.90 U/ml/h at 36 h, which also was 1.5-fold higher than that in the culture with glycine alone.4. The recombinantα-CGTase with a C-terminal His-tag could be purified to homogeneity through a nickel affinity chromatography, but the typical yield of purified enzyme was very low. The recombinant and native enzymes were purified by a combination of ion-exchange and hydrophobic interaction chromatography and the relative high yield was obtained.The purifiedα-CGTase was a monomer in solution. The optimum cyclization reaction temperature of the recombinantα-CGTase was 45°C. It retained 50% of its initial cyclization activity after incubation for 8 h at 40°C, 1.25 h at 45°C, and 0.5 h at 50°C. The nativeα-CGTase had higher optimum temperature (50°C) and thermostability (t1/2, 50°C=0.8 h). The recombinant and native enzymes both showed the highest cyclization activity at pH 5.5 and were quite stable in the pH ranging from 6 to 9.5 and 6 to 10, respectively. The metal cofactor was not required for the function ofα-CGTase. However, the cyclization activity ofα-CGTase was inhibited by Hg2+, Ni2+, Fe2+ or Co2+, while the enzyme could be activated by some bivalent metal ions, especially Ca2+, Ba2+ or Zn2+.At the initial stage of enzyme conversion, the recombinant and nativeα-CGTases producedα-cyclodextrin as a main product. At the later stages, the proportion ofβ-cyclodextrin increased. Finally,β-cyclodextrin was the main product. The recombinant enzyme had a higher preference forα-cyclodextrin production than the native enzyme.The kinetics of theα-CGTase-catalyzed cyclization reaction can be fairly well described by the Hill equation and the Hill coefficient was higher than one, indicating the positive cooperativity of substrate binding.5. The mutations of Asp372 and Tyr89 at subsite ?3 in P. macerans CGTase could change cyclization activities and cyclodextrin product ratios, which indicated that the two residues at subsite ?3 played important roles in cyclodextrin product specificity and subsites ?3 was a key site for cyclodextrin product specificity.The replacement of Asp372 by lysine and Tyr89 by arginine enhanced significantlyα-cyclodextrin specificity of CGTase. Furthermore, the changes in cyclodextrin product specificity for the single mutants D372K and Y89R could be combined in the double mutant D372K/Y89R, which displayed a 1.5-fold increase in the production ofα-cyclodextrin, with a concomitant 43% decrease in the production ofβ-cyclodextrin when compared to the wild-type CGTase.These mutants with higherα-cyclodextrin specificity were more suitable for the industrial production ofα-cyclodextrin than the wild-type enzyme.6. Amino acid residue is one of the main residues located near subsites ?3. The nature of residue 47 has a clear discrimination between the different groups of CGTase, suggesting that the identity of residue 47 may affect cyclodextrin product specificity.The mutations of Lys47 in P. macerans CGTase could change cyclization activities and cyclodextrin productions, indicating the residue had important roles in cyclization reaction and cyclodextrin product specificity.All the mutations reducedα-cyclodextrin forming activity of CGTase, suggesting that Lys47 was very important forα-cyclization reaction, while the increase inβ-cyclodextrin forming activity could be achieved. Especially, the mutations of Lys47 into threonine, serine, or leucine converted P. macerans CGTase fromα-type intoβ/α-type. As a result, all the mutants displayed a shift in product specificity towards the production ofβ-cyclodextrin. Thus, these mutants were more suitable for the industrial production ofβ-cyclodextrin than the wild-type enzyme.
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