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1.
Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans ATCC 21783 was concentrated by ultrafiltration and subsequently purified by hydrophobic interaction chromatography on Octyl Sepharose 4 fast flow. The matrix was able to bind selectively to the enzyme at a very low ammonium sulfate concentration of 0.67 M and enzyme desorption was performed by decreasing gradient of the salt. The overall recovery was 80% with 689-fold purity. CGTases derived from four soil isolates and Toruzyme, the commercial preparation of CGTase, also bound to Octyl Sepharose under similar conditions at 0.67 M and eluted at 0.55–0.5 M of ammonium sulfate. Octyl Sepharose chromatography can thus be used as a platform approach for purification of CGTases from various bacterial sources. Long stretches of sequence predominated by hydrophobic amino acids are reportedly present in the starch binding domains of CGTases. Starch binding experiments indicated the binding of the enzymes to the octyl matrix through these domains. 相似文献
2.
Magnetic porous corn starch was prepared as an affinity adsorbent for the efficient and simple scale-up procedure for one-step purification of cyclodextrin glucanotransferase (CGTase) from Bacillus circulans. Magnetic affinity separation enabled isolation of CGTase from cultivation media (volumes between 10 and 400 mL) with ca 60–70% recovery after elution with alkaline buffers containing soluble starch; the enzyme purification factor was 19–25 in different batches. The majority of ballast proteins were removed during the purification process, which shows high selectivity of the affinity material used. 相似文献
3.
Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS-22 strain, which converts starch predominantly to alpha-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media. 相似文献
4.
The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid
protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60°C. Heterologous recombinant
protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that
the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% β-cyclodextrin (CD) and 10% γ-CD.
This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could
potentially serve as an industrial enzyme for the production of β-CD. 相似文献
5.
Bacillusfirmus strain 37 produces the cyclomaltodextrin glucanotransferase (CGTase) enzyme and CGTase produces cyclodextrins (CDs) through a starch cyclization reaction. The strategy for the cloning and expression of recombinant CGTase is a potentially viable alternative for the economically viable production of CGTase for use in industrial processes. The present study used Bacillus subtilis WB800 as a bacterial expression host for the production of recombinant CGTase cloned from the CGTase gene of B. firmus strain 37. The CGTase gene was cloned in TOPO-TA® plasmid, which was transformed in Escherichia coli DH5α. The subcloning was carried out with pWB980 plasmid and transformation in B. subtilis WB800. The 2xYT medium was the most suitable for the production of recombinant CGTase. The enzymatic activity of the crude extract of the recombinant CGTase of B. subtilis WB800 was 1.33 µmol β-CD/min/mL, or 7.4 times greater than the enzymatic activity of the crude extract of CGTase obtained from the wild strain. Following purification, the recombinant CGTase exhibited an enzymatic activity of 157.78 µmol β-CD/min/mL, while the activity of the CGTase from the wild strain was 9.54 µmol β-CD/min/mL. When optimal CDs production conditions for the CGTase from B. firmus strain 37 were used, it was observed that the catalytic properties of the CGTase enzymes were equivalent. The strategy for the cloning and expression of CGTase in B. subtilis WB800 was efficient, with the production of greater quantities of CGTase than with the wild strain, offering essential data for the large-scale production of the recombinant enzyme. 相似文献
6.
Cyclodextrins (CDs) are used in food, pharmaceutical, and chemical industries, as well as agriculture and environmental engineering.
Cyclodextrin glucanotransferase (CGTase) is an important industrial extracellular enzyme which is used to produce CDs and
oligosaccharides. We previously developed a novel yeast-surface CGTase expression system which was used for the production
of CDs from starch. In the present study, we showed that the presence of CDs may increase the ethanol tolerance of microorganisms.
The cell numbers of Saccharomyces cerevisiae and Escherichia coli in the presence of β-cyclodextrin and ethanol were 1,000-fold and 10-fold higher than that without CDs. The yeast strain
with the immobilized CGTase produced 13 g CDs/l and 1.8 g ethanol/l when it was incubated in yeast medium supplemented with
4% starch. The effect of CDs on microorganisms suggests a potential application for the co-production of CDs and ethanol. 相似文献
7.
Two rapid and easy-to-scale-up methods for the purification of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans were developed: affinity precipitation with starch and aqueous two-phase partition. The first method, optimised by a factorial design, gave an 80% CGTase adsorption at 11% starch and 1.6% ammonium sulphate, and a 65% recovery after elution with 10 m M α-cyclodextrin. The purification factor was 17. Aqueous two-phase partition yielded a 72% CGTase recovery in a two-step procedure; CGTase was obtained in the bottom phase with a purification factor of 37. 相似文献
8.
Two chromatographic processes for purification of cyclodextringlucanotransferase (CGTase) from Bacillus sp. 1070 was carried out. The enzyme has been purified into 9.5 times on Butyl-Toyopearl and followed immobilized metal ion chromatography on Cu(II)-Iminodiacetic (IDA)-agarose. By the application of second purification scheme (chromatography on Butyl-Toyopearl and DEAE-Sephacel) the specific activity of CGTase has folded into 13.5 times. The purity of enzyme was shown to be approximately 90% by SDS-electrophoreses data. It was shown that isolated enzyme has two isoelectric points estimated as 5.1 and 5.3. 相似文献
9.
Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific
activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette)
transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli ( E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design
and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular
recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving
six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant
CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted
extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml,
resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of
about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient
protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage. 相似文献
10.
Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans ATCC 21783 was concentrated by ultrafiltration and subsequently purified by hydrophobic interaction chromatography on Octyl Sepharose 4 fast flow. The matrix was able to bind selectively to the enzyme at a very low ammonium sulfate concentration of 0.67 M and enzyme desorption was performed by decreasing gradient of the salt. The overall recovery was 80% with 689-fold purity. CGTases derived from four soil isolates and Toruzyme, the commercial preparation of CGTase, also bound to Octyl Sepharose under similar conditions at 0.67 M and eluted at 0.55-0.5 M of ammonium sulfate. Octyl Sepharose chromatography can thus be used as a platform approach for purification of CGTases from various bacterial sources. Long stretches of sequence predominated by hydrophobic amino acids are reportedly present in the starch binding domains of CGTases. Starch binding experiments indicated the binding of the enzymes to the octyl matrix through these domains. 相似文献
11.
Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount. 相似文献
12.
Expression of Brevibacillus brevis CD162 cyclodextrin glycosyltransferase (CGTase) gene using pET22b(+) vector in Escherichia coli BL21(DE3) resulted in the formation of inactive inclusion bodies under the usual induction conditions. However, by lowering the induction temperature to 30°C and/or adding 0.5 M mannitol as an osmolyte, the formation of insoluble aggregates was prevented and about a 34-fold increase (8.51 U ml –1) in biologically active soluble form was achieved after 6 h induction. The active CGTase enzyme was estimated to comprise as much as 24% of the total soluble proteins. In addition, other polyols such as glycerol, erythritol, xylitol, sorbitol, and arabitol showed similar effects with mannitol on the production of active CGTase enzyme. 相似文献
14.
A major disadvantage of cyclodextrin production is the limited cyclodextrin product specificity of cyclodextrin glycosyltransferase
(CGTase). Here, we described mutations of Asp372 and Tyr89 at subsite −3 in the CGTase from Paenibacillus macerans strain JFB05-01. The results showed that Asp372 and Tyr89 played important roles in cyclodextrin product specificity of CGTase.
The replacement of Asp372 by lysine and Tyr89 by aspartic acid, asparagine, lysine, and arginine resulted in a shift in specificity
towards the production of α-cyclodextrin, which was most apparent for the mutants D372K and Y89R. 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 of α-cyclodextrin, with a concomitant 43% decrease in the production
of β-cyclodextrin when compared to the wild-type CGTase. Thus, the D372K and Y89R single and double mutants were much more
suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The enhanced α-cyclodextrin specificity
of these mutants might be a result of stabilizing the bent conformation of the intermediate in the cyclization reaction. 相似文献
15.
We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known
enzyme β-/γ-CGTase from Bacillus firmus/ lentus 290-3. However, the product specificity of CGTase825-6 differed from that of β-/γ-CGTase. CGTase825-6 produced γ-cyclodextrin
(CD) as the main product, but degradation of γ-CD was observed with prolonged reaction. The product specificity of the enzyme
was positioned between γ-CGTase produced by Bacillus clarkii 7364 and B. firmus/ lentus 290-3 β-/γ-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671
residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein
structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8
for γ-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool
to produce γ-CD and to study the differences of producing mechanisms between γ-CD and β-CD. 相似文献
16.
Cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19) is an industrially important enzyme, which is used to produce cyclodextrins
(CDs). In this research, we report the use of experimental factorial design to find the best conditions of pH and temperature
for CGTase production by Bacillus circulans var. alkalophilus. The optimized calculated values for the tested variables were, respectively, pH 9.7 and temperature 36 oC, with a CGTase activity of 615 U mL −1. The CGTase production was further studied with the optimized process parameters on submerged cultivations (SC) and solid-state
cultivations (SSC) using soybean industrial fibrous residue (SIFR). The maximum CGTase activity obtained on SC was 1,155 U mL −1 under aerobic conditions. Cell growth and CGTase synthesis in SSC using SIFR as substrate was excellent, with CGTase activity
of 32,776 U g (SIFR) −1. These results strongly support the use of SIFR for CGTase production since it is a non-expensive residue. 相似文献
17.
Cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was adsorbed on the ion exchange resin Amberlite IRA-900. The optimum conditions for the immobilization of the CGTase
were pH 6.0 and 600 U CGTase/g resin, and the maximum yield of immobilization was around 63% on the basis of the amount ratio
of the adsorbed enzyme to the initial amount in the solution. Immobilization of CGTase shifted the optimum temperature for
the enzyme to produce transglycosylated xylitol from 70°C to 90°C and improved the thermal stability of immobilized CGTase,
especially after the addition of soluble starch and calcium ions. Transglycosylated xylitol was continuously produced using
immobilized CGTase in the column type packed bed reactor, and the operating conditions for maximum yield were 10% (w/v) dextrin
(13 of the dextrose equivalent) as the glycosyl donor, 10% (w/v) xylitol as the glycosyl acceptor, 20 mL/h of medium flow
rate, and 60°C. The maximum yield of transglycosylated xylitol and productivity were 25% and 7.82 g·L −1·h −1, respectively. The half-life of the immobilized CGTase in a column type packed bed reactor was longer than 30 days. 相似文献
18.
A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon
Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme
displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant
activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation
activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes
a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family.
The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter
genes have been sequenced ( malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme.
The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed. 相似文献
19.
Cells of the alkalotolerant producer of cyclodextrin glucanotransferase (CGTase) Bacillus circulans ATCC 21783 were used as a model for preparing of magnetic biocatalysts applied for CGTase synthesis in batch and semicontinuous processes. The cell immobilization was carried out with four types of magnetic nano- and microparticles: magnetite microparticles (1–5 μm), entrapped in agar gel beads with bacterial cells (AM-biocatalyst); silanized magnetite (20–40 nm) covalently bound on the cell surface (SM-biocatalyst); and alkaline and citrate ferrofluids (10–20 nm), attached on the cell wall by an ionic interaction (FF-alkaline and FF-citrate biocatalyst). The highest CGTase production was achieved after 96 h of semicontinuous process using SM-biocatalysts (particularly, these composed of 80 mg silanized magnetite and 140 mg bacterial cells) when the specific enzyme activity was 8.4-fold higher compared to that of free cells. Cells modified with magnetic alkaline and citrate ferrofluids exhibited 2.19- and 1.55-fold increase of the specific CGTase activities. Magnetic nanoparticles linked on the cell walls by ionic interactions were partially released during the cultivation, while the covalent bond between the activated magnetite and the cells was very stable. The data obtained demonstrate convincingly the effect of the magnetic technologies for an effective enzyme production. 相似文献
20.
The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25 %) and a mixture of peptone/yeast extract (1 %) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg 2+, Zn 2+, Al 3+ and K +, while it was slightly enhanced by 5 and 9 % with Cu 2+ and Fe 2+ metal ions, respectively. 相似文献
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