CGTase Activity Research Articles

Immobilization of Cyclodextrin glycosyltransferase onto three dimensional- hydrophobic and two dimensional- hydrophilic supports: A comparative study.

Cyclodextrin glycosyltransferase (CGTase) degrades starch into cyclodextrin via enzymatic activity. In this study, we immobilize CGTase from Thermoanaerobacter sp. on two supports, namely graphene nanoplatelets (GNP) consisting of short stacks of graphene nanoparticles and a calcium-based two-dimensional metal organic framework (Ca-TMA). The uptakes of CGTase on GNP and Ca-TMA reached 40 and 21mg g-1 respectively, but immobilized CGTase on Ca-TMA showed a higher specific activity (38 U mg-1 ) than that on GNP (28 U mg-1 ). Analysis of secondary structures of CGTase, shows that immobilization reduces the proportion of β-sheets in CGTase from 56% in the free to 49% and 51.3% for GNP and Ca-TMA respectively, α-helix from 38.5% to 18.1 and 37.5%, but led to increased β-turns from 5.5 to 40% and 11.2% for GNP and Ca-TMA, respectively. Lower levels of conformational changes were observed over the more hydrophilic Ca-TMA compared to hydrophobic GNP, resulting in its better activity. Increased β-turns were found to correlate with lower β-CD production, while more β-sheets and α-helix favored more β-CD. Reusability studies revealed that GNP retains up to 74% of initial CGTase activity, while Ca-TMA dropped to 33% after eight consecutive uses. The results obtained in this work provide insight on the effect of support's surface properties on CGTase performance and can assist in developing robust CGTase-based biocatalysts for industrial application.

Cyclodextrin Glucanotransferase of Alkalophilic Strain <i>Caldalkalibacillus mannanilyticus</i> IB-OR17-B1

Extracellular cyclodextrin glucanotransferase (CGTase, K.F.2.1.19) was characterized for the first time in a strain of bacteria of the species Caldalkalibacillus mannanilyticus IB-OR17-B1. The enzyme was isolated from the culture supernatant using ultrafiltration and affinity adsorption on corn starch. The specific activity of the CGTase was increased in 18-fold as a result of purification with the enzyme yield 56%. The molecular mass of the purified enzyme was 70 kDa according to the denaturing electrophoresis in polyacrylamide gel. The CGTase of C. mannanilyticus IB-OR17-B1 demonstrated a maximal cyclizing activity under pH 8 and temperature 60°C, respectively, and it was stable in the pH range 7–10 and temperatures ≤70°C. The thermal stability of the enzyme under 70°C increased by 10–15% in the presence 5–10 mM of calcium and magnesium salts. The cations of Ag+, Cu2+, Zn2+, Fe2+ and Fe3+ in concentration 5 mM inhibited a CGTase activity by 90, 26, 23, 18 and 11%, respectively. The purified CGTase under optimal conditions and enzyme-substrate ratio 1 U/g converted a potato starch during 24 h to mixture of α-, β- and γ-cyclodextrins with mass ratio 38.8 : 52.6 : 8.6 and yield 42%.

Binding Specificity of a Novel Cyclo/Maltodextrin-Binding Protein and Its Role in the Cyclodextrin ABC Importer System from Thermoanaerobacterales.

Extracellular synthesis of functional cyclodextrins (CDs) as intermediates of starch assimilation is a convenient microbial adaptation to sequester substrates, increase the half-life of the carbon source, carry bioactive compounds, and alleviate chemical toxicity through the formation of CD-guest complexes. Bacteria encoding the four steps of the carbohydrate metabolism pathway via cyclodextrins (CM-CD) actively internalize CDs across the microbial membrane via a putative type I ATP-dependent ABC sugar importer system, MdxEFG-(X/MsmX). While the first step of the CM-CD pathway encompasses extracellular starch-active cyclomaltodextrin glucanotransferases (CGTases) to synthesize linear dextrins and CDs, it is the ABC importer system in the second step that is the critical factor in determining which molecules from the CGTase activity will be internalized by the cell. Here, structure-function relationship studies of the cyclo⁄maltodextrin-binding protein MdxE of the MdxEFG-MsmX importer system from Thermoanaerobacter mathranii subsp. mathranii A3 are presented. Calorimetric and fluorescence studies of recombinant MdxE using linear dextrins and CDs showed that although MdxE binds linear dextrins and CDs with high affinity, the open-to-closed conformational change is solely observed after α- and β-CD binding, suggesting that the CM-CD pathway from Thermoanaerobacterales is exclusive for cellular internalization of these molecules. Structural analysis of MdxE coupled with docking simulations showed an overall architecture typically found in sugar-binding proteins (SBPs) that comprised two N- and C-domains linked by three small hinge regions, including the conserved aromatic triad Tyr193/Trp269/Trp378 in the C-domain and Phe87 in the N-domain involved in CD recognition and stabilization. Structural bioinformatic analysis of the entire MdxFG-MsmX importer system provided further insights into the binding, internalization, and delivery mechanisms of CDs. Hence, while the MdxE-CD complex couples to the permease subunits MdxFG to deliver the CD into the transmembrane channel, the dimerization of the cytoplasmatic promiscuous ATPase MsmX triggers active transport into the cytoplasm. This research provides the first results on a novel thermofunctional SBP and its role in the internalization of CDs in extremely thermophilic bacteria.

Design of a Cyclodextrin Bioproduction Process Using Bacillus pseudofirmus and Paenibacillus macerans

Cyclodextrin (CD) drug delivery systems offer the potential to enhance the desired physicochemical properties and pharmacokinetic parameters of drugs while maintaining their safety. Cyclodextrin-glucosyl-transferase (CGTase) is amongst the most important enzymes used in CD biosynthesis. However, the bioproduction of CDs still faces challenges in terms of optimization and process complexity. This study proposes a novel CD bioproduction system in a batch mode to increase yield and reduce costs. Two bacterial strains were selected: the alkalophilic Bacillus pseudofirmus DSM2517 strain and the neutrophilic Paenibacillus macerans DSM1574 strain. Three different culture media, two temperatures (30 °C and 37 °C), and three scales (shake flasks 20 mL and 100 mL, and bioreactor 3.2 L) were evaluated with respect to bacterial growth kinetics, protein production, and CGTase biosynthesis and activity for β-CD production. Bacterial growth was monitored by measuring optical density (OD600 nm), while CGTase activity was assessed by measuring β-CD production directly in the medium after filtration or in samples after concentration (using a Vivaspin 500® ultrafiltration spin column with a 10 kDa cut-off). β-CD quantification was performed using the phenolphthalein colorimetric method and HPLC. The best conditions for combined growth and protein production, for both microorganisms, in shake flasks were achieved with a medium containing 2% dextrin as the carbohydrate source. Scale-up to the bioreactor displayed improved growth kinetics for both bacteria and higher protein production and CGTase activity for Paenibacillus macerans.

Enhanced excretion of recombinant cyclodextrin glucanotransferase and cell stability of immobilized recombinant Escherichia coli by reducing tryptone concentration

AbstractBACKGROUNDRecombinant protein excretion has become a mainstream strategy in reducing downstream processing costs. However, recombinant protein excretion is often bottlenecked by cell lysis and plasmid stability. In the present study, recombinant Escherichia coli cell immobilization was performed on a hollow fiber membrane. Tryptone concentration in the expression medium (Super Optimal Broth), which was used as a nitrogen source, was varied between 5 and 20 g L−1 to enhance the excretion of recombinant cyclodextrin glucanotransferase (CGTase), plasmid stability, and resistance of cell lysis.RESULTSThe immobilized cells with 5 g L−1 concentration of tryptone improved the plasmid stability with 119% improvement and 69% reduction of cell lysis without remarkably altering the excretion of CGTase compared with the tryptone concentration of 20 g L−1. The immobilized cells showed a 2‐fold increase in excretion of CGTase, a 45% reduction in cell lysis, and a 172% gain in plasmid stability in comparison with the free cells. Moreover, the doubling time increased to 58 and 5 h for the immobilized and free cells, respectively. The immobilized cells recorded 2301.62 U mL−1 of cumulative CGTase activity through seven fermentation cycles using the untreated membrane, marking their excellent reusability.CONCLUSIONThis new technique of recombinant protein expression utilizing an immobilized cell system under low tryptone concentration is an outstanding approach to improve recombinant CGTase excretion and plasmid stability with low cell lysis. © 2023 Society of Chemical Industry (SCI).

Characterization of an Amylolytic Enzyme from Massilia timonae of the GH13_19 Subfamily with Mixed Maltogenic and CGTase Activity.

This work reports the characterization of an amylolytic enzyme from the bacteria Massilia timonae CTI-57. A gene encoding this protein was expressed from the pTrcHis2B plasmid in Escherichia coli BL21 Star™ (DE3). The purified protein had 64kDa, and its modeled structure showed a monomer with the conserved α-amylases structure composed of the domain A with the characteristic (β/α)8-barrel, the small domain B, and the domain C with an antiparallel beta-sheet. Phylogenetic analysis demonstrated that the expressed protein belongs to the GH13_19 subfamily of glycoside hydrolases. The ions Ca2+, Mn2+, Na+, Mg2+, Mo6+, and K+ did activate the purified enzyme, while EDTA and the ions Fe2+,Hg2+, Zn2+, and Cu2+were strong inhibitors. SDS was also a strong inhibitor. The enzyme's optimal pH and temperature were 7.0 and 45°C, respectively, and its Tm was 62.2°C. The KM of the purified enzyme for starch was 13mg/mL, and the Vmax was 0.24μmol of reducing sugars released per min. The characterized enzyme presented higher specificity for maltodextrin and starch and produced maltose as the main starch hydrolysis product. This is the first characterized maltose-forming amylolytic enzyme from the GH13_19 subfamily. The purified enzyme produced β-cyclodextrin from starch and maltodextrin and could be considered a cyclodextrin glucanotransferase (CGTase). This is the first report of a GH13_19 subfamily enzyme with CGTase activity.

Development of an enzymatic encapsulation process for a cycloamylose inclusion complex with resveratrol

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cycloamyloses (CAs), which are large cyclic glucans, and subsequently transforms them to α-, β-, and γ-cyclodextrins. We developed a novel encapsulation process based on the cyclization activity of CGTase and applied it to the formation of CA inclusion complexes with resveratrol (RVT), which has limited bioavailability due to its low water solubility. The encapsulated RVT (CA-RVT) was purified using preparative high-performance liquid chromatography. The water solubility of CA-RVT was 6,000-fold higher than that of RVT. CA-RVT in water demonstrated 98% stability for 1 week at 4 °C. According to radical scavenging activity and anti-inflammatory assays, CA-RVT in aqueous solution exhibited similar activities as an equal amount of RVT in dimethyl sulfoxide, suggesting the limited solubility of RVT can be overcome through CA encapsulation by CGTase, thus enhancing its nutraceutical value as a functional ingredient in the food industry.

Batch CGTase Production with Free and Immobilized <i>Bacillus firmus</i> Strain 37 in Bovine Bone Charcoal

The present study aimed to study the batch production of CGTase (cyclomaltodextrin-glucanotransferase) with Bacillus firmus strain 37 free and immobilized in bovine bone charcoal in batch mode and in a fluidized bed batch reactor, respectively. The bovine bone charcoal is an innovative support material for the immobilization of microorganisms’ producers of enzymes and the use of this microbial support allows its reuse to a significant cost reduction of the process. The batch fermentation with free cells was investigated for 96 h and reached a CGTase activity equal to 0.77 U/mL. When the microorganism was immobilized on bovine bone charcoal (7 g) and cultivated in fluidized bed batch reactor with air supplementation (1 volume of air/volume of medium * minute), the same activity could be achieved in 24 h. The results of enzymatic activity achieved show the potential of CGTase production in a short time with Bacillus firmus strain 37 immobilized in bovine bone charcoal matrix and using air supplementation in the production medium.

Site-saturation mutagenesis of proline 176 in Cyclodextrin Glucosyltransferase from Bacillus sp. Y112 effects product specificity and enzymatic properties

Based on the analysis of amino acid sequence and simulated structure, saturation mutagenesis was performed to explore the role of the site p176 of cyclodextrin glucosytransferase (CGTase) from Bacillus sp. Y112. Compared to the wild-type, mutant P176G showed 10.4 % improvement in conversion from starch to cyclodextrins (CDs), whose β-CD yield increased by 6% and α-CD yield decreased by 8%. Mutants P176L and P176I were increased by 7.9 % and 9.4 % on CDs production, indicating replacement of hydrophobic amino acids significantly improved in cyclization activity. Kinetics studies indicated the substrate affinity of P176G and P176K were increase by 13 % and 14 %, and the catalytic efficiency of P176K was increase by 14 %. In addition, the optimal temperature of mutants transformed from 50℃ to 40℃ and the optimal pH shifted from 10.0 to 8.0. These results indicate that the site P176 plays a critical role in catalytic activity, product specificity and enzymatic properties of CGTase.

Production of the Enzyme Cyclodextrin Glycosyltransferase Using Different Fermentation Techniques

Cyclodextrins produced by cyclodextrin glycosyltransferase (CGTase) are widely used in the pharmaceutical industry to improve the solubility of drug substances as well as protect them against oxidation. The use of this enzyme in the cosmetics industry is also significant. CGTase is an enzyme that belongs to the α-amylase family, which is part of the group of non-Leloir glycosyltransferases. Enzyme-catalysed transglycosylation reactions may involve cyclization, coupling and disproportionation processes. The enzyme CGTase is mostly used to produce cyclodextrins (CDs). CGTase can produce α-, β- and γ-CDs during transglycosylation reactions, depending on the number of glucopyranose units involved (6, 7 or 8). The enzyme CGTase can also be used for enzymatic bioconversion, e.g., in the development of alternative sweeteners, where the bitter aftertaste of the product is reduced during the enzymatic bioconversion of steviol glycosides, thereby obtaining an even sweeter and more advantageous material. In our research, the enzyme CGTase was produced using different fermentation techniques to compare the activity and amount of CGTase produced by each process and optimize the subsequently planned scale-up. In our studies, the strain DSM 13 of Bacillus licheniformis was used, which produced CGTase extracellularly. During the experiments the batch, fed-batch and semi-continuous fermentation techniques were compared in terms of enzymatic production. All cultivation processes were carried out in a desktop lab scale fermenter.

Improved production of cyclodextrin glycosyltransferase from Bacillus stearothermophilus NO2 in Escherichia coli via directed evolution

Cyclodextrin glycosyltransferases (CGTases) are widely used in starch deep processing, so reducing their cost by improving their production is of significant industrial interest. The CGTase from Bacillus stearothermophilus NO2 possesses excellent catalytic properties but suffers from low production in E. coli. In this study, directed evolution was used to create three point mutants (I631T, I641T and K647E) that were produced in E. coli with shake-flask yields 1.7-, 2.1-, and 2.2-fold higher than that of wild-type, respectively. The wild-type and K647E were then produced in a 3-L fermenter. The CGTase activity of the K647E (1904 U mL-1) was 2.0-fold higher than that of the wild-type. The K647E fermentation supernatant could be used directly to prepare 2-O-α-D-glucopyranosyl-L-ascorbic acid, reducing the costs associated with its production. Structural modeling of the three mutants suggested that hydrophilicity, hydrogen bonding, and negative charge may be responsible for their improved production. Since K647 is conserved in the CGTase family, the corresponding residues in the CGTases from Bacillus circulans 251, Paenibacillus macerans, and Anaerobranca gottschalkii were changed to glutamic acid. Productions of the resulting K647E mutants were 2.0-, 1.5-, and 1.0-fold higher than those of their respective wild-types. Electrostatic protein surface analysis suggested that mutations occurring at low negative surface charge may increase CGTase production.

Karakterisasi Enzim Komersial Siklodekstrin Glukanotransferase

The objective of this study is to investigate the characteristic of commercial enzyme Cyclodextrin Glucanotrasferase (CGTase) from Bacillus macerans. The CGTase was purified by dialysis, gel fitration and ion exchange chromatography. Study on Characterization of the enzyme showed that the hydrolytic activity of CGTase was 480 U/mg, the optimum tempetature and pH for enzyme reaction were 450C to 550C and pH 5.0 to 8.0, respectively. The CGTase was relatively stable after heating at 550C for 10 minutes, and maintained its activity at the pH 5.0 to 9.0. The enzyme activity was inhibited by the presence of 1 mM metal ions and cause CGTase lost approximately 40% of its activity. Among the metal ions it was found that Cu2+ was the strongest inhibitor, with presence of 1mM Cu2+ the residual activity of CGTase was 24.4%. Results of purification showed that Specific activities of the enzyme during purification were 269 U/mg (crude enzyme); 955 U/mg (dialysis); 481 U/mg (gel fitrations); and 544 U/mg (ion exchange chromatography).

High-level extracellular secretion and characterization of the thermophilic β-cyclodextrin glucanotranferase from Paenibacillus campinasensis in Escherichia coli.

A strain CGT-1 producing β-cyclodextrin glucanotranferase (β-CGTase) was identified as Paenibacillus campinasensis by morphological analysis and 16s rDNA analysis. The gene coding for β-CGTase was cloned, sequenced, and expressed in Escherichia coli BL21(DE3). Recombinant β-CGTase was purified and its purity evaluated by SDS-PAGE, showing it encodes a mature protein with a molecular mass of 74kDa. The β-CGTase was most active at pH 7.0 and 65°C, respectively. More than 80% activity was retained after incubation at 55°C for 5h. The stability of the enzyme was in a pH range from 5.5 to 10.0. The K m and V max for the enzyme activity on CGTase were 3.75mg/mL and 290.75μmol/min, respectively. The recombinant plasmid pET28a-DacD-cgt-his, pET28a-OmpA-cgt-his, pET28a-OmpT-cgt-his, and pET28a-CGTase-cgt-his were constructed by cloning the signal peptide genes DacD, OmpA, OmpT, and signal peptide derived from cgtase gene into pET28a-cgt-his, respectively. The production of the recombinant β-CGTase with pET28a-DacD-cgt-his reached 60.89U/mL after 72h of culture, which produced an approximately 1.98, 2.93, 4.15 to 9.74-fold higher activity than those containing OmpA, CGTase, OmpT, and the control without signal peptide, respectively. The culture conditions for extracellular production of the recombinant β-CGTase in E. coli BL21(DE3) were optimized. The CGTase activity reached the highest level (37.67U/mL) under the induction of 0.03mM IPTG at OD600 of 0.8 at 30°C after 48h of culture. Optimization of the extracellular secretion of the β-CGTase from Paenibacillus campinasensis in recombinant E. coli laid the foundation for further industrial production and application of β-CGTase.

Enhanced secretion of cyclodextrin glucanotransferase (CGTase) by Lactococcus lactis using heterologous signal peptides and optimization of cultivation conditions

In previous studies of Lactococcus lactis, the levels of proteins secreted using heterologous signal peptides were observed to be lower than those obtained using the signal peptide from Usp45, the major secreted lactococcal protein. In this study, G1 (the native signal peptide of CGTase) and the signal peptide M5 (mutant of the G1 signal peptide) were introduced into L. lactis to investigate the effect of signal peptides on lactococcal protein secretion to improve secretion efficiency. The effectiveness of these signal peptides were compared to the Usp45 signal peptide. The highest secretion levels were obtained using the G1 signal peptide. Sequence analysis of signal peptide amino acids revealed that a basic N-terminal signal peptide is not absolutely required for efficient protein export in L. lactis. Moreover, the introduction of a helix-breaking residue in the H-region of the M5 signal peptide caused a reduction in the signal peptide hydrophobicity and decreased protein secretion. In addition, the optimization of cultivation conditions for recombinant G1-CGTase production via response surface methodology (RSM) showed that CGTase activity increased approximately 2.92-fold from 5.01 to 16.89 U/ml compared to the unoptimized conditions.

Production and characterization of β-cyclodextrin glucanotransferase from Bacillus sp. ND1.

A potent β-CGTase producing bacterium ND1 has been isolated from sugarcane field soil in India. The biochemical, physiologicaland phylogenetic analyses based on 16S rRNA gene suggest that the isolate belongs to Bacillus cereus group. The enzyme β-CGTase produced from isolate ND1 catalyzes production of β-cyclodextrin utilizing starch as a substrate which has diverse applications in various fields. The enzyme production parameters pH, temperature, and substrate concentration were optimized using Central Composite Design (CCD) of Response Surface Methodology (RSM) and were found to be 8.9, 30.55 °C, and 1.88%, respectively for optimal enzyme activity. The crude enzyme was partially purified (29-fold) using ammonium sulphate precipitation followed by ion exchange chromatography. The specific activity of the purified enzyme was found to be 63.53 U mg-1 . The enzyme is monomeric in nature with a molecular weight of 97.4 kD as determined by SDS-PAGE. It is stable in a wide range of pH (6-10) and temperature (40-60 °C) values. The maximum CGTase activity was observed at pH 9 and temperature 50 °C. The Km value was found to be 2.613 ± 0.5 and Vmax was 0.309 ± 0.05 µg min-1 indicating high substrate specificity. Together; these results suggest that the enzyme may be of wide commercial value in various industrial processes.

Immobilized Cells of Bacillus circulans ATCC 21783 on Palm Curtain for Fermentation in 5 L Fermentation Tanks.

Palm curtain was selected as carrier to immobilize Bacillus circulans ATCC 21783 to produce β-cyclodextrin (β-CD). The influence for immobilization to CGTase activity was analyzed to determine the operation stability. 83.5% cyclodextrin glycosyltransferases (CGTase) of the 1st cycle could be produced in the 7th cycle for immobilized cells, while only 28.90% CGTase was produced with free cells. When palm curtain immobilized cells were reused at the 2th cycle, enzyme activities were increased from 5003 to 5132 U/mL, which was mainly due to physical adsorption of cells on palm curtain with special concave surface structure. Furthermore, conditions for expanded culture of immobilized cells in a 5 L fermentation tank were optimized through specific rotation speed procedure (from 350 r/min to 450 r/min with step size of 50 r/min) and fixed ventilation capacity (4.5 L/min), relations between biomass, enzyme activity, pH, and oxygen dissolution was investigated, and the fermentation periods under the two conditions were both 4 h shorter. Compared with free cell, immobilized cell was more stable, effective, and had better application potential in industries.

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells

In prokaryotic cells, 3'-5' exonucleases can attenuate messenger RNA (mRNA) directionally from the direction of the 3'-5' untranslated region (UTR), and thus improving the stability of mRNAs without influencing normal cell growth and metabolism is a key challenge for protein production and metabolic engineering. Herein, we significantly improved mRNA stability by using synthetic repetitive extragenic palindromic (REP) sequences as an effective mRNA stabilizer in two typical prokaryotic microbes, namely, Escherichia coli for the production of cyclodextrin glucosyltransferase (CGTase) and Corynebacterium glutamicum for the production of N-acetylglucosamine (GlcNAc). First, we performed a high-throughput screen to select 4 out of 380 REP sequences generated by randomizing 6 nonconservative bases in the REP sequence designed as the degenerate base "N." Secondly, the REP sequence was inserted at several different positions after the stop codon of the CGTase-encoding gene. We found that mRNA stability was improved only when the space between the REP sequence and stop codon was longer than 12 base pairs (bp). Then, by reconstructing the spacer sequence and secondary structure of the REP sequence, a REP sequence with 8 bp in a stem-loop was obtained, and the CGTase activity increased from 210.6 to 291.5 U/ml. Furthermore, when this REP sequence was added to the 3'-UTR of glucosamine-6-phosphate N-acetyltransferase 1 ( GNA1), which is a gene encoding a key enzyme GNA1 in the GlcNAc synthesis pathway, the GNA1 activity was increased from 524.8 to 890.7 U/mg, and the GlcNAc titer was increased from 4.1 to 6.0 g/L in C. glutamicum. These findings suggest that the REP sequence plays an important function as an mRNA stabilizer in prokaryotic cells to stabilize its 3'-terminus of the mRNA by blocking the processing action of the 3'-5' exonuclease. Overall, this study provides new insight for the high-efficiency overexpression of target genes and pathway fine-tuning in bacteria.

Extractive Bioconversion of Gamma-Cyclodextrin and Recycling of Cyclodextrin Glycosyltransferase in Liquid Biphasic System Using Thermo-Separating Polymer.

An extractive bioconversion conducted on soluble starch with cyclodextrin glycosyltransferase (CGTase) enzyme in ethylene oxide-propylene oxide (EOPO)/potassium phosphates liquid biphasic system (LBS) to extract gamma-cyclodextrin (γ-CD) was examined. A range of EOPO (with potassium phosphates) molecular weights was screen to investigate the effect of the latter on the partioning efficency of CGTase and γ-CD. The results show that the optimal top phase γ-CD yield (74.4%) was reached in 35.0% (w/w) EOPO 970 and 10.0% (w/w) potassium phosphate with 2.0% (w/w) sodium chloride. A theoretical explanation for the effect of NaCl on γ-CD was also presented. After a 2 h bioconversion process, a total of 0.87 mg/mL concentration of γ-CD was produced in the EOPO/ phosphates LBS top phase. After the extraction of top phase from LBS, four continuous repetitive batches were successfully conducted with relative CGTase activity of 1.00, 0.86, 0.45, and 0.40 respectively.

A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production.

Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development of engineered enzymes with improved yield and/or particular product specificity. In this work, we analyzed the role of four residues of the CGTase from Paenibacillus barengoltzii as determinants of product specificity. Single mutations were introduced in the CGTase-encoding gene to obtain mutants A137V, A144V, L280A and M329I and the activity of recombinant proteins was evaluated. The residue at position 137 proved to be relevant for CGTase activity. Molecular dynamics studies demonstrated additionally that mutation A137V produces a perturbation in the catalytic site of the CGTase, which correlates with a 10-fold reduction in its catalytic efficiency. Moreover, this mutant showed increased production of maltooligosaccharides with a high degree of polymerization, mostly maltopentaose to maltoheptaose. Our results highlight the role of residue 137 as a determinant of product specificity in this CGTase and may be applied to the rational design of saccharide-producing enzymes.

Loofa immobilized Bacillus sp. DSM 2523 as a whole cell biocatalyst for production of cyclodextrin glucanotransferase in an airlift reactor with a net draft tube

Cyclodextrin glucanotransferase (CGTase) production was studied using loofa-immobilized Bacillus sp. DSM 2523 and starch substrate. The bacterial cells from 2- and 4-day old cultures were used in the flask studies. Loofa and chitosan were added for cell immobilization and cell flocculation, respectively, and different treatments were considered according to the timing of their addition. The cell responses were evaluated for their degree of cell immobilization and level of CGTase activity. With the use of the selected treatment, testing was carried out in an airlift reactor with a net draft tube having specific geometric properties. The cell capacity for reusability also was examined. The production of CGTase was higher in the second cycle in the reactor operated at 0.25 vvm where the enzyme activity reaching 0.20 U/ml within 2 h. In the flask experiments, CGTase activity reached 0.23 U/ml after 19 h in the second cycle.