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Highlights: [•] We examine extractive bioconversion in aqueous two-phase system (ATPS). [•] The synthesis and recovery of cyclodextrins (CDs) is performed. [•] Repetitive batch of CDs synthesis is performed. [•] ATPS generates a cost-efficient and environmental friendly technique. [ABSTRACT FROM AUTHOR]

Abstract: Cyclodextrin glycosyltransferase catalyses the synthesis of cyclodextrins from starch by intramolecular transglycosylation. However, under certain conditions, the reaction can be directed towards the production of linear dextrins, suppressing the synthesis of cyclodextrins. Thus, the starch acts as donor molecule and low molecular weight oligosaccharides act as acceptors in intermolecular transglycosylation reactions. In this paper the optimal conditions to transform starch into maltooligosaccharides were determined employing glucose and maltose as acceptors and cyclodextrin glycosyltransferase from Bacillus circulans DF 9R as catalyst. Maltooligosaccharides production was optimised in a batch process reaching a yield of 60%. Besides, a continuous process was developed in a membrane reactor, increasing the yield to 72% and multiplying eight times the productivity. In this way, it was possible to obtain a high purity product with higher efficiency, using a cyclodextrin glycosyltransferase. This is even more advantageous when compared with the processes currently used in industry which uses three enzymes. [Copyright &y& Elsevier]

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg-1 protein, 20.0 U mg-1 protein and 11.0 U mg-1 protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl2. Km and Vmax values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co2+, Zn2+, Cu2+, Hg2+, Ba2+, Cd2+, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry. [ABSTRACT FROM AUTHOR]

The production of cyclodextrins (CDs) by cyclodextrin glycosyltransferase (CGTase) from Bacillus clarkii 7364 was studied. Forty-seven percent ( w/ w) conversion rate to γ-CD was obtained in the process performed by reacting 5 U per gram of starch CGTase with 15 % ( w/ v) soluble starch in 0.025 M sodium phosphate-NaOH buffer (pH 12) at 55 °C in the presence of 2 % ( w/ v) glycyrrhizic acid. Meanwhile, the ratio of γ:β-CD was 89:11, with negligible formation of α-CD. Under these conditions, there is a significant increase in overall production of CDs and a marked change in product selectivity for γ-CD. The possible mechanisms were discussed upon different product profiles with respect to the size and amount of CDs synthesized at different reaction conditions. The approach described here can be easily applied to an enzymatic process for the production of γ-CD on an industrial scale, and such high selectivity, at high conversions, is especially attractive from a commercial perspective. [ABSTRACT FROM AUTHOR]

Abstract: In this work, the enzymatic properties of immobilized cyclodextrin glycosyltransferase (CGTase) of Thermoanaerobacter sp. were investigated and compared with the soluble form of the enzyme. CGTase was immobilized on mesoporous silica microspheres synthesized using polyethylene glycol 400 as swelling agent, silanized with 3-aminopropyltrimethoxysilane (APTMS), and activated with glutaraldehyde prior to immobilization. This innovative approach for support preparation produced high yields of immobilization (83%) and activity recoveries (73%), which are the highest so far reported for CGTase. The soluble enzyme (CGTase) and its immobilized form (ImCGTase) showed similar values for the optimal pH activity, while optimal reaction temperatures were found to be 100°C and 80°C, respectively. The immobilized enzyme showed similar values for K m and thermal stabilities with the soluble form, while its V max was lower. The immobilized enzyme was tested in repeated batches in order to simulate recovery and reuse, keeping about 60% of the initial catalytic activity after 15 cycles, showing its good chemical and mechanical resistance. [Copyright &y& Elsevier]

A Gram-positive, endospore-forming, alkali-tolerant bacterial strain, designated CGII, was isolated from the wastewater of a cassava flour mill in the state of São Paulo, Brazil and submitted to phylogenetic studies and biochemical tests. The 16S rRNA gene sequence indicated the highest degree of genomic similarity with MLB2 strains of Bacillus lehensis (100%). A two-level central composite rotatable design was then employed to optimize the medium composition and culture conditions for the production of the enzyme cyclodextrin glycosyltransferase (CGTase) in shake-flask and bioreactors. CCTase activity was measured under different production conditions, such as culture medium, agitation and aeration. Highest enzyme production by B. lehensis was achieved in 72 h with a maximal activity of 134.05 U mL. The response surface method demonstrated that the proposed model achieved a good level of agreement with experimental data, with a correlation coefficient of 0.910, thereby confirming the adequate reliability of the model. [ABSTRACT FROM AUTHOR]

Abstract: The bacteria with hydrolysis activity to glucomannan were isolated from the rhizosphere of Amorphophallus konjac through enrichment cultivation. One strain with strong activity in degrading glucomannan was identified preliminarily as Paenibacillus azotofixans YUPP-5 according to the sequence analysis of 16S rDNA. This strain is able to hydrolyze many polysaccharide with β-1,4 linkage, including glucomannan, galactomannan, xylan, carboxymethyl cellulose, and chitin. One hydrolytic enzyme band of approximately 70kDa was examined from the supernatants of YUPP-5 by using zymogram with mixture polysaccharides as substrate. The encoding gene had an open reading frame of 2157bp, which deduced cyclodextrin glycosyltransferase (CGTase), including 718 amino acids with a signal peptide in the N-terminal region. When the gene was expressed in Escherichia coli BL21, the recombinant CGTase exhibited strong activity in degrading polysaccharides with β-1,4 linkage, and in forming cyclodextrin by using carboxymethyl cellulose as substrate. This CGTase exhibited some new functions. Finally, the hydrolytic oligosaccharides from galactomannan or glucomannan were detected by thin layer chromatography. Pentasaccharide, tetrasaccharide, trisaccharide, and disaccharide could be examined as reaction time went on. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Enzymatic synthesis of (−)-epicatechin (EC) glucosides was performed through the transglucosylation reaction catalyzed by the cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01. The enzyme showed the same product specificity for the three donor substrates, starch, β-cyclodextrin and maltoheptaose (G7). Using β-cyclodextrin as the glucosyl donor, several EC glucoside products were obtained at an overall minimal yield of 18.1%. The structures of the four main products were elucidated by MS and NMR techniques as (−)-EC-3′-O-α-d-glucopyranoside (EC3A), (−)-EC-3′-O-α-d-diglucopyranoside (EC3B), (−)-EC-3′-O-α-d-triglucopyranoside (EC3C) and (−)-EC-4′-O-α-d-glucopyranoside (EC4A). Of these, EC3A was the major product while EC4A, unique for this CGTase, was formed in the lowest amount. The water solubility and stability against UV irradiation of EC3A were significantly higher than that of EC. Although the antioxidant activity was 1.5-fold lower, the advantage of the enhanced solubility and stability makes the EC3A glucoside more beneficial as food ingredient than its parent EC. [Copyright &y& Elsevier]

Abstract: In this works, the polymer–salt aqueous two phase system (ATPS) which is polyethylene–glycol (PEG) with sodium citrate was constructed to purify the enzyme CGTase from fermentation broth. Impacts of parameters such as phase composition, tie-line length (TLL), volume ratio (V R), crude sample loading, pH and the addition of sodium chloride (NaCl) on the partition behavior of cyclodextrin glycosyltransferase (CGTase) were investigated. The study exhibited that the optimum system for the purification of the enzyme CGTase was achieved on the 19.0% PEG and 11.5% citrate system with TLL of 38.89% (w/w), V R of 2.0, with additional 4% (w/w) NaCl and 20% crude load at pH 7.0. CGTase from Bacillus sp. was partially purified by the ATPS up to 16.3-fold with a yield of 70%. [Copyright &y& Elsevier]

Abstract: Bacillus macerans cyclodextrin glycosyltransferase (CGTase) was used to convert dodecyl-β-maltoside (DDM) to dodecyl-β-maltooctaoside (DDMO) using α-cyclodextrin (α-CD) or starch as glycosyl donors. At 300mM α-CD, varied DDM concentration and 60°C, the reaction obeyed Michaelis–Menten kinetics with a K m value of 18mM and a V max value of 100U/mg enzyme. However, at 25mM α-CD the reaction rate decreased with increasing DDM concentration (5–50mM), and when the α-CD concentration was varied at fixed DDM concentration an S shaped curve was obtained. The deviations from Michaelis–Menten kinetics were interpreted as being caused by formation of inclusion complexes between α-CD and DDM and by micellation of DDM. To achieve a high reaction rate, a high concentration of free α-CD is necessary, since α-CD in the form of a complex has low reactivity. When starch is used as glycosyl donor in the CGTase catalyzed alkyl glycoside elongation reaction, it is thus important to choose reaction conditions under which the cyclization of starch to α-CD is efficient. [Copyright &y& Elsevier]

In this work, the influence of oxygen mass transfer rates on the production of cyclodextrin glycosyltransferase (CGTase) by the alkaliphilic bacterium Bacillus circulans ATCC 21783 was investigated. Experimental design and response surface methodology were applied to optimize agitation speed and air flow rate in batch cultivations, in order to identify their significant effects and interactions with the synthesis of CGTase. Results were expressed as the volumetric mass transfer rates of oxygen (ka, [per hour]). The maximal CGTase productivity of 155 U mL h was achieved with ka of 48 h. CGTase production was also studied in fed-batch cultures using the optimized parameters obtained in the batch experiments. The maximal CGTase productivity on fed-batch cultivations was 137 U mL h with feeding rates of starch at 0.17 g L h. [ABSTRACT FROM AUTHOR]

Abstract: A transglycosylation reaction between an alkyl polyglycoside and α-cyclodextrin catalysed by cyclodextrin glycosyltransferase (CGTase) from Bacillus macerans was investigated. The reaction products were identified by comparison with standards generated by CGTase catalysed modification of pure alkyl glycosides using HPLC–ELSD and –MS analysis. The main products were alkyl glucopyranosides (substrates present in the alkyl polyglycoside) glycosylated with 6 (primary coupling products) or 12 (secondary coupling products) glucose residues. Both α and β anomers were glycosylated. [Copyright &y& Elsevier]

Abstract: Bacillus macerans cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) was covalently immobilised on Eupergit C and used in a packed-bed reactor to investigate the continuous production of long-carbohydrate-chain alkyl glycosides from α-cyclodextrin (α-CD) and n-dodecyl-(1,4)-β-maltopyranoside (C12G2β). The effects of buffer ion strength and pH, and enzyme loading on the immobilisation yield and the enzyme activity were evaluated. Approximately 98% of the protein and 33% of the total activity were immobilised. At pH 5.15, the enzymatic half-life was 132min at 60°C and 18min at 70°C. The immobilised enzyme maintained 60% of its initial activity after 28 days storage at 4°C. The degree of conversion was controlled by simple regulation of the flow rate through the reactor, making it possible to optimise the product distribution. It was possible to achieve a yield of the primary coupling product n-dodecyl-(1,4)-β-maltooctaoside (C12G8β) of about 50%, with a ratio between the primary and the secondary coupling product of about 10. Thermoanaerobacter sp. CGTase (Toruzyme 3.0L) immobilised on Eupergit C had good operational stability at 60 and 70°C thus showing the advantages of using more thermostable enzymes in biocatalysis. However, this enzyme was unsuitable for the production of C12G8β due to extensive disproportionation reactions, giving a broad product range. [Copyright &y& Elsevier]

Cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 and its recombinant enzyme exhibit three isoforms (I, II, and III) with the same apparent size but different charge. Here, we demonstrate for the first time that the deamidation of labile Asns causes the change in molecular forms of CGTase. The faster increase in number of isoforms was observed upon incubation in deamidation buffer at the more alkaline pH. The increase in levels of isoform II and III over time correlated with the increase in isoaspartate, a unique deamidation product. The predicted labile Asns were individually mutated to Asp, then the selected mutant and wild type isoforms were tryptic digested and labile Asns were investigated by MALDI-TOF. From the results, Asn427 was the most susceptible residue for deamidation, followed by Asn336, Asn415, and Asn567. In addition, Gln389 might also share a role. The advantage of using appropriate CGTase isoform in cyclodextrin production is reported. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the gene expression and characteristics of a fusion protein that contains a cyclodextrin glycosyltransferase produced by bacteria Paenibacillus sp. A11. The study involves the purification of a fusion protein into homogeneity using starch absorption and Ni-NTA affinity chromatography. Moreover, it observes that the fusion enzyme can catalyze the synthesized cyclodextrins.

Cyclodextrins are cyclic maltooligosaccharides composed of glucose units linked by α(1-4) glucosidic bonds. There are mainly three types of cyclodextrins named according to the number of glucose units that are α-CD, β-CD, and γ-CD having 6, 7 and 8 glucose units, respectively. They are geometrically conical cylinders with a hydrophobic internal cavity and hydrophilic outer surface. Due to this structure, they are able to form inclusion complexes with a variety of hydrophobic molecules by changing their physical and chemical properties. Thus, they are extensively used in food, pharmaceutical, cosmetics, chemical, agricultural and textile industries. [ABSTRACT FROM AUTHOR]

Abstract: The cgt gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90h of induction, the activity of α-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant α-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45°C, and half-lives are approximately 8h at 40°C, 1.25h at 45°C and 0.5h at 50°C. The recombinant α-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca2+, Ba2+, and Zn2+ in a concentration-dependent manner, while it is dramatically inhibited by Hg2+. The kinetics of the α-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation. [Copyright &y& Elsevier]

The targeting of recombinant proteins for secretion to the culture medium of Escherichia coli presents significant advantages over cytoplasmic or periplasmic expression. However, a major barrier is inadequate secretion across two cell membranes. In the present study, we attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine could promote extracellular secretion of the recombinant α-CGTase for which one potential mechanism might be the increase in membrane permeability. However, further analysis indicated that glycine supplementation resulted in impaired cell growth, which adversely affected overall recombinant protein production. Significantly, delayed supplementation of glycine could control cell growth impairment exerted by glycine. As a result, if the supplementation of 1% glycine was optimally carried out at the middle of the exponential growth phase, the α-CGTase activity in the culture medium reached 28.5 U/ml at 44 h of culture, which was 11-fold higher than that of the culture in regular terrific broth medium and 1.2-fold higher than that of the culture supplemented with 1% glycine at the beginning of culture. [ABSTRACT FROM AUTHOR]

Abstract: The alkalophilic Bacillus sphaericus strain 41 was isolated from soybean-soil culture, using a highly alkaline pH medium containing 1% Na2CO3. The cyclodextrin glycosyltransferase (CGTase) from this microorganism was purified up to 315-fold with a yield of 31%, by biospecific affinity-column chromatography using Sepharose 6B gel and β-cyclodextrin (β-CD) as the ligand. The molecular weight of the purified enzyme was estimated to be 59kDa by SDS-PAGE. In addition to the cyclization, the CGTase showed disproportionation and coupling activities. For cyclization activity, the optimal pH was 6.0 and the temperature was 65°C. The enzyme showed pH stability in the range of 6.0–7.0. Thermal deactivation was noticeable above 70°C, and the enzyme was highly stable below 65°C. The activation and deactivation energy for the production of β-CD were 9.4kcal/mol and 28.0kcal/mol, respectively. The influence of substrate concentration on the initial rate of CD production was studied, and the kinetic parameters were determined. The K m was 0.0008mol/L and V max was 0.0631mol of β-CD/(Lh), using maltodextrin as substrate. The CGTase was strongly inhibited by the products, and produced a level of CDs reaching 22g/L with a β-CD ratio of 54%. This enzyme produced α-, β- and γ-CD in the ratio of 0.40:1:0.45. [Copyright &y& Elsevier]

Abstract: Activity characteristics and kinetic aspects of a cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans DF 9R were studied. A mixture of α-, β- and γ-cyclodextrins (CDs), glucose, maltose and negligible amounts of longer linear dextrins were produced from gelatinized amylose, amylopectin and starch from different sources. In the coupling reaction, CDs were the substrates in the presence of acceptors such as maltose and/or longer oligosaccharides. From oligosaccharides formed by three or more glucose units, this enzyme produced linear chains of several lengths which were then cyclized. CGTase catalytic efficiency was compared employing an analytical grade starch and cassava starch for food use. Since the results obtained were similar for both starches, the use of an economic starch is an advantage. CGTase was inhibited by the substrate and its own products. Starch concentrations over 20mg/mL inhibited the cyclizing activity. CDs behaved as competitive inhibitors and maltose as an uncompetitive inhibitor while maltotriose showed a mixed inhibition pattern. Limit dextrins showed a scarce inhibitory effect on enzyme activity. CD production could be improved with an ultrafiltration membrane reactor for continuous removal of the products; the starch concentration should be maintained below an inhibitory concentration and limit dextrins would remain in the reactor without affecting enzyme activity. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Cyclodextrin glycosyltransferases (CGTases E.C.2.4.1.19) catalyze cyclomaltooligosaccharides (cyclodextrins) production, an important industrial process. We herein report structural features of Bacillus circulans DF 9R cyclodextrin glycosyltransferase including its sequence and several aspects of enzyme structure–function relationship. Protein ethoxyformylation, under our experimental conditions, indicated that only one out of the 13 enzyme histidines was modified leading to a drastic drop in cyclizing and hydrolytic activity. Besides, tryptic digestion of the 14C ethoxyformylated protein and studies of the peptide mixture showed that histidine 233 is the most reactive histidine residue. This is the first cyclodextrin glycosyltransferase with a known primary structure and a glutamine instead of glycine residue at position 179 in the highly conserved −6 subsite, shown to be involved in substrate binding. The presence of glycine at that position was considered as a requirement for such binding following the induced-fit mechanism already proposed. Moreover, the enzyme has all the features previously described for an α- or α/β-cyclodextrin producer. [Copyright &y& Elsevier]

Abstract: A novel thermophilic anaerobic, rod-shaped, non-spore forming, gram positive bacterium was isolated from an oil field in Turkey, that produces cyclodextrin glycosyltransferase (CGTase) from starch. According to the some morphological, biochemical and 16S rRNA analysis, the strain belongs to the genus Thermoanaerobacter. The strain mainly utilizes starch and derivatives, glucose and fructose as carbon source between 45 and 75°C, optimally at 65°C. Optimum pH for growth is 7.5. 16S RNA studies indicate that the bacterium has a similarity of 98.3% to homoacetogenic Thermoanaerobacter kivui and the main fermentation product is acetic acid as in the case with homoacetogenic bacteria. The main difference between the bacterium and T. kivui concerns the utilization of starch. Based on the phylogenetic and biochemical analysis, it is proposed that the species are a new member of the genus Thermoanaerobacter. The strain has CGTase activity optimum at 80°C and pH 7.0–8.0. [Copyright &y& Elsevier]

Carvacrol has strong antibacterial and antiinflammatory effects and has been increasingly employed in the food and pharmaceutical industries. However, carvacrol is volatile, easily oxidized, and difficult to dissolve in water. In this study, carvacrol was encapsulated with mixed cyclodextrins during enzymatic cyclodextrin synthesis and microencapsulated carvacrol was obtained by spray drying. The encapsulation efficiency and encapsulation yield of microencapsulated carvacrol were 92.16% and 72.30%, respectively. The encapsulation yield for the pure β-cyclodextrin/carvacrol inclusion complex (38.96%) was lower than that of microencapsulated carvacrol (50.44%) when the guest/host ratio was 1:4. No significant difference (P > 0.05) in the amount of encapsulated carvacrol was found when the guest/host ratio was less than 1:6. Microencapsulated carvacrol appeared in scanning electron micrographs as spherical particles with obvious invaginations. Determination of the inclusion formation constant, NMR analysis, and molecular modeling revealed that cavity size affected the ability of cyclodextrins to include carvacrol. Finally, the rate constants (k) of carvacrol release from microencapsulated carvacrol (1.09–8.53 × 10−4) at various temperatures and relative humidities were systematically lower than those of β-cyclodextrin/carvacrol inclusion complexes (5.20–199 × 10−4). This study transcended traditional essential oil-cyclodextrin inclusion formation by developing a mixed-cyclodextrin inclusion compound that achieved better encapsulation and superior release. • Carvacrol increased the yield of enzymatically produced cyclodextrins. • Mixed cyclodextrins encapsulated carvacrol with high encapsulation yield and superior release. • Their cavity sizes affected the ability of cyclodextrins to include carvacrol. [ABSTRACT FROM AUTHOR]

Cyclodextrins are cyclic α-1,4-glucans that are produced from starch or starch derivates using cyclodextrin glycosyltransferase (CGTase). The most common forms are α-, β-, and γ-cyclodextrins. This mini-review focuses on the enzymatic production, unique properties, and applications of γ-cyclodextrin as well as its difference with α- and β-cyclodextrins. As all known wild-type CGTases produce a mixture of α-, β-, and γ-cyclodextrins, the obtaining of a CGTase predominantly producing γ-cyclodextrin is discussed. Recently, more economic production processes for γ-cyclodextrin have been developed using improved γ-CGTases and appropriate complexing agents. Compared with α- and β-cyclodextrins, γ-cyclodextrin has a larger internal cavity, higher water solubility, and more bioavailability, so it has wider applications in many industries, especially in the food and pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Abstract: Cyclodextrins (CD) are cyclic oligosaccharides with multiple applications in the food, pharmaceutical, cosmetic, agricultural and chemical industries. In this work, the conditions used to produce CD with cyclodextrin glycosyltransferase from Bacillus circulans DF 9R were optimized using experimental designs. The developed method allowed the partial purification and concentration of the enzyme from the cultural broth and, subsequently, the CD production, using the same cassava starch as enzyme adsorbent and as substrate. Heat-treatment of raw starch at 70°C for 15min in the presence of adsorbed cyclodextrin glycosyltransferase allowed the starch liquefaction without enzyme inactivation. The optimum conditions for CD production were: 5% (w/v) cassava starch, 15 U of enzyme per gram of substrate, reaction temperature of 56°C and pH 6.4. After 4h, the proportion of starch converted to CD reached 66% (w/w) and the weight ratio of α-CD:β-CD:γ-CD was 1.00:0.70:0.16. [Copyright &y& Elsevier]

Abstract: A new CGTase was obtained from Bacillus firmus, strain 7B, isolated from oat soil culture, using a high alkaline pH medium containing 1% Na2CO3. The enzyme was characterized in soluble form, for pH 5–11, temperature from 30 to 85°C, using a 1% maltodextrin substrate solution and appropriate buffers. It produced mainly β-CD and the cell-free supernatant had a precipitating activity measured by the trichloroethylene method that is a 100-fold greater than that of the enzyme of Bacillus firmus, strain 37, previously studied by our group. The molecular weight of the pure protein was measured as 56,230Da with SDS-PAGE. The optimum temperature for the enzyme activity was 50°C and it was most active at pH 6.0. Thermal deactivation was noticeable above 65°C and the enzyme was highly stable below 60°C. The influence of substrate or product concentration on the initial rate of CD production was studied and the kinetic parameters were determined. The enzyme showed cyclization activity on different raw and hydrolyzed starches and hydrolyzed cornstarch gave the highest activity. [Copyright &y& Elsevier]

Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction. To manipulate the product specificity of the Paenibacillus sp. A11 and Bacillus macerans cyclodextrin glycosyltransferases towards the preferential formation of γ-cyclodextrin (CD8), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies. The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD8 showed pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases. However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were higher than those of the native cyclodextrin glycosyltransferases. When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD8 synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased. Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced CD6 : CD7 : CD8 : ≥ CD9 ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40 °C with starch substrates. In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD8 and ≥ CD9, and the overall cyclodextrin yield was increased by 12% compared to the native enzymes. The crosslinked imprinted cyclodextrin glycosyltransferases also showed higher stability in organic solvents, retaining 85% of their initial activity after five cycles of synthesis reactions. [ABSTRACT FROM AUTHOR]

Abstract: This paper presents the immobilization of the Thermoanaerobacter cyclomaltodextrin glucanotransferase (CGTase) enzyme into cross-linked 6% agarose beads activated by high density of linear aldehyde groups (glyoxyl-agarose) that allow the establishment of multi-attachment enzyme-support bonds. The immobilization conditions were 25°C, pH 10 and 5h of contact time. The immobilization yield was almost 100% and the activity recovery was ca. 32%. The biocatalyst at 85°C was capable of producing cyclodextrins (CDs) from dextrin or soluble starch (both at 1% (w/v)) at a greater rate than the soluble enzyme. In addition, the biocatalyst maintained 90% of its initial activity after 5h at 85°C. The maximum conversion of dextrin to β-CD and γ-CD (total mass of produced CDs/substrate initial mass×100) was 29% both for the soluble and immobilized enzymes. Using starch as substrate the maximum starch conversion to β-CD and γ-CD was 29% and 38%, for the immobilized and soluble enzyme, respectively. The β-CD selectivity yield [mass of β-CD produced/(mass of β-CD produced+mass of γ-CD produced)×100] increased from 67.9% for the free enzyme to 85.4% for the immobilized CGTase. [Copyright &y& Elsevier]

Abstract: Minichaperone, a monomeric polypeptide binding domain fragment of GroEL, has chaperone activities in vivo and in vitro. Fed-batch cultures to optimize production of poly-arginine fused minichaperone (MiniELR10) in recombinant Escherichia coli were done to produce dry cell weight of 75.2g/L, MiniELR10 expression level of 30.7%, and MiniELR10 concentration of 11.1g/L in yeast extract supplemented conditions. MiniELR10 was simply purified through a single step of cation exchange chromatography with high purity. In a prewashing step and an adsorption process by controlling NaCl concentration, removal of contaminant intracellular E. coli proteins and selective binding of MiniELR10 on a cation exchanger were achieved without loss of the target protein. The purified MiniELR10 was efficiently immobilized on the cation exchanger and applied to the refolding of Bacillus macerans cyclodextrin glycosyltransferase (CGTase), which was expressed as inclusion body in recombinant E. coli. The refolding yield of CGTase was enhanced by 71% by the immobilized MiniELR10 compared with a conventional refolding method. Thus, minichaperone immobilized by poly-arginine fusion could assist refolding of heterologous proteins expressed as inclusion body. [Copyright &y& Elsevier]

Cyclodextrin glycosyltransferase (CGTase) is an enzyme that produces cyclodextrins from starch by an intramolecular transglycosylation reaction. Cyclodextrins have been shown to have a number of applications in the food, cosmetic, pharmaceutical, and chemical industries. In the current study, the production of CGTase by Paenibacillus campinasensis strain H69-3 was examined in submerged and solid-state fermentations. P. campinasensis strain H69-3 was isolated from the soil, which grows at 45°C, and is a Gramvariable bacterium. Different substrate sources such as wheat bran, soybean bran, soybean extract, cassava solid residue, cassava starch, corn starch, and other combinations were used in the enzyme production. CGTase activity was highest in submerged fermentations with the greatest production observed at 48–72 h. The physical and chemical properties of CGTase were determined from the crude enzyme produced from submerged fermentations. The optimum temperature was found to be 70–75°C, and the activity was stable at 55°C for 1 h. The enzyme displayed two optimum pH values, 5.5 and 9.0 and was found to be stable between a pH of 4.5 and 11.0. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]