Production Of Cyclodextrin Glycosyltransferase Research Articles

Genetic enhancement of Bacillus cyclodextrin glycosyltransferase production

Genetic enhancement of Bacillus cyclodextrin glycosyltransferase production

Byproducts from cassava industry: alternative substrates for cyclodextrin glycosyltransferase production by alkalophilic Bacillus trypoxylicola SM–02

Bioproductos de la industria de la yuca: sustratos alternativos para la producción de ciclodextrina glucosiltransferasa por alcalófilo Bacillus trypoxylicola SM-02 En el presente trabajo estudiamos el uso licor de maíz fermentado (LMF), harina de cáscara de yuca (HCY) y aguas residuales de yuca para la producción de ciclodextina glicosiltransferasa (CGTase) por un nuevo aislado alcalófilo de Bacillus trypoxylicola SM-02 en fermentación sumergida. Los experimentos se realizaron por Diseño Central Compuesto Rotativo 22 totalizando 11 ensayos. La mayor actividad enzimática de 352.53 U/mL se obtuvo con 1.5 g de HCY y 0.6 g de LMF. La temperatura y el pH óptimos fueron 55 ºC y pH 8.0, respectivamente. CGTase mostró una actividad relativa superior al 50% durante 120 min. a la temperatura óptima. Solo el CaCl2 mostró actividad positiva para CGTasa. Los resultados apuntaron a un buen potencial de B. trypoxylicola SM-02 para la producción de CGTasa usando substratos residuales. In the present work was studied the use of cassava peel flour (CPF), corn steep liquor (CSL), and cassava wastewater as substrates to produce cyclodextrin glycosyltransferase (CGTase) from a new alkalophilic isolate of Bacillus trypoxylicola SM-02 by submerged fermentation. The experiments were performed as a Central Composite Design 22, totalizing 11 assays. An enzymatic activity of 352.53 U/mL was obtained using 1.5 g of CPF and 0.6 g of CSL. The optimum temperature and pH of CGTase was 55 °C and 8.0, respectively. The CGTase depicted a relative activity of more than 50% for 120 min at the optimum temperature. The only salt that positively influenced the CGTase activity was CaCl2. The results are indicative of a potential role of B. trypoxylicola SM-02 in the production of CGTase using residual substrates.

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.

Optimization of Cyclodextrin Glycosyltransferase Production from Sorghum

A central composite experimental design was used to evaluate optimal conditions of the medium component, pH and temperature for cyclodextrin glycosyltransferase production from sorghum by Bacillus circulans (American Type Culture Collection). Sorghum concentration, initial pH and temperature were optimized using the response surface method. Maximal CGTase production of 216.94 U.mL-1 was obtained after 48 hours of fermentation when the concentrations of the medium were 0.16% sifted sorghum, 0.5% triptone, 0.5% yeast extract, 1% sodium carbonate, 0.1% K2HPO4, 0.02% MgSO4•7 H2O, initial pH of 8.0 and temperature of 35°C under rotational shaking. Further studies aimed at increasing the fermentation scale using optimal conditions for the high enzymatic production and high volumetric productivity are needed to improve the fermentation process described in the present study.

Production of cyclodextrin glycosyltransferase by immobilized Bacillus sp. on chitosan matrix.

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol-sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15Uml(-1) (36h), 47.50Uml(-1) (36h) and 68.36Uml(-1) (36h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15Uml(-1) (18h) on cassava, 79.17Uml(-1) (12h) on potato and 55.37Uml(-1) (in 6h and max 77.75Uml(-1) in 36h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.

Cyclodextrin glycosyltransferase production by free cells of Bacillus circulans DF 9R in batch fermentation and by immobilized cells in a semi-continuous process.

Cyclodextrin glycosyltransferase (CGTase) catalyzes starch conversion into cyclic or linear oligosaccharides, important industrial products for the complexation of non-polar substances. In this work, conditions to increase CGTase production from Bacillus circulans strain DF 9R were optimized by two systems. On one hand, free cells were grown in batch fermentation experiments to optimize aeration and pH. The highest activity (1.47 ± 0.21 U ml(-1)) was achieved after 48 h of growth, aeration of 1.5 vvm and pH regulated to 7.6. On the other hand, bacterial cells were immobilized on loofa and synthetic sponge, and used for CGTase production in a semi-continuous process. An initial biomass of 30 mg of lyophilized cells and an immobilization time of 24 h with loofa or synthetic sponge were enough to achieve increased production of CGTase: 0.91 ± 0.10 and 0.95 ± 0.11 U ml(-1), respectively. Sponges with immobilized bacteria were reused in 12 successive cycles. Besides, in our conditions, CGTase was not adsorbed onto the supports used for immobilization, which ensured the total recovery of the enzyme from the culture medium. The two CGTase production processes studied showed similar productivity and could be potentially scaled up.

Production of Cyclodextrin Glycosyltransferase (CGTase) by Bacillus lehensis S8 using Sago Starch as Carbon Source

Production of Cyclodextrin Glycosyltransferase (CGTase) by Bacillus lehensis S8 using Sago Starch as Carbon Source

The Influence of Oxygen Volumetric Mass Transfer Rates on Cyclodextrin Glycosyltransferase Production by Alkaliphilic Bacillus circulans in Batch and Fed-Batch Cultivations

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 (kla, [per hour]). The maximal CGTase productivity of 155 U mL−1 h−1 was achieved with kla of 48 h−1. 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−1 h−1 with feeding rates of starch at 0.17 g L−1 h−1.

Cyclodextrin glycosyltransferase production by new Bacillus sp. strains isolated from brazilian soil

Cyclodextrin glycosyltransferase production by new Bacillus sp. strains isolated from brazilian soil

Cyclodextrin glycosyltransferase production by new Bacillus sp. strains isolated from brazilian soil.

Three strains of Bacillus sp. (BACRP, BACNC-1 and BACAR) were isolated from soil adhered to cassava husk. CGTase specific activity for the three isolated strains was higher when cultivated at 40°C. Potato starch, cassava starch, maltodextrin and glucose were used as carbon source and growth temperatures varied from 25 to 55°C. The three isolates presented higher CGTase specific activity when cultivated with potato starch at 40°C. Isolated BACRP and BACAR presented specific activity of 4.0×10–3 and 2.2×10–3 U/mg prot at pH 7.0, respectively, when cultivated in mediums added with NaCl 2%; at pH 10,0 their activities were of 3.4×10–3 and 3.0×10–3 U/mg prot, respectively, in the same concentration of NaCl. On the other hand, the isolated BACNC-1 presented activity specific of 2.4×10–3 U/mg prot when cultivated at pH 7.0 added of NaCl 1%, and at pH 10.0 the specific activity was of 3.4×10–3 U/mg prot without NaCl addition. This work also showed the presence of cyclodextrins formed during fermentation process and that precipitation with acetone or lyophilization followed by dialysis was efficient at removing CDs (cyclodextrins), thus, eliminating interference in the activity assays. The enzyme produced by the BACAR strain was partially purified and β-CD was liberated as a reaction product.

Optimization of Cyclodextrin Glycosyltransferase Production by Response Surface Methodology Approach

The aim of the study was to optimized cyclodextrin glycosyltransferase (CGTase) production using local isolated strain MK6 which was identified as Bacillus sp. Optimum activity obtained at temperature of 70 °C and the enzyme shows a wide range of pH stability ranging from 4-10 when stored at 4 °C for 24 h and temperature stability ranging from 30-80 °C at 1 h incubation period. The CGTase activity was even maintained at 0.4 U mL-1 at 90 °C for 40 min incubation. Prior to optimization of CGTase production, selection for the best carbon source through detection using modified phenolphthalein method containing different types of starch were performed. Sago starch gave significant result and was used for further optimization using statistical analysis namely Response Surface Methodology (RSM) approach. The optimal calculated values were 3.34% sago starch, initial pH of 10.15 and agitation speed of 187 rpm; with predicted activity of 2.07 U mL-1 of CGTase. These predicted optimal parameters were confirmed in the laboratory and the final CGTase activity obtained was very close to the predicted value of 2.56 U mL-1.

Production of cyclodextrin glycosyltransferase by alkaliphilic Bacillus circulans in submerged and solid-state cultivation

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 degrees C, 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.

Evaluation of Solid and Submerged Fermentations for the Production of Cyclodextrin Glycosyltransferase by <i>Paenibacillus campinasensis</i> H69-3 and Characterization of Crude Enzyme

Evaluation of Solid and Submerged Fermentations for the Production of Cyclodextrin Glycosyltransferase by <i>Paenibacillus campinasensis</i> H69-3 and Characterization of Crude Enzyme

Enhanced secretion of heterologous cyclodextrin glycosyltransferase by a mutant of Bacillus licheniformis defective in the D-alanylation of teichoic acids.

To examine whether inactivation of the dlt operon and increased charge density of the wall enhances secretion of heterologous proteins in industrial strains of Bacillus licheniformis. The dltA gene of B. licheniformis was cloned, sequenced and mutated by inserting a chloramphenicol acetyl transferase (cat) gene cassette. The mutation facilitated growth in the late exponential growth phase, increased endogenous autolysis and decreased resistance to a cationic peptide, polylysine. It was observed that dltA mutation increased the production of cyclodextrin glycosyltransferase (CGTase) by 1.5- to sevenfold depending on the growth phase, but decreased the production of penicillinase by twofold. The results suggest that the d-alanylation of teichoic acids is an element that can be used to improve the production of some secretory proteins in industrial applications based on this important industrial microorganism.

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production fromKlebsiella pneumoniae pneumoniae AS-22

Production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae pneumoniae AS-22 was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, like carbon, nitrogen, phosphorus, and mineral sources as well as initial pH and temperature, were tested on enzyme production. The optimum concentrations of the selected media components were determined using statistical experimental designs. Two level fractional factorial designs in five variables, namely, dextrin, peptone, yeast extract, ammonium dihydrogen orthophosphate, and magnesium sulphate concentrations were constructed. The optimum medium composition thus found consisted of 49.3 g/L dextrin, 20.6 g/L peptone, 18.3 g/L yeast extract, 6.7 g/L ammonium dihydrogen orthophosphate, and 0.5 g/L magnesium sulphate. The maximum CGTase activity obtained was 21.4 U/mL in 28 h of incubation. The cell growth and CGTase production profiles were studied with the optimized medium in shake flasks and in 1-L fermenters. It was observed that the enzyme production was growth associated both in shake flask and in fermenter, although it was slower in shake flask. The maximum CGTase activity obtained in the fermenter was 32.5 U/mL in 16 h. The optimized medium resulted in about 9-fold increase in the enzyme activity as compared to that obtained in the basal medium in shake flask as well as in fermenter.

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS‐22

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS‐22

Cyclodextrins are likely to induce cyclodextrin glycosyltransferase production inBacillus macerans

Cyclodextrin glycosyltransferase (CGTase) activity was monitored inBacillus macerans culture fluids up to 56 h incubation time using glucose (G1), maltose (G2), maltotriose (G3), maltoheptaose (G7), α-,β-,γ-cyclodextrins (CDs) and soluble starch as carbon sources. Highest maximum specific growth rates (µmax) were observed with glucose, γ-CD and soluble starch (µmax values were 0.86, 0.74 and 0.69/h, respectively) while the maximum viable cell numbers were always within the range of 2.3–7.1×1011 CFU/mL independently of the carbon source used. Highest CGTase production was found in the presence of soluble starch and G7 (55.0 and 35.4 nkat/mL, respectively), these saccharides being easily transformed to CDs by CGTase. Moreover, when culture media were supplemented with cyclic malto-oligosaccharides the CGTase activities were about twice higher (19.6–20.6 nkat/mL) than those obtained with the linear G2 and G3 saccharides (8.9 and 11.3 nkat/mL, respectively) which give rise only to negligible quantities of CDs. CDs, which are the major end products of the action of CGTase, are regarded thus as the likely physiological inducers of the enzyme.

Evaluation of solid, slurry, and submerged fermentations for the production of cyclodextrin glycosyl transferase by Bacillus cereus

Cyclodextrin glycosyl transferase, a multifunctional extracellular enzyme used for the preparation of cyclodextrins, was produced by Bacillus cereus, an indigenous strain. Solid substrate fermentations with wheat bran and slurry fermentations consisting of 10% wheat bran were compared with submerged fermentations using defined nutrient medium. Solid state fermentation displayed the maximum activity of 59 units ml −1 in 24 h when compared to slurry fermentation (58 units ml −1) and submerged fermentation (40 units ml −1). The use of wheat bran enhanced the activity by 50%. The promoting action of wheat bran was evident, with the supplementation of wheat bran extract in submerged fermentation resulting in 2.7-fold (110 units ml −1) higher activity in comparison to submerged fermentation.

Production of cyclodextrin glycosyltransferase by batch and chemostat culture of Bacillus macerans in chemically defined medium

AbstractA chemically defined medium for cultivation of Bacillus macerans is reported. Growth rates and cyclodextrin glycosyltransferase (CGT) titres obtained were similar to those obtained using media containing potato extract. The proteins in supernatant fluids from a batch culture were separated by disc electrophoresis and results obtained showed that CGT was produced after growth ceased, in agreement with results of activity measurements. The maximum growth rate in the chemostat considerably exceeded that in batch culture; this anomalous effect is unexplained. High CGT titres were produced at low dilution rates (0.03 to 0.05 h−1) but residual starch was present at higher dilution rates and CGT synthesis was repressed. Enzyme titres obtained in chemostat cultures at D = 0.03 h−1 using defined medium containing 13 g starch/1 were 2.75 times greater than the maximum obtainable by batch cultivation and about 20 times greater than those reported by other workers using medium containing diced potato and CaCO3. A two‐stage chemostat cultivation was performed using dilution rates of 0.1 h−1 and 0.033 h−1 in the first and second stages, respectively. The CGT activity in the second stage increased by 57 per cent when a maintenance feed of starch was supplied at 0.08 g g−1 dry biomass h−1. Only negligible CGT titres were obtained when a dilution rate of 0.5 h−1 was used in the first stage. For reasons not understood, DM medium would not support biomass yields greater than 5 g 1−1. This limitation was not due to production of an inhibitor, or to deficiency of N, Fe, Zn, Mn, thiamine or biotin.

Production of cyclodextrin glycosyltransferase by Bacillus macerans in batch cultures

AbstractSatisfactory growth of Bacillus macerans and high activities of cyclodextrin glycosyltransferase (CGT) were obtained in media containing potato extract. The appearance of CGT in culture supernatant fluids during the stationary phase of growth was not due to autolysis, since intracellular levels of the enzyme were very low throughout the cultivation. When cell growth was limited by the starch concentration in the medium, CGT activity remained low during the logarithmic phase of growth but high activities appeared suddenly in the supernatant fluid when growth ceased. Negligible activities were produced by cultures growing in a nitrogen‐deficient medium in which starch was present in excess. CGT therefore appears to be a true extracellular enzyme, produced in response to exhaustion of the starch from the medium. A simple fractionaltion procedure was developed for concentrating the CGT from culture supernatant fluids. This enzyme preparation was used to convert 20% (w/v) starch solutions to cyclodextrins in 24 h and the cyclodextrins recovered were equivalent to 55% of the original starch.