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CGTase production, mainly by Bacillus sp. Although, waxy corn starch CS is used industrially to obtain CGTase and CDs because of its high amylopectin content, alternative sources such as amaranth starch AS could be used to accomplish those purposes. The maximum CGTase specific activity Cyclodextrins CDs are synthesized from starch and related carbohydrates such as amylose, amylopectin and maltooligosaccharide by cyclodextrin glycosyltransferase CGTase, E.
CGTase catalyzes the CDs formation from starch via inter- and intramolecular transglycosylation reactions, which include cycization, disproportionation, coupling, and hydrolysis.
CGTases usually produce a mixture of CDs, glucose, maltose, and other oligosaccharides with varying polymerisation degrees. CDs have a unique structure of hydrophobic cavity of different diameter smaller than 0. Furthermore, CDs are typical host molecules and may encapsulate a great variety of molecules to form crystalline inclusion complexes. Thus, the formation of the inclusion complexes modifies the physical and chemical properties of the host molecule, mostly in terms of water solubility.
In this sense, CDs are important ingredients as molecular encapsulators for applications in food, cosmetic, and pharmaceutical industries Sivakumar and Shakilabanu, For instance, topical application of ferulic acid FA may be useful for preventing skin cancer, but its application on the skin is limited by the poor stability of FA.
The problem may be overcome by the use of CDs to form stable inclusion complexes to increase the stability of the active principle, and improve its solubility, bioavailability and delivery on the skin. Due to the importance of CDs and the derivatives, their safety and toxicological profiles have been reviewed. The reason for this lies in the ease of its production and subsequent low price more than 10, tons produced annually with an average bulk price of approximately 5 USD per kg.
CGTase is produced by bacteria, which can be found in various places such as soil, waste plantation, hot springs and even in deep sea mud. These bacteria are mostly Bacillus sp. However, Klebsiella pneumoniae, Micrococcus luteus, Thermococcus sp. The bacterial strain Bacillus macerans is the most frequently used source of the CGTase enzyme, but B. CGTase produced by B. During the past 2 decades, 51 different CGTase crystal structures, isolated from bacteria, have been published.
According to the different CD specificities. Paenibacillus macerans, Bacillus circulans, Alkaliphillic Bacillus sp. Production of CGTase by B. Therefore, this study was also conducted to know the specificity of CGTase from B.
Other strategy could be that used by Zhou et al. CGTase production can be improved by manipulating fermentation conditions such as pH, temperature, concentrations of nutrients and compositions of the production media carbon and nitrogen sources. Sivakumar and Shakilabanu found that maltose was the best carbon source and yeast extract was the best nitrogen source for CGTase production using B.
Optimization of culture conditions of CGTase production by B. They found that fermentation time and K 2 HPO 4 level were crucial factors in order to improve enzyme production process. Recently, the continuous operation has been chosen over the batch system, because it offers a greater process control, high productivity and an improvement of quality and yield. Molecules of amylose and amilopectin starch fractions are organized into quasicrystalline macromolecular aggregates called starch granules.
The size, shape, and structure of these granules vary substantially among botanical sources. Various types of starch can be used as substrate for CDs production, such as starch of potato and tapioca among others.
Amylopectin gives higher yield of CDs because the reaction with CGTase begins at the non-reducing end of this branched molecule. Many efforts have been made to improve the production of CDs. Until now, there have been several reports on factors affecting CD production by CGTases from several microorganisms. Some reports have focused on the kinetics of CGTase, but most of them have only focused on the effect of substrate concentation. Amaranth is a pseudo-cereal consumed mainly in Mexico and in Central and South America.
AS is of a waxy or glutinous kind and consists of spherical, angular or poligonal granules with an exceptionally small size, ranging from 0. Therefore, amaranth starch granules have high susceptibility to amylases because of their exceptionally high amylopectin content Kong et al.
Urban et al. CGTase was obtained using soluble corn starch as substrate, by a 3-day cultivation in submerged fermentation SmF under aerobic conditions. However, the growth kinetic parameters of bacteria and enzyme activity at fermentation conditions were not evaluated. Therefore, the aim of this work was, in the first part of the study, to characterize the CGTase production by B.
Grain of Amaranthus hypochondriacus L. Starch isolation from the amaranth grain was made by the alkaline method described by Villarreal et al. Flour 25 g was steeped in a 1N NaOH solution in a magnetic shaking heater at room temperature for 1 h. Then they were re-suspended in distilled water and adjusted pH to 7. The retained fiber portion was milled, washed and filtered using distilled water.
The resulted suspension was centrifuged, the supernatant discarded, as well as, the top layer of scrapped starch dark until an imperceptible dark layer was left. The total starch content was determined by the method described by Holm et al. The glucose content in the sample was computed by least squares linear regression. The starch content was calculated on a dry matter basis according to the following formula:. The yield and recovery of the starch obtained were estimated according to the following formulae:.
Briefly, starch samples were completely dispersed by heating in dimethyl sulphoxide. Lipids were removed by precipitating the starch in ethanol, recovering the precipitated starch. The concentration of amylose in the starch sample was estimated as the ratio of absorbance of GOPOD at nm of the supernatant of the precipitated sample with Con A, regard to the total starch sample.
The AS used in this study yielded The starch content of the amylaceous extract was These values were very similar to those displayed by amylose and amylopectin in A. CGTase was obtained using B. This bacterium was obtained from the strains collection of the School of Chemistry that belongs to the National Autonomous University of Mexico.
The pH of the medium was adjusted to 7. For inoculum preparation, the biomass from the agar plate was transferred to a mL Erlenmeyer flask, with 50 mL of a medium at pH 7. The medium was added with 0. The medium pH was adjusted to 7.
The fermenter was inoculated with 9. Sample of 3 mL was taken every 12 h. Finally, the biomass was determined by dry weight. The solution of Velhurst-Pearl equation is as follows:.
Then the reaction was stopped by a thermal shock. Afterwards 0. The aliquots were removed after incubation and assayed for the cyclization activity of CGTase. After incubation for 10 min, the cyclization activity was measured. The isolation of the CGTase was performed according to a previous method described by Gheetha and More , with minor modifications.
Afterwards, the water was changed every 2 h. The proteins were eluted by using the previous buffer at 0. The 2 mL fractions were collected and monitored at nm. The fractions with maximum absorbance were pooled and concentrated using a Sephadex G column, equilibrated with 5 mM Tris-HCl buffer pH 8.
Then the fractions 2 mL were collected in order to assess the enzymatic activity. After incubation of the mixture reaction, the cyclization activity of CGTase was measured. The native AS isolated from grain of A. Corn starch CS was also used as a positive control of the trial. The method carried out by Ibrahim et al. The enzymatic reaction was stopped by boiling it for 10 min and after that, the reaction mixture was submerged in cold water for 10 min.
The CDs produced were measured by mass spectrometry MS. The following settings were used: electrospray ionization ESI in positive mode. The dry gas nitrogen flow rate was set at 4. Collision energy varied in the range of 25—30 eV. Data processing was carried out with Chromeleon 6. Next the samples were introduced directly to the electrospray source of the MS using an LC pump and the mobile phase at a flow rate 3. HRMS high resolution measurements provided by a TOF analyzer in order to enable the processing of the elemental composition of the registered ions.
The kinetic growth parameters of B. Lag phase was practically negligible; the total growth time of B. Values of pH were very similar between cultures initial pH was 7. It remained stable until 84 h of fermentation and finally reached pH 9. The time course production of CGTase in relation to the growth phases of B. The enzyme synthesis using AS and CS as carbon source, began at the early exponential phase and the maximum CGTase specific activity was obtained after 36 h of cultivation, with spontaneous increase in cell biomass yield Figure 1A.
Thereafter, the CGTase activity gradually decreased with the prolongation of the fermentation periods up to 96 h.
The YF mutant had the highest affinity which was indicated by the lowest K m of Increasing hydrophobicity around the catalytic center appeared to favor the cyclization activity of the mutants. Cyclodextrin glycosyltransferase EC 2. Catalysis of cyclization is the primary function of CGTase Leemhuis et al. Contrarily, mutant AV CGTase in Bacillus circulans strain improved the hydrolytic activity but reduced cyclization activity Leemhuis et al.
The enzyme displayed many activities related to the degradation and transformation of starch. Gene encoding H. Sequence analysis revealed an open reading frame of bp that encodes a protein of amino acids. The CGTase is secreted to the extracellular medium by the Tat pathway. The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme.
This peculiar enzyme is capable of catalyzing more than one reaction with the most important being the synthesis of non-reducing cyclic dextrins known as cyclodextrins starting from starch , amylose , and other polysaccharides. CGTase is an enzyme common to many bacterial species, in particular of the Bacillus genus e. All of the CGTases can catalyze up to four reactions: cyclization, coupling, disproportionation and hydrolysis. All these activities share the same catalytic mechanism which is common to all glycosyl-hydrolases. The coupling reaction can be easily described as the reverse process of cyclization: the enzyme cleaves a cyclodextrin to produce a linear dextrin which is subsequently joined to a linear oligosaccharide. Disproportionation is very similar to coupling, but the cleaved dextrin is not a cyclodextrin, but a linear oligosaccharide that is then joined to a second oligosaccharide. CGTase also has a weak hydrolyzing activity which consists in cleaving the longer polysaccharidic chains into shorter fragments.
CGTase production, mainly by Bacillus sp. Although, waxy corn starch CS is used industrially to obtain CGTase and CDs because of its high amylopectin content, alternative sources such as amaranth starch AS could be used to accomplish those purposes. The maximum CGTase specific activity Cyclodextrins CDs are synthesized from starch and related carbohydrates such as amylose, amylopectin and maltooligosaccharide by cyclodextrin glycosyltransferase CGTase, E. CGTase catalyzes the CDs formation from starch via inter- and intramolecular transglycosylation reactions, which include cycization, disproportionation, coupling, and hydrolysis. CGTases usually produce a mixture of CDs, glucose, maltose, and other oligosaccharides with varying polymerisation degrees. CDs have a unique structure of hydrophobic cavity of different diameter smaller than 0.