Β-cyclodextrin Production Research Articles

Production of β-cyclodextrin by immobilized CGTase in packed bed bioreactor through the medium component by employing Taguchi methodology

This study investigates the optimization of submerged culture conditions for the production of extracellular cyclodextrin glucanotransferase (CGTase) enzyme by Bacillus macerans ATCC 8244, employing Taguchi orthogonal array (OA) experimental design methodology. Five critical factors - starch, peptone, temperature, pH, and agitation speed - were examined at two levels using an OA layout of L8 for the experimental design. Analysis revealed a significant enhancement in CGTase yield, with the modified culture conditions resulting in a 21.2% increase from 513.416 U/ml to 622.5 U/ml. The successful application of Taguchi's design methodology underscores its efficacy in optimizing enzyme production processes. This study highlights Taguchi's design potential as a valuable tool for improving CGTase production efficiency while minimizing resource consumption. Additionally, the utilization of a packed bed bioreactor (PBR) facilitated continuous production of β-cyclodextrin (β-CD), demonstrating stability over 10 days with only a slight decrease in productivity. The Km,app value was determined to be 23.4 mg starch/ml, indicating a slight increase.

Cycas revoluta leaves: As a potential flavonoids source for targeted regulation of immune-related markers in lung adenocarcinoma

Cycas revoluta Thunb. (CR) is a widely distributed ornamental and economical plant, and its seeds are important sources of starch and β-cyclodextrin production. However, the medicinal value of CR leaves should also be considered, because flavonoids in CR leaves could represent a possible substance basis for the treatment of lung adenocarcinoma (LUAD). Oxidative stress (OS) and inflammatory immune response (IIR) have been reported to be associated with tumorigenesis and tumor progression. In this study, for the first time, Ultra-high-performance liquid chromatography-quadruple-electrostatic field orbitrap high resolution mass spectrometry (UPLC-Q Exactive Orbitrap-MS) method was used to systematically analyze the chemical profile of CR leaves, and characteristic fragment ions and fragmentation patterns of flavonoids obtained from CR. leaves was analyzed. Subsequently, network pharmacology and molecular docking methods were applied to explore the underlying central targets and related pathways of the targeted regulation of OS and IIR by flavonoids in CR leaves. Consequently, A total of 62 components were characterized from CR. including 10 flavonoids, which were (-)-epigallocatechin, amentoflavone, bilobetin, catechin, ginkgetin, hinokiflavone, naringenin, podocarpusflavone A, sciadopitysin, and vitexin respectively. The hub targets of these 10 flavonoids from CR in treating LUAD was narrowed in Fms-like tyrosine kinase 3 (FLT3), Cyclin A2 (CCNA2), Cyclin B1 (CCNB1), Insulin-like growth factor-binding protein 3 (IGFBP3), Peroxisome proliferator-activated receptor-γ (PPARG), Catalase (CAT), and Myeloid cell leukemia-1 (MCL1). Among them, amentoflavone and podocarpusflavone A have shown great therapeutic potential, while CAT and CCNA2 can be used as potential markers for future studies of flavonoids in CR leaves against LUAD from the perspective of OS and IIR. Taken together, the results showed that flavonoids in CR leaves are potential sources of anti-LUAD compounds by targeting OS and IIR, which will offer a scientific insight for the exploitation and application of CR leaves as functional resources and also provide methodological references for the deeper development of other cash crops.

CONVENTIONAL PRODUCTION OPTIMIZATION OF CYCLODEXTRIN GLUCOSYL TRANSFERASE BY A NOVEL ISOLATE OF BACILLUS SP. PBS1 FROM POTATO RHIZOSPHERE

The cyclodextrin glucosyltransferase enzyme (CGTase) is an industrially crucial enzyme for the production of β-cyclodextrin (β-CD). CGTase has a high propensity to produce a mixture of cyclodextrins (CDs). From the industrial perspective, a CGTase that produces only one type of CD is of critical importance. Bacillus sp. PBS1 produced CGTase that converts starch solely into β-CD. The isolated strain PBS1 was found to close similarity with alkaliphilic Bacillus sp. based on biochemical, morphological, and phylogenetic analysis of its 16s rRNA gene sequencing. The selection and optimization of media ingredients are warranted for the best possible production of β-CD. These steps were carried out by conventional optimization strategies. The presence of glucose, maltose, lactose, sucrose, galactose, mannitol, nitrates, urea, metal salts, and K2HPO4 led to the suppression of CGTase production. The improved enzyme production was observed in peptone, soluble starch, magnesium sulfate, and Na2CO3. The organism produces maximum CGTase (93.42 ± 2.4 U/ml) at 96-hour incubation in the optimized production medium containing 8% starch, 2% peptone, 0.06% MgSO4.7H2O, 0.5% Na2CO3, and having pH of 9.3. The optimization of the medium led to ~16% improvement in CGTase production by Bacillus sp. PBS1.

Immobilization of β-cyclodextrin glycosyltransferase on gelatin enhances β-cyclodextrin production

The β-cyclodextrin glycosyltransferase (β-CGTase) from Bacillus circulans STB01 was immobilized on gelatin with an immobilization yield of 100 % and an activity recovery of 23.1 %. Compared with the free β-CGTase, the immobilized exhibited a broader pH optimum with two peaks (pH 5.5 and pH 8.5) as well as significantly enhanced thermostability and pH stability. Immobilized β-CGTase retained 85.5 % of its activity after storage for 50 days at 4 ℃ and 91.9 % of its activity after 20 cycles of enzyme activity assay. β-Cyclodextrin inhibition experiments showed that immobilized β-CGTase was less susceptible to product inhibition than free β-CGTase. As a result, immobilized β-CGTase delivered a 9.7 % increase of product yield in a single use and the total yield after 10 times of use was 9.2-fold of the yield using free enzyme. According to the results, immobilization can greatly improve the utility of β-CGTase as a catalyst for the industrial production of β-cyclodextrin.

Biosynthesis of raw starch degrading β-cyclodextrin glycosyltransferase by immobilized cells of Bacillus licheniformis using potato wastewater

The study was sought to enhance the synthesis of thermal stable β-cyclodextrin glycosyltransferase (β-CGTase) using potato wastewater as a low-cost medium and assess the degree to which it is efficient for industrial production of β-cyclodextrin (β-CD) from raw potato starch. Thermophilic bacteria producing β-CGTase was isolated from Saudi Arabia and the promising strain was identified as <i>Bacillus licheniformis</i> using phylogenetic analysis of the 16S rRNA gene. Alginate-encapsulated cultures exhibited twice-fold of β-CGTase production more than free cells. Scanning electron microscopy (SEM) of polymeric capsules indicated the potential for a longer shelf-life, which promotes the restoration of activity in bacterial cells across semi-continuous fermentation of β-CGTase production for 252 h. The optimal conditions for β-CGTase synthesis using potato wastewater medium were at 36 h, pH of 8.0, and 50°C with 0.4% potato starch and 0.6% yeast extract as carbon and nitrogen sources, respectively. The purified enzyme showed a specific activity of 63.90 U/mg with a molecular weight of ∼84.6 kDa as determined by SDS-PAGE analysis. The high enzyme activity was observed up to 60°C, and complete stability was achieved at 75°C. High levels of activity and stability were shown at pH 8.0, and the pH range from 7.0–10.0, respectively. The enzyme has an appreciable affinity for raw potato starch with a Km of 5.7 × 10⁻⁶ M and a Vmax of 87.71 µmoL/mL/min. β-CD production was effective against 25 U/g of raw potato starch. The outcomes demonstrated its feasibility to develop a fermentation process by integrating the cost-effective production of β-CGTase having distinctive properties for β-CD production with ecofriendly utilization of potato wastewater.

Enhancement of ��-Cyclodextrin Production and Fabrication of Edible Antimicrobial Films Incorporated with Clove Essential Oil/��-cyclodextrin Inclusion Complex

Edible films containing antimicrobial agents can be used as safe alternatives to preserve food products. Essential oils are well-recognized antimicrobials. However, their low water solubility, volatility and high sensitivity to oxygen and light limit their application in food preservation. These limitations could be overcome by embedding these essential oils in complexed product matrices exploiting the encapsulation efficiency of β-cyclodextrin. This study focused on the maximization of β-cyclodextrin production using cyclodextrin glucanotransferase (CGTase) and the evaluation of its encapsulation efficacy to fabricate edible antimicrobial films. Response surface methodology (RSM) was used to optimize CGTase production by Brevibacillus brevis AMI-2 isolated from mangrove sediments. This enzyme was partially purified using a starch adsorption method and entrapped in calcium alginate. Cyclodextrin produced by the immobilized enzyme was then confirmed using high performance thin layer chromatography, and its encapsulation efficiency was investigated. The clove oil/β-cyclodextrin inclusion complexes were prepared using the coprecipitation method, and incorporated into chitosan films, and subjected to antimicrobial testing. Results revealed that β-cyclodextrin was produced as a major product of the enzymatic reaction. In addition, the incorporation of clove oil/β-cyclodextrin inclusion complexes significantly increased the antimicrobial activity of chitosan films against Staphylococcus aureus, Staphylococcus epidermidis, Salmonella Typhimurium, Escherichia coli, and Candida albicans. In conclusion, B. brevis AMI-2 is a promising source for CGTase to synthesize β-cyclodextrin with considerable encapsulation efficiency. Further, the obtained results suggest that chitosan films containing clove oils encapsulated in β-cyclodextrin could serve as edible antimicrobial food-packaging materials to combat microbial contamination.

Cyclodextrin glucosyltransferase immobilization on polydopamine-coated Fe3O4 nanoparticles in the presence of polyethyleneimine for efficient β-cyclodextrin production

An enzymatic approach to β-cyclodextrin synthesis attracts the attention of food engineers and chemists because of the broad applications of β-cyclodextrin in the food industry and the demand for a green process for β-cyclodextrin production. Achieving a high recovery of cyclodextrin glycosyltransferase through immobilization and a high proportion of β-cyclodextrin in a product mixture of cyclodextrins would improve the purification process of β-cyclodextrin. In this study, a recombinant cyclodextrin glycosyltransferase (CGTase) was immobilized on polydopamine-Fe3O4 (PDA-Fe3O4) nanoparticles in the presence of polyethyleneimine (PEI). After optimization of the preparation of the PDA-Fe3O4 nanoparticles and the CGTase immobilization step, 77.9% of the CGTase was recovered. The immobilized CGTase exhibited good thermal and operational stabilities, and gave a ratio of 88.9% β-cyclodextrin when the reaction temperature and kinetic parameters were optimized. Immobilized CGTase was recycled nine times and retained 19.1% β-activity and 0% γ-activity. This study has established a reliable approach to the high recovery of CGTase during efficient β-cyclodextrin production.

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.

Double mutations enhance β-cyclization activity of cyclodextrin glycosyltransferase from Bacillus circulans

A major drawback to the industrial production of β-cyclodextrin is the limited β-cyclization activity of cyclodextrin glycosyltransferase (CGTase). Here, we construct mutants of the β-CGTase from Bacillus circulans strain STB01 that contain single substitutions at Tyr89 and double substitutions at Tyr89 and Asp577. The results show that the double mutants Y89G/D577R, Y89D/D577R, and Y89N/D577R display enhanced β-cyclization activity, and have higher β-cyclization activity than that of the three single Tyr89 mutants. The double mutant Y89D/D577R exhibited the highest β-cyclization activity and β-cyclodextrin production, increasing 35.1% and 12.4% compared with those of the wild-type CGTase, respectively. The β-cyclization activity of double mutant Y89D/D577R is also higher than that of the single mutant D577R, which had the highest β-cyclization activity among the mutants prepared in our previous studies. The enhanced β-cyclization activity of these mutants may be a result of intermolecular interactions that stabilize intermediates in the β-cyclization reaction. Thus, double mutant Y89D/D577R is much more suitable for industrial β-cyclodextrin production than the wild-type enzyme.

β-Cyclodextrin Production by Cyclodextrin Glucanotransferase from an Alkaliphile Microbacterium terrae KNR 9 Using Different Starch Substrates.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an important member of α-amylase family which can degrade the starch and produce cyclodextrins (CDs) as a result of intramolecular transglycosylation (cyclization). β-Cyclodextrin production was carried out using the purified CGTase enzyme from an alkaliphile Microbacterium terrae KNR 9 with different starches in raw as well as gelatinized form. Cyclodextrin production was confirmed using thin layer chromatography. Six different starch substrates, namely, soluble starch, potato starch, sago starch, corn starch, corn flour, and rice flour, were tested for CD production. Raw potato starch granules were found to be the best substrate giving 13.46 gm/L of cyclodextrins after 1 h of incubation at 60°C. Raw sago starch gave 12.96 gm/L of cyclodextrins as the second best substrate. To achieve the maximum cyclodextrin production, statistical optimization using Central Composite Design (CCD) was carried out with three parameters, namely, potato starch concentration, CGTase enzyme concentration, and incubation temperature. Cyclodextrin production of 28.22 (gm/L) was achieved with the optimized parameters suggested by the model which are CGTase 4.8 U/L, starch 150 gm/L, and temperature 55.6°C. The suggested optimized conditions showed about 15% increase in β-cyclodextrin production (28.22 gm/L) at 55.6°C as compared to 24.48 gm/L at 60°C. The degradation of raw potato starch granules by purified CGTase was also confirmed by microscopic observations.

Enhancing the α-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite -7.

Cyclodextrin glycosyltransferases (CGTases) (EC 2.4.1.19) catalyze the conversion of starch or starch derivates into mixtures of α-, β-, and γ-cyclodextrins. Because time-consuming and expensive purification procedures hinder the widespread application of single-ingredient cyclodextrins, enzymes with enhanced specificity are needed. In this study, we tested the hypothesis that the α-cyclodextrin selectivity of Paenibacillus macerans α-CGTase could be augmented by masking subsite -7 of the active site, blocking the formation of larger cyclodextrins, particularly β-cyclodextrin. Five single mutants and three double mutants designed to remove hydrogen-bonding interactions between the enzyme and substrate at subsite -7 were constructed and characterized in detail. Although the rates of α-cyclodextrin formation varied only modestly, the rate of β-cyclodextrin formation decreased dramatically in these mutants. The increase in α-cyclodextrin selectivity was directly proportional to the increase in the ratio of their kcat values for α- and β-cyclodextrin formation. The R146A/D147P and R146P/D147A double mutants exhibited ratios of α-cyclodextrin to total cyclodextrin production of 75.1% and 76.1%, approximately one-fifth greater than that of the wild-type enzyme (63.2%), without loss of thermostability. Thus, these double mutants may be more suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The production of β-cyclodextrin by these mutants was almost identical to their production of γ-cyclodextrin, which was unaffected by the mutations in subsite -7, suggesting that subsite -7 was effectively blocked by these mutations. Further increases in α-cyclodextrin selectivity will require identification of the mechanism or mechanisms by which these small quantities of larger cyclodextrins are formed.

Asp577 mutations enhance the catalytic efficiency of cyclodextrin glycosyltransferase from Bacillus circulans

The amino acid residue Asp 577 is located in calcium-binding site III (CaIII) of the cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) from Bacillus circulans STB01. In the present study, the effects of replacing Asp577 with glycine, alanine, valine, leucine, and isoleucine on the catalytic efficiency of this CGTase were investigated. Two of these replacements, D577G and D577A, increased the β-cyclization activity of CGTase. Kinetic studies showed that the Km values of D577G and D577A were 36.1% and 18.0% lower and the kcat/Km values were 43.9% and 23.0% higher than those of the wild-type enzyme, respectively. These mutations increased both the affinity of CGTase for maltodextrin and the catalytic efficiency of the cyclization reaction. Furthermore, although D577G and D577A only slightly enhanced β-cyclodextrin production, compared with the wild-type enzyme, their higher β-cyclization activities resulted in a significant reduction in the amount of mutant protein required during the cyclodextrin production process. Thus, the two mutants are more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhancement of catalytic efficiency may be due to the smaller size of the glycine and alanine side chains, which may weaken the impact of this residue on CaIII.

Enhanced thermal stability of cyclodextrin glycosyltransferase in alginate–gelatin mixed gel beads and the application for β-cyclodextrin production

Abstract This study aimed to enhance thermal stability of cyclodextrin glycosyltransferase (CGTase) in alginate–gelatin mixed gel beads. Comparing two immobilization techniques, entrapment technique was more suitable for CGTase immobilization because it gave higher immobilization efficiency and thermal stability than did adsorption technique. Four important parameters for immobilization by entrapment including alginate, gelatin, and CaCl 2 concentrations and bead size were optimized through response surface methodology. The optimal condition for immobilization efficiency was controversial with that for thermal stability. The best compromise between these two responses was: 3% (w/v) alginate, 3% (w/v) gelatin, 176.64 mM CaCl 2 and 2 mm bead-diameter. With this condition, CGTase was efficiently entrapped in the beads and its half-life at 50 °C was about 20 times longer than that of the free CGTase. The reusability of the entrapped CGTase was further enhanced when using glutaraldehyde as cross-linking agent and restabilizing the beads in CaCl 2 solution after each use. The entrapped CGTase could produce β- cyclodextrin at the highest yield of 8.6 g/L when using 4% starch solution.

Continuous production of β-cyclodextrin by cyclodextrin glycosyltransferase immobilized in mixed gel beads: Comparative study in continuous stirred tank reactor and packed bed reactor

Cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. C26 was highly stable when it was immobilized in mixed alginate–gelatin gel beads. Enzymatic synthesis of β-cyclodextrin (β-CD) from starch by immobilized CGTase was performed in a continuous stirred-tank reactor (CSTR) and a packed-bed reactor (PBR). A comparative study on reaction kinetics found that the apparent maximum production rate (Vmax,app) for reaction in CSTR (0.5742gL−1h−1) was higher than that in PBR (0.33gL−1h−1) likely due to the higher mass transfer rate in CSTR. The lower apparent Michaelis–Menten constant (Km,app) in PBR (31.88gL−1) compared to that in CSTR (58.3gL−1) indicate that the enzyme in PBR required lower amount of substrate to become half-saturated. The appropriate dilution rate for operation in both reactors was the same at 0.75h−1. To effectively produce β-CD, an integrated CSTR-PBR was developed. Through this integrated system, the final β-CD concentration and yield were improved from 6.10gL−1 and 15.3% in a single CSTR up to 10.6gL−1 and 26.5%, respectively. Moreover, the immobilized CGTase had good operational stability for 96h of continuous use. This study has shown that the integrated CSTR-PBR was effective for continuous production of β-CD from starch and may greatly contribute to developing industrialized production of β-CD.

Mutations at calcium binding site III in cyclodextrin glycosyltransferase improve β-cyclodextrin specificity

Cyclodextrin glycosyltransferases (CGTases, EC 2.4.1.19) are industrially important enzymes that produce cyclodextrins from starch by intramolecular transglycosylation. In this study, the effects of amino acid residue at position 315 in calcium binding site III (CaIII) on product specificity of CGTase were investigated by replacing Ala315 in the CGTase from Bacillus circulans STB01 with arginine, aspartic acid, threonine, leucine and valine. The cgt gene, which encodes this enzyme, was expressed in B. subtilis WB600 alongside site-directed mutants A315R, A315D, A315T, A315L and A315V. The results showed that CaIII plays an important role in cyclodextrin product specificity. Replacement of Ala315 by charged amino acid residues enhanced β-cyclodextrin specificity, compared with the wild-type CGTase. Mutations A315R and A315D resulted in an approximately 10% increase in β-cyclodextrin activity. Furthermore, under conditions resembling the industrial production processes, the mutants A315R and A315D displayed obvious increases in the production of β-cyclodextrin, indicating they were much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhancement of β-cyclodextrin specificity for the mutants might be due to the stability of CaIII by charged amino acid substitutions.

Mutations in cyclodextrin glycosyltransferase from Bacillus circulans enhance β-cyclization activity and β-cyclodextrin production.

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) is used to produce cyclodextrins, which are cyclic glucans with many industrial applications. In the present study, the effects of the amino acid residue at position 577, which is located in calcium-binding site III (CaIII), on cyclization activity and cyclodextrin production were investigated by replacing Asp577 in CGTase from Bacillus circulans STB01 with glutamate, arginine, lysine, and histidine. The results showed that mutations D577E and D577R significantly increased the β-cyclization activity. The D577R mutant, in particular, displayed a 30.7% increase in the β-cyclization activity when compared to the wild-type CGTase. Furthermore, under conditions resembling industrial production processes, the D577R and D577E mutants displayed 9.1 and 2.0% enhancement in β-cyclodextrin production, respectively. More importantly, the higher β-cyclization activities resulted in a significant reduction in the amount of mutant protein required during the process. Thus, the two mutants were much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme.

Mutations enhance β-cyclodextrin specificity of cyclodextrin glycosyltransferase from Bacillus circulans

The effects of amino acid residue at position 31 in the neighborhood of calcium binding site I (CaI) on product specificity of cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) were investigated by replacing Ala31 in the CGTase from Bacillus circulans STB01 with arginine, proline, threonine, serine and glycine. The results showed that the mutations A31R, A31P, and A31T resulted in the increases in β-cyclodextrin-forming activity and β-cyclodextrin production, indicating that these mutations enhanced β-cyclodextrin specificity of the CGTase. Especially the mutant A31R displayed approximately 26% increase in β-cyclodextrin production with a concomitant 41% decrease in α-cyclodextrin production when compared to the wild-type CGTase. Thus, it was much more suitable for the industrial production of β-cyclodextrin than the wild-type enzyme. The enhanced β-cyclodextrin specificity of the mutants might be a result of stabilizing CaI, which also suggested that CaI might play an important role in cyclodextrin product specificity of CGTase.

Modeling and Optimization of β-Cyclodextrin Production by Bacillus licheniformis using Artificial Neural Network and Genetic Algorithm

Background: The complexity of the fermentation processes is mainly due to the complex nature of the biological systems which follow the life in a non-linear manner. Joined performance of artificial neural network (ANN) and genetic algorithm (GA) in finding optimal solutions in experimentation has found to be superior compared to the statistical methods. Range of applications of β-cyclodextrin (β-CD) as an enzymatic derivative of starch is diverse, where the complex performance of cyclodextrin glucanotransferase (CGTase) as the involved enzyme is not well recognized. Objectives: The aim of the present work was to use ANN systems with different training algorithms and defined architectures joined with GA, in order to optimize β-CD production considering temperature of the reaction mixture, substrate concentration, and the inoculum’s pH as the input variables. Materials and Methods: Commercially Neural Power, version 2.5 (CPC-X Software, 2004) was used for the numerical analysis according to the specifications provided in the software. β-CD concentration was determined spectrophotometrically according to phenolphthalein discoloration technique, described in the literature. Results: Randomly obtaining the experimental data for β-CD production in a fermentation process, could get explainable order using the ANN system coupled with GA. Changes of the β-CD as the function of each of the three selected input variables, were best quantified with use of the ANN system joined with the GA. The performance of the IBP learning algorithm was highly favorable (10300 epoch’s number within 5 second, with the lowest RMSE value) while the sensitivity analysis of the results which was carried out according to the weight method, were indicative of the importance of input variables as follows: substrate concentration < temperature < inoculum’s pH. For instance, small changes in the system’s pH are associated with the large variation in the β-CD production as has been described by the suggested model. Conclusions: Production of β-CD (enzymatic derivative of starch) by B. licheniformis was satisfactorily described based on multivariate data analysis application of the ANN system and the experimental data were optimized by considering ANN plus the GA where the IBP was used as the training method and with use of three neurons as the constructed variables in the hidden layer of the test network.

Continuous production of β-cyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan

Cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on glutaraldehyde-activated chitosan spheres and used in a packed bed reactor to investigate the continuous production of β-cyclodextrin (β-CD). The optimum temperatures were 75°C and 85°C at pH 6.0, respectively for free and immobilized CGTase, and the optimum pH (5.0) was the same for both at 60°C. In the reactor, the effects of flow rate and substrate concentration in the β-CD production were evaluated. The optimum substrate concentration was 4% (w/v), maximizing the β-CD production (1.32g/L) in a flow rate of 3mL/min. In addition, the biocatalyst had good operational stability at 60°C, maintaining 61% of its initial activity after 100 cycles of batch and 100% after 100h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.

β-cyclodextrin production by the cyclodextrin glucanotransferase from Paenibacillus illinoisensis ZY-08: cloning, purification, and properties

The gene encoding the cyclodextrin glucanotransferase (CGTase, EC2.4.1.19) of Paenibacillus illinoisensis was isolated, cloned, sequenced and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34-residues. The deduced amino acid sequence of the CGTase from P. illinoisensis ZY-08 exhibited highest identity (99 %) to the CGTase sequence from Bacillus licheniformis (P14014). The four consensus regions of carbohydrate converting domain and Ca(2+) binding domain could be identified in the sequence. The CGTase was purified by using cold expression vector, pCold I, and His-tag affinity chromatography. The molecular weight of the purified enzyme was about 74 kDa. The optimum temperature and pH of the enzyme were 40 °C and pH 7.4, respectively. The enzyme activity was increased by the addition of Ca(2+) and inhibited by Ba(2+), Cu(2+), and Hg(2+). The K m and V max values calculated were 0.48 mg/ml and 51.38 mg of β-cyclodextrin/ml/min. The ZY-08 and recombinant readily converted soluble starch to β-cyclodextrin but ZY-08 did not convert king oyster mushroom powder and enoki mushroom powder. However the recombinant CGTase converted king oyster mushroom powder and enoki mushroom powder to β-cyclodextrin.