Cyclodextrin Glycosyltransferase Research Articles

Produção, purificação e aumento da performance de ciclodextrina glicosiltransferases para produção de ciclodextrinas

Biospecific affinity chromatography was used to purify three cyclodextrin glycosyl transferases (CGTases) obtained from microorganisms isolated of soil. The cyclodextrins (CDs) production by CGTases was evaluated using starches from different sources. CDs were measured through the Complexation Theory and by HPLC. CGTase from Bacillus firmus strain 7B showed the best production (30 mmol/L of β-CD and 4.3 mmol/L of γ-CD), and its cultivation conditions were optimized. The maximum enzymatic activity was achieved using lung peptone, soluble starch and agitation speed of 160 rpm. Studied CGTases were shown quite interesting for the industrial production of CDs.

Formation of cyclodextrins with cyclodextrin glucosyltransferase stimulated with polarized light

After illumination with white, linearly polarized light (WLPL), cyclodextrin glycosyltransferase produced mixture of alpha-, beta-, and gamma-cyclodextrins (CD) with higher overall yield than did that enzyme when nonilluminated. The illumination also influenced the ratio of those CD and that effect depended on concentration of enzyme and illumination time. At a high enzyme concentration (0.64 U/cm(3)), regardless the illumination time, formation of beta-CD predominated. The highest yield of beta-CD was afforded after 1 h illumination and 2 h illumination led to a significant increase in the yield of gamma-CD. Three-month storage of enzyme illuminated with WLPL did not reduce its enhanced activity.

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

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

The Polarized Light‐Induced Enzymatic Formation and Degradation of Biopolymers

AbstractSummary: White, linearly polarized transformed starch amylopectin into amylose. Moreover, it activated α‐amylolysis of starch, hydrolysis of xylane with xylanase, hydrolyses of chitin with chitinase, chitosan with chitosanase, and, interestingly, influenced production of cyclodextrins with cyclodextrin glycosyltransferase. Effect of duration of illumination of cyclodextrin glucosyltransferase with polarized light had certain effect upon the yield and isomer ration of three isomeric cyclodextrins. Application of the polarized light required 1–2 hour illumination of the enzymes in a small reaction vessel followed by admixture of so activated enzymes to a bioreactor. Further reactions did not require any illumination.

Rapid Access to Genes of Biotechnologically Useful Enzymes by Partial Genome Sequencing: The Thermoalkaliphile Anaerobranca gottschalkii

Anaerobranca gottschalkii strain LBS3^T is an extremophile living at high temperature (up to 65°C) and in alkaline environments (up to pH 10.5). An assembly of 696 DNA contigs representing about 96% of the 2.26-Mbp genome of A. gottschalkii has been generated with a low-sequence-coverage shotgun-sequencing strategy. The chosen sequencing strategy provided rapid and economical access to genes encoding key enzymes of the mono- and polysaccharide metabolism, without dilution of spare resources for extensive sequencing of genes lacking potential economical value. Five of these amylolytic enzymes of considerable commercial interest for biotechnological applications have been expressed and characterized in more detail after identification of their genes in the partial genome sequence: type I pullulanase, cyclodextrin glycosyltransferase (CGTase), two α-amylases (AmyA and AmyB), and an α-1,4-glucan-branching enzyme.

Extracellular expression, purification, and characterization of the recombinant cyclodextrin glycosyltransferase from Peanibacillus macerans

Extracellular expression, purification, and characterization of the recombinant cyclodextrin glycosyltransferase from Peanibacillus macerans

Structure–function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

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.

A novel thermophilic anaerobic bacteria producing cyclodextrin glycosyltransferase

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.

Estudos de parâmetros que influenciam na produção da enzima CGTase de Bacillus firmus, cepa nº 37

The cyclodextrins (CDs) are cyclic oligosaccharides formed by residues of glucopyranose linked by α-1,4. The most common are the α-, β- and γ-CD that present 6, 7 and 8 units of glucopyranose, respectively. They are produced from starch by the action of the enzyme cyclodextrin glycosyltransferase (CGTase). Frequently, β-CD is produced in larger amount. A study of optimization of the production of the CGTase from Bacillus firmus , strain no. 37 (β-CGTase) was performed. The production of the enzyme occurred during the phase of exponential growth of the microorganism and the maximum activity was observed within three days of cultivation at 37oC. The best production of the enzyme was obtained with inoculum of optical density between 0.5 and 1.0 (660 nm). The use of the maltodextrin for production of the enzyme provided an enzymatic activity at 31% lower than the substrate, soluble starch. Therefore, the substrate maltodextrin is not appropriate to improve the production of the studied enzyme.

Cyclodextrin production in hollow fiber membrane reactor system: Effect of substrate preparation

Production and separation of cyclodextrins (CDs) were assessed by employing an enzymatic membrane reactor (EMR) system. Comparison between gelatinized and ungelatinized tapioca starch was conducted to identify the pre-eminent type of tapioca starch via its reaction kinetic and the behavior of the ultrafiltration membrane in an EMR system. Results showed that the Michaelis constant value ( K m) for gelatinized tapioca starch was 47% lesser than the ungelatinized tapioca starch, which indicates higher affinity and strong substrate binding with the employed cyclodextrin glycosyltransferase (CGTase). However, gelatinized tapioca starch generated high membrane fouling, which demonstrated 60% higher hydraulic resistances and 7% lower percentage of flux recovery than ungelatinized tapioca starch, due to the cake deposition associated with large swollen tapioca starch molecules.

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.

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.

The Cyclodextrin Glycosyltransferase of Paenibacillus pabuli US132 Strain: Molecular Characterization and Overproduction of the Recombinant Enzyme

The gene encoding the cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was 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 enzyme exhibited the highest identity (94%) to the β-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37°C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19°C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed Vmax and Km values of 253 ± 36 μmol of β-cyclodextrin/mg/min and 0.36 ± 0.18 g/L, respectively.

Metodologia de seleção de cepas para produção etodologia da ciclodextrina glicosiltransferase e para purificação da enzima

Cyclodextrins (CDs) are maltooligosaccharides produced from starch by cyclodextrin glycosyltransferase (CGTase) enzyme. This research aimed at establishing method for strains selection for CGTase production and enzyme purification. The microorganisms were selected from 53 analyses of starch cultures soils on plates containing specific culture medium for strains selection that produce CGTase. The enzymes were obtained by culturing these microorganisms in liquid medium. The enzyme activity was determined with photospectrometric methods and precipitation with trichloroethylene. The strain isolated from oat soil was the one that showed the highest activity [0.1864 µmol of s-CD (min mL)-1]. This strain was used for enzyme production in laboratory scale and purification by biospecific affinity chromatography. The strain selected in this research opens new perspectives for enzymes production and CDs in industrial scale.

Production and single step purification of cyclodextrin glycosyltransferase from alkalophilic Bacillus firmus by ion exchange chromatography

Production and purification of starch digesting cyclodextrin glycosyl transferase (CGTase) from alkalophilic Bacillus firmus was investigated. Fermentation was carried out in 14 l bioreactor at 28 °C using a medium containing dextrin, yeast extract, peptone, (NH 4)H 2PO 4 and MgSO 4·7H 2O. The extracellular enzyme was concentrated by tangential flow ultrafiltration. The concentrated enzyme was chromatographed using DEAE-sepharose and phenyl sepharose. DEAE-sepharose could be used to purify CGTase in a single step with 23.1 fold purification and 80.6% recovery. The enzyme obtained had homogeneity and the molecular weight was 76 kDa confirmed by SDS-PAGE.

γ-Cyclodextrin: a review on enzymatic production and applications

Cyclodextrins are cyclic alpha-1,4-glucans that are produced from starch or starch derivates using cyclodextrin glycosyltransferase (CGTase). The most common forms are alpha-, beta-, and gamma-cyclodextrins. This mini-review focuses on the enzymatic production, unique properties, and applications of gamma-cyclodextrin as well as its difference with alpha- and beta-cyclodextrins. As all known wild-type CGTases produce a mixture of alpha-, beta-, and gamma-cyclodextrins, the obtaining of a CGTase predominantly producing gamma-cyclodextrin is discussed. Recently, more economic production processes for gamma-cyclodextrin have been developed using improved gamma-CGTases and appropriate complexing agents. Compared with alpha- and beta-cyclodextrins, gamma-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.

Production and characterization of a new cyclodextrin glycosyltransferase from Bacillus firmus isolated from Brazilian soil

A new CGTase was obtained from Bacillus firmus, strain 7B, isolated from oat soil culture, using a high alkaline pH medium containing 1% Na 2CO 3. 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 56,230 Da 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.

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.

Cloning and Expression of Cyclodextrin Glycosyltransferase Gene from Paenibacillus sp. T16 Isolated from Hot Spring Soil in Northern Thailand

Gene encoding cyclodextrin glycosyltransferase (CGTase), from thermotolerant Paenibacillus sp. T16 isolated from hot spring area in northern Thailand, was cloned and expressed in E. coli (JM109). The nucleotide sequences of both wild type and transformed CGTases consisted of 2139 bp open reading frame, 713 deduced amino acids residues with difference of 4 amino acid residues. The recombinant cells required 24 h culture time and a neutral pH for culture medium to produce compatible amount of CGTase compared to 72 h culture time and pH 10 for wild type. The recombinant and wild-type CGTases were purified by starch adsorption and phenyl sepharose column chromatography and characterized in parallel. Both enzymes showed molecular weight of 77 kDa and similar optimum pHs and temperatures with recombinant enzyme showing broader range. There were some significant difference in pH, temperature stability and kinetic parameters. The presence of high starch concentration resulted in higher thermostability in recombinant enzyme than the wild type. The recombinant enzyme was more stable at higher temperature and lower pH, with lower K(m) for coupling reaction using cellobiose and cyclodextrins as substrates.

A recombinant cyclodextrin glycosyltransferase cloned from Paenibacillus sp. strain RB01 showed improved catalytic activity in coupling reaction between cyclodextrins and disaccharides

Recombinant cyclodextrin glycosyltransferase (CGTase) was obtained by cloning the PCR gene fragment from thermotolerant Paenibacillus sp. strain RB01 screened from hot spring area in Thailand and cloned into the Escherichia coli expression vector. The nucleotide sequence was analyzed and aligned. Nucleotide sequence of the recombinant CGTase contained an open reading frame of 2139 bp encoding 713 amino acid residues. The recombinant required one-third of culture time and neutral pH to produce CGTase compared to wild type. CGTases from both wild type and transformant were purified in parallel by starch adsorption and DEAE cellulose column. Their biochemical properties such as molecular weight, optimum pH and temperature were quite similar. However, the recombinant enzyme showed improved catalytic activity in the coupling reaction between cyclodextrins (CDs) and some disaccharides. Among several sugars tested with excess βCD, cellobiose was the best substrate followed by leucrose. Very low activity was observed with trehalose, lactose and mellibiose. Sucrose and raffinose showed no activity. The K m and other kinetic parameters of recombinant enzyme were determined for cellobiose and several cyclodextrin derivatives. Recombinant CGTase showed lower K m for βCD and its derivatives, with improved activity compared to wild type enzyme.