Cyclodextrin glucanotransferase synthesis by semicontinuous cultivation of magnetic biocatalysts from cells of Bacillus circulans ATCC 21783

Cells of the alkalotolerant producer of cyclodextrin glucanotransferase (CGTase) Bacillus circulans ATCC 21783 were used as a model for preparing of magnetic biocatalysts applied for CGTase synthesis in batch and semicontinuous processes. The cell immobilization was carried out with four types of magnetic nano- and microparticles: magnetite microparticles (1–5 μm), entrapped in agar gel beads with bacterial cells (AM-biocatalyst); silanized magnetite (20–40 nm) covalently bound on the cell surface (SM-biocatalyst); and alkaline and citrate ferrofluids (10–20 nm), attached on the cell wall by an ionic interaction (FF-alkaline and FF-citrate biocatalyst). The highest CGTase production was achieved after 96 h of semicontinuous process using SM-biocatalysts (particularly, these composed of 80 mg silanized magnetite and 140 mg bacterial cells) when the specific enzyme activity was 8.4-fold higher compared to that of free cells. Cells modified with magnetic alkaline and citrate ferrofluids exhibited 2.19- and 1.55-fold increase of the specific CGTase activities. Magnetic nanoparticles linked on the cell walls by ionic interactions were partially released during the cultivation, while the covalent bond between the activated magnetite and the cells was very stable. The data obtained demonstrate convincingly the effect of the magnetic technologies for an effective enzyme production.     

Integrated immobilized cell reactor-adsorption system for β-cyclodextrin production: a model study using PVA-cryogel entrapped Bacillus agaradhaerens cells

Production of cyclodextrins (CDs) by immobilized cells of the alkaliphilic Bacillus agaradhaerens LS-3C with integrated product recovery was studied. The microorganism was entrapped in polyvinyl alcohol-cryogel beads and used as a convenient source of immobilized cyclodextrin glycosyltransferase (CGTase). On activation by incubation in the cultivation medium containing 1% (w/v) starch, the entrapped cells multiplied and secreted CGTase with an activity of 2–3 mg -cyclodextrin h^–1 g^–1 beads. The immobilized biocatalyst exhibited maximum activity at pH 9 and 50 °C, and formed cyclodextrins comprising 92–94% -CD and remaining -CD. The cyclodextrin product from the immobilized cell bioreactor was continuously recovered by adsorption to Amberlite XAD-4 in a recycle batch mode. The product adsorption was facilitated at low temperature while hot water was used for elution.     

α-环状糊精葡萄糖基转移酶的固定化及其性质研究

研究了一种α-环糊精葡萄糖基转移酶的固定化和固定化酶的性质。通过对戊二醛浓度、酶量和交联时间各单因素的考察,确定了最佳的固定化条件。与游离酶相比,以DEAE纤维素为载体的固定化酶最适pH向酸性偏移,最适温度不变,pH稳定性和热稳定性都有所提高。在40℃、150r/min下反应3h,转化率可以达到32%。固定化酶可以连续使用4次以上。固定化酶在4℃、5mmol/L CaCl2溶液里保存18d,还剩余80%以上的活力。     

Synthesis of Cyclodextrin glycosyl transferase by immobilized cells of Bacillus circulans

The cells of Bacillus circulans (ATCC 21783) immobilized in sodium alginate gel matrix were able to synthesize the extracellular enzyme, Cyclodextrin glycosyl transferase (CGTase, E.C. 2.4.1.19) which is industrially employed for the preparation of cyclodextrins. Optimization for the maximum production of enzyme was carried out by varying the cell density (3.3–53.5 kg/m³) in the gel and the incubation temperature (30°–42°C). The CGTase activity was found to be the highest (45 units/cm³) with maximum cell loading at 37°C. The reusability of immobilized cells was ascertained by repeated batch experiments. The enzyme activity exhibited was in the range of 50 to 55 units/cm³ in each batch. The continuous synthesis of CGTase by immobilized cells has been demonstrated by operating a fluidized bed reactor at a dilution rate 1.1 · 10^–4 sec^–1 for a period of 15 days. The enzyme activity has decreased to 42.5 units/cm³ from an initial value of 61 units/cm³ during continuous operation.The authors are grateful to Dr. A.D. Damodaran, Director, Regional Research Laboratory, Trivandrum for his keen interest and encouragement and to Department of Biotechnology, Government of India, New Delhi for financial support.     

Effect of different substrates and their preliminary treatment on cyclodextrin production

Summary Various kinds of substrates were tested for cyclodextrin production with cyclodextrin glucanotransferase (CGTase) from Bacillus megaterium. The enzyme formed cyclodextrin from different kinds of starch, dextrins, amylose, and amylopectin. However, the highest degree of conversion was obtained from starch. Corn starch appeared to be the best substrate – the cyclodextrin yield was 50.9%. The effect of molecular mass and preliminary treatment of starch with α-amylase on the CD yield was investigated. It was proved that CGTase preferred native starch with high molecular mass and low dextrose equivalent. The preliminary treatment with α-amylase occurred to be inefficient and unnecessary since it did not lead to an increase in the CD yield. Some of the substrates were treated with pullulanase. The effect of debranching was highest in the case of corn starch: the cyclodextrin yield increased by 10%.     

Cyclodextrin production and genetic characterization of cyclodextrin glucanotranferase of <Emphasis Type="Italic">Paenibacillus graminis</Emphasis>

Paenibacillus graminis strains were described recently as cyclodextrin (CD) producers. Cyclodextrins are produced by cyclodextrin glucanotransferase (CGTase) which has not been characterized in P. graminis. Similar amounts of α- and β-CDs were produced by P. graminis (MC22.13) and P. macerans (LMD24.10^T). Primers were designed to sequence the gene encoding CGTase from P. graminis. A phylogenetic tree was constructed and P. graminis CGTase protein showed to be closer (79.4% protein identity) to P. macerans |P31835|. Hybridization studies suggested that the gene encoding CGTase is located in different positions in the genomes of P. macerans and P. graminis.     

Optimisation of batch culture conditions for cyclodextrin glucanotransferase production from Bacillus circulans DF 9R

Background  

The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related α-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans.     

Molecular cloning and characterization of a novel γ-CGTase from alkalophilic Bacillus sp.

We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known enzyme β-/γ-CGTase from Bacillus firmus/lentus 290-3. However, the product specificity of CGTase825-6 differed from that of β-/γ-CGTase. CGTase825-6 produced γ-cyclodextrin (CD) as the main product, but degradation of γ-CD was observed with prolonged reaction. The product specificity of the enzyme was positioned between γ-CGTase produced by Bacillus clarkii 7364 and B. firmus/lentus 290-3 β-/γ-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671 residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8 for γ-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool to produce γ-CD and to study the differences of producing mechanisms between γ-CD and β-CD.     

Rapid Affinity Purification Processes for Cyclodextrin Glycosyltransferase from <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">circulans</Emphasis>

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 mM 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.     

A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity

Low reaction yields and the high cost of obtaining a single type of pure CD make γ-CD costly. Using rational design and with the aid of 3D modeling structures, recombinant CGTase from Bacillus sp. G1 was molecularly engineered with the aim of producing a higher percentage of γ-CD. A single mutation at subsite −3, denoted H43T, was found to increase γ-CD production from 10% to approximately 39% using tapioca starch. This novel increment was probably the result of reduced steric hindrance to the formation of γ-CD because of the shortened side chain together with the shortened loop at positions 86–89, at substrate-binding subsite −3. A mutation (Tyr188 → Trp) and a deletion at loop 139–144 showed little effect on product specificity; however, mutagenesis at these sites affected cyclization, coupling and hydrolysis activities as well as the kinetic properties of the mutant CGTase. Based on rational design, three further mutations of the mutant H43T (denoted H43T/Δ(139–144)/S134T/A137V/L138D/V139I, H43T/S85G and H43T/Y87F) were constructed and produced γ-CD with yields of 20%, 20% and 39%, respectively. The mutant H43T/Δ(139–144)/S134T/A137V/L138D/V139I had very low cyclization and coupling activities, however their hydrolysis activity was retained. Double mutation (H43T/S85G) caused the enzyme to exhibit higher starch hydrolysis activity, approximately 26 times higher than the native CGTase G1. Although the mutants H43T and H43T/Y87F could produce the same percentage (39%) of γ-CD, the latter was more efficient as the total amount of CD produced was higher based on the V_max and k_cat values.     

Performance of Aspergillus niger B 03 β-xylosidase immobilized on polyamide membrane support

The dynamics of β-xylosidase biosynthesis from Aspergillus niger B 03 was investigated in laboratory bioreactor. Maximum xylosidase activity 5.5 U/ml was achieved after 80 h fermentation at medium pH 4.0. The isolated β-xylosidase was immobilized on polyamide membrane support and the basic characteristics of the immobilized enzyme were determined. Maximum immobilization and activity yield obtained was 30.0 and 6.8%, respectively. A shift in temperature optimum and pH optimum was observed for immobilized β-xylosidase compared to the free enzyme. Immobilized enzyme exhibited maximum activity at 45 °C and pH 4.5 while its free counterpart at 70 °C and pH 3.5, respectively. Thermal stability at 40 and 50 °C and storage stability of immobilized β-xylosidase were investigated at pH 5.0. Kinetic parameters K_m, V_max and K_i were determined for both enzyme forms. Free and immobilized β-xylosidase were tested for xylose production from birchwood xylan. The substrate was preliminarily depolymerized with xylanase to xylooligosaccharides and the amount of xylose obtained after their hydrolysis with free and immobilized β-xylosidase was determined by HPLC analysis. Continuous enzyme hydrolysis of birchwood xylan was performed with xylanase and free or immobilized β-xylosidase. The maximum extent of hydrolysis was 25 and 30% with free and immobilized enzyme, respectively. Immobilized preparation was also examined for reusability in 20 consecutive cycles at 40 °C.     

Intermolecular transglycosylating reaction of cyclodextrin glucanotransferase immobilized on capillary membrane

The intermolecular transglycosylating reaction of cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) immobilized on a capillary membrane was investigated using low molecular weight substrates such as cyclodextrin (CD), maltooligosaccharide (MOS), and a CD-MOS mixture. The immobilized CGTase catalyzed the conversion reaction of α-CD to β-CD and MOS or β-CD to α-CD and MOS within a short residence time. The conversion ratio increased as the amount of immobilized CGTase increased. The addition of glucose, maltose, and sucrose as acceptors in the substrate solution containing CD resulted in the acceleration of CD degradation compared with only CD substrate. Furthermore, the MOS substrate (degree of polymerization =2–6) was disproportionated with a conversion ratio exceeding 70% by the immobilized CGTase. These data demonstrate that immobilized CGTase can catalyze intermolecular transglycosylation between low molecular substrates in a few minutes by regulating the amount of immobilized enzyme and the residence time. This might contribute to our comprehension of CGTase-immobilized bioreactors for CD production as well as to the development of new glycosides through its excellent transglycosylation ability.     

Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst

The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.

The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.     

Agar gel immobilization ofBacillus brevis cells for production of thermostable α-amylase

Growing cells of a thermophilic strain ofBacillus brevis, producer of thermostable α-amylase, were immobilized by entrapment in agar gel. Optimum immobilization conditions for effective α-amylase production in batch fermentations were established (gel concentration 3%, initial biomass concentration in the gel 0.8% (W/V), and preculture age—late exponential phase). The dynamics of α-amylase synthesis by the biocatalysts obtained under the optimal conditions was compared with that of free cells and the operational stability of the biocatalysts was studied in semicontinuous cultivation experiments. Maximum α-amylase yields (252% of the control) were achieved after the second cycle of cultivation. Scanning electron microscopy was used to characterize the bacteria entrapped in agar gel.     

Overexpression of cyclodextrin glycosyltransferase gene from Brevibacillus brevis in Escherichia coli by control of temperature and mannitol concentration

Expression of Brevibacillus brevis CD162 cyclodextrin glycosyltransferase (CGTase) gene using pET22b(+) vector in Escherichia coli BL21(DE3) resulted in the formation of inactive inclusion bodies under the usual induction conditions. However, by lowering the induction temperature to 30°C and/or adding 0.5 M mannitol as an osmolyte, the formation of insoluble aggregates was prevented and about a 34-fold increase (8.51 U ml^–1) in biologically active soluble form was achieved after 6 h induction. The active CGTase enzyme was estimated to comprise as much as 24% of the total soluble proteins. In addition, other polyols such as glycerol, erythritol, xylitol, sorbitol, and arabitol showed similar effects with mannitol on the production of active CGTase enzyme.     

Cyclodextrin production by Bacillus firmus strain 37 immobilized on inorganic matrices and alginate gel

Production of β-cyclodextrin (β-CD) by Bacillus firmus strain 37 cells, immobilized by adsorption on silica–titania (SiO₂/TiO₂) and silica–manganese dioxide (SiO₂/MnO₂) matrices, was optimized for temperature, substrate concentration and initial biomass. The immobilization process was most efficient at 60 °C with 10% maltodextrin and 1.0 g of cells, resulting, after a 5-day assay, in a β-CD production of 11.7 ± 0.1 mM for cells immobilized on SiO₂/TiO₂ and 11.2 ± 0.1 mM in SiO₂/MnO₂. Entrapment in alginate gel resulted in a maximum β-CD production of 4.1 ± 0.1 mM, which was maintained constantly until the end of a 10-day assay. During this same period, free cells produced 8.3 ± 0.2 mM, and cells immobilized on SiO₂/TiO₂ and SiO₂/MnO₂, 16.7 ± 0.4 and 17.3 ± 0.5 mM, respectively. β-CD production by cells immobilized in calcium alginate in four repetitive cycles of 5 days each, showed an increase up to the third cycle, reaching 4.8 ± 0.2 mM, while production by free cells started falling from the second cycle. In this same assay, cells immobilized on SiO₂/TiO₂ and SiO₂/MnO₂, showed the best β-CD production results at the end of the first cycle, with a gradual fall occurring due to the desorption of cells thereafter.     

Immobilisation of cyclodextrin glucanotransferase from <Emphasis Type="Italic">Bacillus circulans</Emphasis> ATCC 21783 on purified seasand

Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans (ATCC 21783) was immobilised on a silica-based support: purified seasand. Although adsorption of 98% was achieved, considerable desorption was encountered. This problem was minimised by crosslinking the adsorbed enzyme with glutaraldehyde. The immobilised enzyme after crosslinking could be used repeatedly for cyclodextrin (CD) production in a batch process. The activity retention was 80% at the end of the eighth cycle. The immobilised enzyme showed a shift in the pH optimum towards the alkaline side and also an improvement in the pH stability compared to the free enzyme. It catalysed the formation of β-CD as a major product. A significant amount of α-CD production was also observed on prolonged incubation. Electronic Publication     

Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity

The thermoalkalophilic Bacillus halodurans JB 99 cells known for production of novel thermostable alkaline keratinolytic protease were immobilized in calcium alginate matrix. Batch and repeated batch cultivation using calcium alginate immobilized cells were studied for alkaline protease production in submerged fermentation. Immobilized cells with 2.5% alginate and 350 beads/flask of initial cell loading showed enhanced production of alkaline protease by 23.2% (5,275 ± 39.4 U/ml) as compared to free cells (4,280 ± 35.4 U/ml) after 24 h. In the semicontinuous mode of cultivation, immobilized cells under optimized conditions produced an appreciable level of alkaline protease in up to nine cycles and reached a maximal value of 5,975 U/ml after the seventh cycle. The enzyme produced from immobilized cells efficiently degraded chicken feathers in the presence of a reducing agent which can help the poultry industry in the management of keratin-rich waste and obtaining value-added products.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Magnetic porous corn starch for the affinity purification of cyclodextrin glucanotransferase produced by Bacillus circulans

Magnetic porous corn starch was prepared as an affinity adsorbent for the efficient and simple scale-up procedure for one-step purification of cyclodextrin glucanotransferase (CGTase) from Bacillus circulans. Magnetic affinity separation enabled isolation of CGTase from cultivation media (volumes between 10 and 400 mL) with ca 60–70% recovery after elution with alkaline buffers containing soluble starch; the enzyme purification factor was 19–25 in different batches. The majority of ballast proteins were removed during the purification process, which shows high selectivity of the affinity material used.