The residue 179 is involved in product specificity of the <Emphasis Type="Italic">Bacillus circulans</Emphasis> DF 9R cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferases (CGTases) are important enzymes in biotechnology because of their ability to produce cyclodextrin (CD) mixtures from starch whose relative composition depends on enzyme source. A multiple alignment of 46 CGTases and Shannon entropy analysis allowed us to find differences and similarities that could be related to product specificity. Interestingly, position 179 has Gly in all the CGTases except in that from Bacillus circulans DF 9R which possesses Gln. The absence of a side chain at that position has been considered as a strong requirement for substrate binding and cyclization process. Therefore, we constructed two mutants of this enzyme, Q179L and Q179G. The activity and kinetic parameters of Q179G remained unchanged while the Q179L mutant showed a different CDs ratio, a lower catalytic efficiency, and a decreased ability to convert starch into CDs. We show that position 179 is involved in CGTase product specificity and must be occupied by Gly—without a side chain—or by amino acid residues able to interact with the substrate through hydrogen bonds in a way that the cyclization process occurs efficiently. These findings are also explained on the basis of a structural model.     

利用高效阴离子色谱快速检测筛选环糊精糖基转移酶产生菌>

利用高效阴离子色谱快速直接地检测微生物发酵液中的环糊精成分,尤其是大环环糊精的组成,进而创造了一种能快速准确地从土壤中筛选产环糊精糖基转移酶菌种的方法。共分离了149个产胞外淀粉水解酶的微生物菌株,利用高效阴离子交换色谱共检测了其中11株菌,其中6株主要产CD6 ,5株主要产CD7,主要产CD8的没有。在直接鉴定产生环糊精糖基转移酶菌株的过程中,也可以定量检测各种环糊精包括大环糊精（CD大于8）的含量。     

Enzyme modification of starch granules: formation and retention of cyclomaltodextrins inside starch granules by reaction of cyclomaltodextrin glucanosyltransferase with solid granules

Cyclomaltodextrin glucanosyltransferase (CGTase) was adsorbed into starch granules and allowed to react at 37 degrees C. The reaction was conducted with the granules removed from an aqueous environment, but containing 50% w/w water inside the granule. Reaction for 20 h gave a maximum of 1.4%, w/w of cyclodextrins (CDs) inside the granule. Waxy maize and maize starches gave the highest amounts of CDs (1.3 and 1.4%, respectively), with tapioca and amylomaize-7 starches giving about 50% less (0.9 and 0.6%, respectively). Reaction of a combination of CGTase and isoamylase with solid starch granules gave a 2.6-fold increase in the formation of CDs, with a maximum yield of 3.4 and 100% retention inside waxy maize starch granules.     

Cyclodextrin glycosyltransferase from Bacillus circulans DF 9R: Activity and kinetic studies

Activity characteristics and kinetic aspects of a cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans DF 9R were studied. A mixture of α-, β- and γ-cyclodextrins (CDs), glucose, maltose and negligible amounts of longer linear dextrins were produced from gelatinized amylose, amylopectin and starch from different sources. In the coupling reaction, CDs were the substrates in the presence of acceptors such as maltose and/or longer oligosaccharides. From oligosaccharides formed by three or more glucose units, this enzyme produced linear chains of several lengths which were then cyclized. CGTase catalytic efficiency was compared employing an analytical grade starch and cassava starch for food use. Since the results obtained were similar for both starches, the use of an economic starch is an advantage. CGTase was inhibited by the substrate and its own products. Starch concentrations over 20 mg/mL inhibited the cyclizing activity. CDs behaved as competitive inhibitors and maltose as an uncompetitive inhibitor while maltotriose showed a mixed inhibition pattern. Limit dextrins showed a scarce inhibitory effect on enzyme activity. CD production could be improved with an ultrafiltration membrane reactor for continuous removal of the products; the starch concentration should be maintained below an inhibitory concentration and limit dextrins would remain in the reactor without affecting enzyme activity.     

Optimization of cyclodextrin production from sago starch

Cyclodextrin (CD) is synthesized by bacterial cyclodextrin glycosyltransferase (CGTase) and is widely used in food, pharmaceutical, cosmetic, and agricultural industries. In this study, Bacillus circulans CGTase was partially purified by ammonium sulfate precipitation at 50-70% saturation. The optimum pH and temperature for CD production from sago starch were found to be in the ranges of 4.5-5.0 and 55-60 degrees C, respectively. beta-CD was the predominant product, constituting 65% of all CD products. The beta-CD produced using partially purified and crude CGTase were compared and found to have no significant difference in yield and productivity. The appropriate proportion of CGTase to sago starch for beta-CD production was determined by response surface methodology. The most appropriate enzyme:substrate ratio was 50 U g sago starch(-1) CGTase and 60 g l(-1) sago starch.     

The effect of cyclodextrins on the ethanol tolerance of microorganisms suggests potential application

Cyclodextrins (CDs) are used in food, pharmaceutical, and chemical industries, as well as agriculture and environmental engineering. Cyclodextrin glucanotransferase (CGTase) is an important industrial extracellular enzyme which is used to produce CDs and oligosaccharides. We previously developed a novel yeast-surface CGTase expression system which was used for the production of CDs from starch. In the present study, we showed that the presence of CDs may increase the ethanol tolerance of microorganisms. The cell numbers of Saccharomyces cerevisiae and Escherichia coli in the presence of β-cyclodextrin and ethanol were 1,000-fold and 10-fold higher than that without CDs. The yeast strain with the immobilized CGTase produced 13 g CDs/l and 1.8 g ethanol/l when it was incubated in yeast medium supplemented with 4% starch. The effect of CDs on microorganisms suggests a potential application for the co-production of CDs and ethanol.     

Ultrafiltration system for cyclodextrin production in repetitive batches by CGTase from <Emphasis Type="Italic">Bacillus firmus</Emphasis> strain 37

This study aimed to improve the yield of cyclodextrins (CDs) production in repetitive batches. An innovative ultrafiltration system was used to remove the inhibitory products that accumulated in the medium and to recover the enzyme. The assays were performed with the CGTase from Bacillus firmus strain 37 in purified, semi-purified, and crude extract forms. Maltodextrin (10 % w/v) and corn starch (5 % w/v) were used as substrates. After eight repetitive 24-h batches, the yield of β-CD obtained with the purified enzyme and the corn starch substrate was 0.54 mmol/L/h, which was 36 % greater than that observed with the 10 % maltodextrin substrate. The crude CGTase extract with the corn starch substrate showed a productivity of 0.38 mmol/L/h, which was 29 % lower than using the purified enzyme and the corn starch substrate but 7 % higher than using the purified enzyme and the maltodextrin substrate. The crude extract, assayed with the corn starch substrate in the presence of 10 % ethanol reached 0.43 mmol/L/h productivity, which was 12 % higher compared to the assay without ethanol. The semi-purified enzyme was assayed with the corn starch substrate in the presence of 10 % ethanol for eight batches lasting 12 h and an excellent selectivity for the β-CD was obtained, reaching a mean percentage of 96.0 %. Therefore, this ultrafiltration system enabled several batches of CD production, with efficient removal of products inhibitory to the CGTase and recovery of the enzyme. The possibility of industrial application of this system is promising.     

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%.     

Site-saturation mutagenesis of proline 176 in Cyclodextrin Glucosyltransferase from Bacillus sp. Y112 effects product specificity and enzymatic properties

Based on the analysis of amino acid sequence and simulated structure, saturation mutagenesis was performed to explore the role of the site p176 of cyclodextrin glucosytransferase (CGTase) from Bacillus sp. Y112. Compared to the wild-type, mutant P176G showed 10.4 % improvement in conversion from starch to cyclodextrins (CDs), whose β-CD yield increased by 6% and α-CD yield decreased by 8%. Mutants P176L and P176I were increased by 7.9 % and 9.4 % on CDs production, indicating replacement of hydrophobic amino acids significantly improved in cyclization activity. Kinetics studies indicated the substrate affinity of P176G and P176K were increase by 13 % and 14 %, and the catalytic efficiency of P176K was increase by 14 %. In addition, the optimal temperature of mutants transformed from 50℃ to 40℃ and the optimal pH shifted from 10.0 to 8.0. These results indicate that the site P176 plays a critical role in catalytic activity, product specificity and enzymatic properties of CGTase.     

Cloning,extracellular expression and characterization of a predominant β-CGTase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. G1 in <Emphasis Type="Italic">E. coli</Emphasis>

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60°C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% β-cyclodextrin (CD) and 10% γ-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of β-CD.     

A novel process of cyclodextrin production by the use of specific adsorbents: Part II. A new reactor system for selective production of α-cyclodextrin with specific adsorbent

We have developed a novel process of α-cyclodextrin (α-CD) production by using a new adsorbent that is characterized by its exceedingly powerful selectivity for α-CD compared with other CDs. α-CD production was carried out in a closed reactor system that was composed of a main reactor, wherein liquefied starch was converted to CDs by cyclodextrin glucosyltransferase (CGTase: EC 2.4.1.19), and a column packed with the adsorbent. While the reaction mixture was circulated in the system, α-CD was selectively adsorbed in the column and its concentration in the mixture of the main reactor was kept at a low level. This low concentration of α-CD stimulated the conversion of starch to CDs and as a result, enhanced its yield based on added starch. When 8.3 % (w/v) of liquefied starch was used in the reactor system, the yield of α-CD was 22.2% and α-CD occupied 58.7 % of the reaction mixture of total CDs synthesized. Meanwhile, in a batch system without the adsorbent, the yield of α-CD and its fraction were 10.8% and 45.0%, respectively. After the conversion reaction, and following the preliminary washing with water through the column. α-CD was easily eluted with hot water, resulting in a high purity of about 95%.     

Enzymatic production of cyclodextrins from milled corn starch in an ultrafiltration membrane bioreactor

Cyclodextrin glycosyltransferase (CGTase) was found to be severely inhibited by cyclodextrins. In order to increase the conversion yield by reducing product inhibition and reuse the CGTase in the production of cyclodextrins from milled corn starch, an ultrafiltration membrane bioreactor system was employed. In a batch operation with ultrafiltration, the conversion yield was increased 57% compared with that without ultrafiltration. Operating conditions for the continuous production of cyclodextrins in the membrane bioreactor were optimized by taking into consideration the filtration rate and the conversion yield as follows: initial starch concentration, 7% (w/v); starch feeding rate, 240 mg/h; CGTase loading, 350 units/initial gram starch. When cyclodextrins were continuously produced in the membrane bioreactor under optimized conditions, 340 units of CGTase was require to produce 1 g of cyclodextrins for 48 h, while in the case of conventional batch operation, 1 g of cyclodextrins was produced for 24 h by 1410 units of CGTase. (c) 1993 John Wiley & Sons, Inc.     

Production of cyclodextrin glucanotransferase from an alkalophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. by pH-stat fed-batch fermentation

Batch and fed-batch fermentation processes were employed to culture an alkalophilic Bacillus sp. for the production of cyclodextrin glucanotransferase (CGTase). CGTase production was repressed by glucose and induced by soluble starch. By fed-batch fermentation, a CGTase activity up to 56 unit ml⁻¹ with 65 g dry cells l⁻¹ were achieved. The CGTase activity and cell density were increased 360 and 510%, respectively, from those values achieved with batch fermentation.     

Sequencing,cloning, and heterologous expression of cyclomaltodextrin glucanotransferase of Bacillus firmus strain 37 in Bacillus subtilis WB800

Bacillusfirmus strain 37 produces the cyclomaltodextrin glucanotransferase (CGTase) enzyme and CGTase produces cyclodextrins (CDs) through a starch cyclization reaction. The strategy for the cloning and expression of recombinant CGTase is a potentially viable alternative for the economically viable production of CGTase for use in industrial processes. The present study used Bacillus subtilis WB800 as a bacterial expression host for the production of recombinant CGTase cloned from the CGTase gene of B. firmus strain 37. The CGTase gene was cloned in TOPO-TA^® plasmid, which was transformed in Escherichia coli DH5α. The subcloning was carried out with pWB980 plasmid and transformation in B. subtilis WB800. The 2xYT medium was the most suitable for the production of recombinant CGTase. The enzymatic activity of the crude extract of the recombinant CGTase of B. subtilis WB800 was 1.33 µmol β-CD/min/mL, or 7.4 times greater than the enzymatic activity of the crude extract of CGTase obtained from the wild strain. Following purification, the recombinant CGTase exhibited an enzymatic activity of 157.78 µmol β-CD/min/mL, while the activity of the CGTase from the wild strain was 9.54 µmol β-CD/min/mL. When optimal CDs production conditions for the CGTase from B. firmus strain 37 were used, it was observed that the catalytic properties of the CGTase enzymes were equivalent. The strategy for the cloning and expression of CGTase in B. subtilis WB800 was efficient, with the production of greater quantities of CGTase than with the wild strain, offering essential data for the large-scale production of the recombinant enzyme.

     

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha-cyclomaltodextrin (alpha-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991--1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95 degrees C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed beta-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to alpha-CD. However, the CBP exhibited higher thermostability in the presence of alpha-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.     

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 (μ_max) were observed with glucose, γ-CD and soluble starch (μ_max 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.     

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.     

Organic solvent and pH induced alteration of product specificity of CGTase

Cyclodextrin glucanotransferase [CGTase, E.C.2.4.1.19] is an extracellular enzyme, which catalyzes the formation of α−, β−, γ− CDs from starch. Their proportions of formations depend on enzyme sources and reaction conditions. To understand what determines the product specificity of CGTases, we examined the alteration of product specificity of CGTase fromBacillus macerans by organic solvents and pH. At acidic pH range less than pH 6 where the enzyme was unstable, the ratio of α−/β-CD production was increased 4 times more than that at neutral pH range. As we increased the concentration of 2-butanol, α−/β-CD ratio was proportionally increased but/ratio remained constant. The α−/β-CD ratio of products was increased in the reaction media which yielded low products.     

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.     

Effect of PEG and other additives on cyclodextrin production by Bacillus macerans cyclomaltodextrin-glycosyl-transferase

Summary The effect of PEG and other polyols additives on cyclodextrins (CDs) production by Bacillus macerons cyclomaltodextrin-glycosyl-transferase (CGTase) was investigated. Mannitol, glycerol and PEG-200 (20%,v/v) enhanced the enzymatic production yield regardless of substrate concentration. Furthermore, the PEG-200 addition increased the thermostability of the CGTase.