Engineering of Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae for Improved Cyclodextrin Production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.     

Sequence analysis,cloning and extracellular expression of cyclodextrin glucanotransferase gene from the alkaliphilic Bacillus pseudalcaliphilus 8SB in Escherichia coli

The cgt-gene from the alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was isolated and sequenced. An open reading frame (ORF) of 2112 bp encoding a polypeptide of 704 amino acids, composed of a 29-amino acid signal sequence and a 675-amino acid mature enzyme was found. The established low level of homology with nucleotide sequences of other Bacillus CGTases (less than 82%) suggested that the cgt-gene from Bacillus pseudalcaliphilus 8SB encodes a new enzyme. The cgt-gene was cloned as a PCR amplicon and thereby the construction of genome library was avoided. This is the first evidence for the use of pJET vector as an expression vector. The opportunity to apply its T7 promoter for efficient extracellular production of heterologous proteins in Escherichia coli BL21 (DE3) was demonstrated. The expression of extracellular recombinant CGTase improved 23-fold, concerning β-CGTase activity and 4.5-fold concerning γ-CGTase activity after IPTG induction and glycine supplementation was achieved.     

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.     

Engineering of cyclodextrin glucanotransferase on the cell surface of Saccharomyces cerevisiae for improved cyclodextrin production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.     

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.     

Enhancement of enzymatic production of cyclodextrins by adding polyethylene glycol or polypropylene glycol

Enzymatic production of cyclodextrins (CDs) from soluble starch was studied using either Bacillus macerans or Bacillus ohbensis cyclomaltodextrin glucanotransferase (CGTase). The production yield of CDs was found to be increased up to 1.5–2 times by the addition of low molecular weight polyethylene glycol (PEG 400) or polypropylene glycol (PPG 425) to the reaction medium. Such results were interpreted as being due to a conformational change of the substrate as well as reduction of hydrolytic activity of the enzyme in the presence of these additives.     

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.     

Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization

Enhancing the production of α-cyclodextrin glycosyltransferase (α-CGTase) is a key aim in α-CGTase industries. Here, the mature α-cgt gene from Paenibacillus macerans JFB05-01 was redesigned with systematic codon optimization to preferentially match codon frequencies of Escherichia coli without altering the amino acid sequence. Following synthesis, codon-optimized α-cgt (coα-cgt) and wild-type α-cgt (wtα-cgt) genes were cloned into pET-20b(+) and expressed in E.?coli BL21(DE3). The total protein yield of the synthetic gene was greater than wtα-cgt expression (1,710?mg?L^?1) by 2,520?mg?L^?1, with the extracellular enzyme activity being improved to 55.3?U?mL^?1 in flask fermentation. ΔG values at -3 to +50 of the pelB site of both genes were ?19.10?kcal?mol^?1. Functionally, coα-CGTase was equally as effective as wtα-CGTase in forming α-cyclodextrin (α-CD). These findings suggest that preferred codon usage is advantageous for translational efficiency to increase protein expression. Finally, batch fermentation was applied, and the extracellular coα-CGTase enzyme activity was 326?% that of wtα-CGTase. The results suggest that codon optimization is a reasonable strategy to improve the yield of α-CGTase for industrial application.     

Expression of Bacillus macerans cyclodextrin glucanotransferase gene in Saccharomyces cerevisiae

Cyclodextrin glucanotransferase (CGTase) gene of Bacillus macerans was subcloned down-stream of yeast ADH1 promoter and expressed in Saccharomyces cerevisiae. Most of the CGTase expressed was in the extracellular medium with a maximum activity of about 0.28 unit ml^–1 after 48 h cultivation. The recombinant CGTase was secreted as an N-linked-glycosylated form and predominantly produced -cyclodextrin from 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.     

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.     

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.

     

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 Ca²⁺ 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 Ca²⁺ and inhibited by Ba²⁺, Cu²⁺, and Hg²⁺. 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.     

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.     

Carbohydrate-Binding Module–Cyclodextrin Glycosyltransferase Fusion Enables Efficient Synthesis of 2-O-d-Glucopyranosyl-l-Ascorbic Acid with Soluble Starch as the Glycosyl Donor

In this study, we achieved the efficient synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) from soluble starch by fusing a carbohydrate-binding module (CBM) from Alkalimonas amylolytica α-amylase (CBM_Amy) to cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans. One fusion enzyme, CGT-CBM_Amy, was constructed by fusing the CBM_Amy to the C-terminal region of CGTase, and the other fusion enzyme, CGTΔE-CBM_Amy, was obtained by replacing the E domain of CGTase with CBM_Amy. The two fusion enzymes were then used to synthesize AA-2G from soluble starch as a cheap and easily soluble glycosyl donor. Under the optimal conditions, the AA-2G yields produced using CGTΔE-CBM_Amy and CGT-CBM_Amy were 2.01 g/liter and 3.03 g/liter, respectively, which were 3.94- and 5.94-fold of the yield from the wild-type CGTase (0.51 g/liter). The reaction kinetics of the two fusion enzymes were analyzed and modeled to confirm the enhanced specificity toward soluble starch. It was also found that, compared to the wild-type CGTase, the two fusion enzymes had relatively high hydrolysis and disproportionation activities, factors that favor AA-2G synthesis. Finally, it was speculated that the enhancement of soluble starch specificity may be related to the changes of substrate binding ability and the substrate binding sites between the CBM and the starch granule.     

Cyclodextrin glycosyltransferase: a key enzyme in the assimilation of starch by the halophilic archaeon Haloferax mediterranei

A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.     

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.     

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.     

Purification and properties of Bacillus coagulans cyclomaltodextrin glucanotransferase

Cyclomaltodextrin glucanotransferase (CGTase), produced in a culture filtrate by Bacillus coagulans, was purified to a single, homogeneous protein. It has a monomeric structure with a molecular weight of 65,000, isoelectric point of 4.6, and contains 2 mol of Ca²⁺ per mol of the enzyme. The enzyme was most active at pH 6.0 and at 70°C. It did not lose its activity by heat treatment at 70°C for 10 min in the presence of CaCl₂ in the pH range of 5.5∼9.5, and by incubation in the pH range of 5.0∼10.5 at 4°C for one month. The enzyme converted about 60% of potato starch to cyclodextrins for 20 h at 50°C, and the ratio of α-: β-: γ-cyclodextrin produced was 8.1:8.9:1.0 B. coagulans CGTase was compared with B. macerans CGTase which was purified by the same method.