Isolation of highly purified cyclodextrin glucanotransferase from Bacillus sp. 1070

Two chromatographic processes for purification of cyclodextringlucanotransferase (CGTase) from Bacillus sp. 1070 was carried out. The enzyme has been purified into 9.5 times on Butyl-Toyopearl and followed immobilized metal ion chromatography on Cu(II)-Iminodiacetic (IDA)-agarose. By the application of second purification scheme (chromatography on Butyl-Toyopearl and DEAE-Sephacel) the specific activity of CGTase has folded into 13.5 times. The purity of enzyme was shown to be approximately 90% by SDS-electrophoreses data. It was shown that isolated enzyme has two isoelectric points estimated as 5.1 and 5.3.     

Mutations affecting the activity of the cyclodextrin glucanotransferase of Paenibacillus pabuli US132: insights into the low hydrolytic activity of cyclodextrin glucanotransferases

The cyclodextrin glucanotransferase from Paenibacillus pabuli US132 (US132 CGTase) was engineered using a rational approach in an attempt to provide it with anti-staling properties comparable to those of the commercial maltogenic amylase (Novamyl). The study aimed to concurrently decrease the cyclization activity and increase the hydrolytic activity of US132 CGTase. A five-residue loop (PAGFS) was inserted, alone or with the substitution of essential residues for cyclization (G180, L194 and Y195), mimicking the case of Novamyl. The findings indicate that, unlike the case of the CGTase of Thermoanerobacterium thermosulfurigenes strain EM1 whose initial high hydrolytic activity was exceptional, these mutations completely abolished the cyclization and hydrolytic activities of the US132 CGTase. This suggests that those mutations are not able to convert conventional CGTases, whose hydrolytic activities are very weak, into hydrolases. Accordingly, and for the first time, a structural barrier at subsite ?3 was advanced as an influential factor which might explain the low hydrolytic activity of conventional CGTases.     

The evolution of cyclodextrin glucanotransferase product specificity

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, β- and γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Hans Leemhuis acknowledges financial support from the Netherlands Organization for Scientific Research (NWO).     

Immobilization of cyclodextrin glucanotransferase on capillary membrane

A bioreactor system with the enzyme immobilized on a capillary membrane is a promising tool for the mass production of valuable substances, because of the good productive efficiency. To investigate the kinetics of immobilized cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) on a capillary membrane in a bioreactor system, the amount of immobilized CGTase and the operating conditions, such as pressure and the reaction temperature, were examined under a constant substrate concentration (1.0%) and a constant flow rate (0.12 m/s). When the CGTase was immobilized at a concentration of 0.04 to 0.62 mg per membrane area (cm²), the decrease in the immobilized amount of CGTase resulted in an increase in the cyclodextrin production rate (g of CD/h·m²; CPR) and the CPR correlated well with the flux of the CGTase-immobilized membrane. Although a higher reaction temperature caused an increase in the CPR within a short operating time of the bioreactor, repeated operation at 60°C led to a reduction in the CPR due to the denaturation of the immobilized CGTase. The percentage of cyclodextrin (CD) to total sugar obtained in the permeate was slightly more than 60% under most operating conditions, but immobilization of the excess amount of CGTase (0.42–0.62 mg/cm²) reduced the CD yield as well as the ratio of α-CD to β-CD, suggesting that it led to a CGTase side-reaction such as intermolecular transglycosylation. These data suggest that the conditions under which the bioreactor with 0.04–0.40 mg/cm² was operated; a reaction temperature of 50°C, a residence time of 1–2 min and adjustable pressure, could be employed to obtain a high CPR using a large scale CGTase-immobilized membrane bioreactor.     

Glucosylation of sucrose laurate with cyclodextrin glucanotransferase

Sucrose monolauroyl esters were found to serve as substrates for cyclodextrin glucanotransferase (CGTase)-catalyzed transglucosidation reactions, affording new sucrose esters that have an additional 1-3 glucose residues on the pyranose ring of the sucrose moiety in the ester.     

Enzymatic synthesis of piceid glycosides by cyclodextrin glucanotransferase

Novel glycosides of piceid (3,4′-5-trihydroxy stilbene 3-O-β-d-glucoside) were produced by the transglycosylation reactions of cyclodextrin glucanotransferase (CGTase) from Bacillus macerans, with piceid (PicG₁) and maltodextrin as the acceptor and donor substrates, respectively. The reactions were performed at 40 °C with 2.56 mM piceid (0.1% w/v) and maltodextrin (5% w/v) in 0.02 M citrate phosphate buffer, pH 6.0 containing 5% (v/v) methanol for 6 h. Glucose, maltose, sucrose, maltotriose and α-cyclodextrin (α-CD) were also used to analyze their ability to function as donor substrates, for the glycosylation of piceid. Among the different donor substrates used, the maximum transfer efficiency (TE) of glycosylation of piceid was observed for α-cyclodextrin (78.9%) followed by maltodextrin (72.1%). The partially purified piceid glycoside products (PicG₂ and PicG₃) were identified by mass spectrometry.     

Characterisation of cyclodextrin glucanotransferase fromBacillus circulans ATCC 21783 in terms of cyclodextrin production

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) fromBacillus circulans ATCC 21783 was purified by ultrafiltration and a consecutive starch adsorption. Total enzyme yield of 75.5% and purification factor of 13.7 were achieved. CGTase was most active at 65°C, possessed two clearly revealed pH-optima at 6.0 and 8.6 and retained from 75 to 100% of its initial activity in a wide range of pH, between 5.0 and 11.0. The cyclising activity was enhanced by 1 mM CaCl₂ or 4 mM CoCl₂. The enzyme was thermostable up to 70°C, and 64% of the original activity remained at 70°C after 30 min heat treatment. Up to 41% conversion into cyclodextrins was obtained from 40 g l^?1 starch without using any additives. This CGTase produced two types of cyclodextrins, beta and gamma, in a ratio 73:27 after 4 h reaction time at 65°C. This feature of the enzyme could be of interest for industrial cyclodextrin production.     

An improved method of photometric determination of cyclodextrin glucanotransferase activity

The method of spectrophotometric determination of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) activity with the use of phenolphthalein as a colored reagent has been improved. This technique includes an enzymatic reaction at 40°C for 60 min in 2% starch, with subsequent supplementation of the reaction mixture (0.5 ml) with the phenolphthalein reagent (3.0 ml) prepared in 0.1 M potassium carbonate buffer (pH 11.0) according to a special procedure, and measurement of the optical density of the obtained mixture at 553 nm. The activity was calculated using the exponential growth equation that connects a drop in the optical density and the degree of dilution of the enzyme. The described technique is suitable for working in a sufficiently broad range of specific activity of β-CGTase and does not require precise adjustment of the degree of dilution of solutions analyzed.     

An improved method of photometric determination of cyclodextrin glucanotransferase activity

The method of spectrophotometric determination of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) activity with the use of phenolphthalein as a colored reagent has been improved. This technique includes an enzymatic reaction at 40 degrees C for 60 min in 2% starch, with subsequent supplementation of the reaction mixture (0.5 ml) with the phenolphthalein reagent (3.0 ml) prepared in 0.1 M potassium carbonate buffer (pH 11.0) according to a special procedure, and measurement of the optical density of the obtained mixture at 553 nm. The activity was calculated using the exponential growth equation that connects a drop in the optical density and the degree of dilution of the enzyme. The described technique is suitable for working in a sufficiently broad range of specific activity of beta-CGTase and does not require precise adjustment of the degree of dilution of solutions analyzed.     

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.     

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.     

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.     

Crystallization of and preliminary crystallographic data for Bacillus stearothermophilus cyclodextrin glucanotransferase

Cyclodextrin glucanotransferase from Bacillus stearothermophilus TC-91 has been crystallized from an ammonium sulfate solution by the dialysis equilibrium method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with cell dimensions of a = 125.5 A, b = 88.1 A, and c = 81.5 A. The crystals appear to be suitable for X-ray structure analysis, diffracting to at least 2.1 A and being resistant to radiation damage.     

Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli

The gene encoding cyclodextrin glucanotransferase (CGTase) was successfully cloned from B. macerans by PCR. A recombinant plasmid pCS005 with a gene encoding the Lpp-OmpA-CGTase trifusion protein was constructed and transformed into E. coli for the surface display of CGTase. Results of immunoblotting analysis and protease accessibility on the fractionated cell membranes confirmed that the Lpp-OmpA-CGTase trifusion protein was successfully anchored on the outer membrane of E. coli. However, only 50% of the membrane-anchored trifusion proteins were displayed on the outer surface of E. coli with the remaining 50% un-translocated. The low efficiency of surface display is attributed to the large size of CGTase. Only a trace amount of CGTase activity was detected for both the whole cells and the cell debris fractions. Because the results of the protease accessibility study suggested that the trypsin-resistant conformation of CGTase was preserved in the membrane-anchored CGTase, we believe that the lack of enzyme activity is mainly due to the inaccessibility of the CGTase active site, near the N-terminus, for substrate molecules. It can be estimated that the critical size for surface display of protein in E. coli is approximately 70 kDa.     

Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-alpha-glucoside formation

In this study, we characterized cyclodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus in L-ascorbic acid-2-O-alpha-D-glucoside (AA-2G) formation and compared its enzymological properties with those of rat intestinal and rice seed alpha-glucosidases which had the ability to form AA-2G. CGTase formed AA-2G efficiently using alpha-cyclodextrin (alpha-CD) as a substrate and ascorbic acid (AA) as an acceptor. Several AA-2-oligoglucosides were also formed in this reaction mixture, and they could be converted to AA-2G by the additional treatment of glucoamylase. The optimum temperature for AA-2G formation was 70 degrees C and its optimum pH was around 5.0. CGTase also utilized beta- and gamma-CDs, maltooligosaccharides, dextrin, amylose, glycogen and starch as substrates, but not any disaccharides except maltose. CGTase showed the same acceptor specificity as two alpha-glucosidases, whereas its hydrolyzing activity towards AA-2G was very low compared with those of alpha-glucosidases. Cleavage profiles of AA-2-oligoglucosides by CGTase present a possible mechanism for AA-2G formation that CGTase transfers a glucose-hexamer to an acceptor at the first step and then a glucose is stepwisely removed from the non-reducing end of the product through glucoamylase-like action of this enzyme. These results indicate that CGTase is able to synthesize AA-2G more efficiently than rat and rice alpha-glucosidases and utilization of this enzyme makes the mass production of AA-2G possible.     

A novel glucanotransferase from a Bacillus circulans strain that produces a cyclomaltopentaose cyclized by an alpha-1,6-linkage

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an alpha-1,6-linkage [ICG5; cyclo-{-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS-PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 degrees C, and stable from pH 4.5 to 9.0 at up to 35 degrees C. The addition of 1 mM Ca(2+) enhanced the thermal stability of the enzyme up to 40 degrees C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular alpha-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular alpha-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.     

Altered phospholipid composition in mutants of Escherichia coli sensitive or resistant to organic solvents.

Mutants of Escherichia coli with altered resistance to low molecular weight organic solvents were isolated. Solvent-resistant mutants showed a decrease in the ratio of phosphatidylethanolamine to the anionic phospholipids (phosphatidylglycerol and cardiolipin) relative to the wild-type, whereas solvent-sensitive strains showed an increase. Reversion studies on representative mutants demonstrated that the phenotypic response to solvents and the changes in phospholipid composition were genetically associated. The fatty acid and lipopolysaccharide compositions of the various mutants showed no significant differences from the parental strain. The lesions in two of the solvent-sensitive mutants (DC7 and DC9) and one of the resistant mutants (DC11) were mapped by cotransduction with phage P1 and shown to lie very close to the pss locus at 56 min on the Escherichia coli map.     

Paenibacillus profundus sp. nov., a deep sediment bacterium that produces isocoumarin and peptide antibiotics

A novel bacterial strain Sl 79^T was isolated from a deep surface sediment sample obtained from the Sea of Japan and investigated by phenotypic and molecular methods. The bacterium Sl 79^T was Gram-positive, facultatively anaerobic, spore-forming, motile and able to form two different types of colonies. It contained the major menaquinone MK-7 and anteiso-C_15:0 followed by iso-C_15:0 as predominant fatty acids. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Sl 79^T belonged to the genus Paenibacillus where it clustered to Paenibacillus apiarius NRRL NRS-1438^T with a sequence similarity of 97.7 % and sharing sequence similarities below than 96.7 % to other validly named Paenibacillus species. Strain Sl 79^T was found to possess a remarkable inhibitory activity against indicatory microorganisms. On the basis of combined spectral analyses, strain Paenibacillus sp. Sl 79^T was established to produce isocoumarin and novel peptide antibiotics. On the basis of DNA–DNA relatedness, phenotypic and phylogenetic data obtained, it was concluded that strain Sl 79^T represents a novel species, Paenibacillus profundus sp. nov. with the type strain Sl 79^T = KMM 9420^T = NRIC 0885^T.     

Catalysis of protease/cyclodextrin complexes in organic solvents: Effects of reaction conditions and cyclodextrin structure on catalytic activity of proteases

α-Chymotrypsin (CT) was lyophilized from an aqueous solution in the presence of hydroxypropyl-β-cyclodextrin (HP-β-CyD). The enzyme preparation was used as a catalyst for transesterification between N-acetyl-l-tyrosine ethyl ester and methanol in a mixed solvent of acetonitrile/water (97/3 (v/v)). The enzyme preparation had much higher catalytic activity than free CT. The activity increased with an increase of HP-β-CyD/CT ratio and reached a maximum activity at the weight ratio of 4. Also, the activity of HP-β-CyD/CT increased with an increase in water content in the reaction media, and the maximum activity was obtained at 5–10% water. The fluorescence spectroscopic analysis suggested that the co-lyophilization with HP-β-CyD increased the structural stability of CT in acetonitrile/water. Upon co-lyophilization with HP-β-CyD, the activity of CT increased in any of the solvents used, but the activity depended strongly on the nature of the organic solvents. The catalytic activity of subtilisin Carlsberg (STC) also increased by co-lyophilization with α-, β-, γ-CyD or tri-O-methyl-β-CyD. α-CyD gave the best result, while HP-β-CyD diminished the activity of STC.