Amylomaltase from Thermus filiformis: expression in Saccharomyces cerevisiae and its use in starch modification.

AIM: To express amylomaltase from Thermus filiformis (TfAM) in a generally recognized as safe (GRAS) organism and to use the enzyme in starch modification. METHODS AND RESULTS: TfAM was expressed in Saccharomyces cerevisiae, using 2% (w/v) galactose inducer under GAL1 promoter. The enzyme was thermostable with high disproportionation and cyclization activities. The main large-ring cyclodextrin (CD) products were CD24-CD29, with CD26 as maximum at all incubation times. TfAM was used to modify cassava and pea starches, the amylose content decreased 18% and 30%, respectively, when 5% (w/v) starch was treated with 0·5 U TfAM g-1 starch. The increase in short branched chain (DP, degree of polymerization, 1-5) and the broader chain length distribution pattern which extended to the longer chain (DP40) after TfAM treatment were observed. The thermal property was changed, with an increase in retrogradation of starch as suggested by a lower enthalpy. CONCLUSIONS: TfAM was successfully expressed in S. cerevisiae and was used to make starches with new functionality. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the expression of AM in the GRAS yeast and the production of a modified starch gel from pea starch to improve the versatility of starch for food use.

Domain shuffling of cyclodextrin glucanotransferases for tailored product specificity and thermal stability.

Cyclodextrin glucanotransferases (CGTases) convert α-1,4-glucans to cyclic oligosaccharides (cyclodextrins, CD), which have found applications in the food and the pharmaceutical industries. In this study, we used two CGTases with different cyclization activities, product specificities, and pH and temperature optima to construct chimeric variants for the synthesis of large-ring CD. We used (a) a synthetic thermostable CGTase mainly forming α- and ß-CD (CD6 and CD7) derived from Geobacillus stearothermophilus ET1/NO2 (GeoT), and (b) a CGTase with lower cyclization activity from the alkaliphilic Bacillus sp. G825-6, which mainly synthesizes γ-CD (CD8). The A1, B, A2, and CDE domains of the G825-6 CGTase were replaced with corresponding GeoT CGTase domains by utilizing a megaprimer cloning approach. A comparison of the optimum temperature and pH, thermal stability, and CD products synthesized by the variants revealed that the B domain had a major impact on the cyclization activity, thermal stability, and product specificity of the constructed chimera. Complete suppression of the synthesis of CD6 was observed with the variants GeoT-A1/B and GeoT-A1/A2/CDE. The variant GeoT-A1/A2/CDE showed the desired enzyme properties for large-ring CD synthesis. Its melting temperature was 9 °C higher compared to the G825-6 CGTase and it synthesized up to 3.3 g·L-1 CD9 to CD12, corresponding to a 1.8- and 2.3-fold increase compared to GeoT and G825-6 CGTase, respectively. In conclusion, GeoT-A1/A2/CDE may be a candidate for the further development of CGTases specifically forming larger CD.

A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production.

Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development of engineered enzymes with improved yield and/or particular product specificity. In this work, we analyzed the role of four residues of the CGTase from Paenibacillus barengoltzii as determinants of product specificity. Single mutations were introduced in the CGTase-encoding gene to obtain mutants A137V, A144V, L280A and M329I and the activity of recombinant proteins was evaluated. The residue at position 137 proved to be relevant for CGTase activity. Molecular dynamics studies demonstrated additionally that mutation A137V produces a perturbation in the catalytic site of the CGTase, which correlates with a 10-fold reduction in its catalytic efficiency. Moreover, this mutant showed increased production of maltooligosaccharides with a high degree of polymerization, mostly maltopentaose to maltoheptaose. Our results highlight the role of residue 137 as a determinant of product specificity in this CGTase and may be applied to the rational design of saccharide-producing enzymes.

Y418 in 410s loop is required for high transglucosylation activity and large-ring cyclodextrin production of amylomaltase from Corynebacterium glutamicum.

Amylomaltase catalyzes α-1,4 glucosyl transfer reaction to yield linear or cyclic oligosaccharide products. The aim of this work is to investigate functional roles of 410s loop unique to amylomaltase from Corynebacterium glutamicum (CgAM). Site-directed mutagenesis of Y418, the residue at the loop tip, was performed. Y418A/S/D/R/W/F - CgAMs were characterized and compared to the wild-type (WT). A significant decrease in starch transglucosylation, disproportionation and cyclization activities was observed. Specificity for G3 substrate in disproportionation reaction was not changed; however, Y418F showed an increase in preference for longer oligosaccharides G5 to G7. The catalytic efficiency of Y418 mutated CgAMs, except for Y418F, was significantly lower (up to 8- and 12- fold for the W and R mutants, respectively) than that of WT. The change was in the kcat, not the Km values which were around 16-20 mM. The profile of large-ring cyclodextrin (LR-CD) product was different; the principal product of Y418A/D/S was shifted to the larger size (CD36-CD40) while that of the WT and Y418F peaked at CD29-CD33. The product yield was reduced especially in W and R mutants. Hence Y418 in 410s loop of CgAM not only contributes to transglucosylation activities but also controls the amount and size of LR-CD products through the proposed hydrophobic stacking interaction and the suitable distance of loop channel for substrate entering. This is the first report to show the effect of the loop tip residue on LR-CD product formation.

Pseudopolyrotaxane Formation in the Synthesis of Cyclodextrin Polymers: Effects on Drug Delivery, Mechanics, and Cell Compatibility.

Numerous groups have reported the use of cyclodextrin (CD)-based polymers for drug delivery applications due to their capacity to form inclusions with small molecule drugs, delaying the rate of drug release beyond that of diffusion alone (termed "affinity-based" drug delivery). Herein we demonstrate synthesis and characterization of a new family of CD-based polymers, some as pseudopolyrotaxanes, generated under mild (aqueous, room temperature) conditions. The formation of these new affinity polymers results in broad mechanical properties. Three diglycidylether cross-linkers which vary in length from 0 to 10 ethylene glycol units were examined. Pseudopolyrotaxane formation was found only with the highest-length cross-linker, noted first by a sharp change in both material properties and then confirmed by chemical signature. Materials were thoroughly evaluated by NMR, DSC, DMA, TGA, XRD, and FTIR. Cross-linker choice was also tested for impact on drug loading and delivery capacity, using antibiotics as model drugs. Chemically similar polymers without showing affinity rapidly saturated in loading experiments, while affinity materials showing high capacity for drug loading, even beyond the solubility limit of the drugs. When using the polymers with these new cross-linkers, affinity-based drug delivery is maintained: the materials are capable of antibiotic delivery, and clearance of Staphylococcus aureus, at least an order of magnitude better than diffusion-only control polymers. In cell compatibility studies, CD-based polymers were shown to have low overt cell toxicity and even resisted cell adhesion, presumably due to their highly hydrated state.

Elicitation, an Effective Strategy for the Biotechnological Production of Bioactive High-Added Value Compounds in Plant Cell Factories.

Plant in vitro cultures represent an attractive and cost-effective alternative to classical approaches to plant secondary metabolite (PSM) production (the "Plant Cell Factory" concept). Among other advantages, they constitute the only sustainable and eco-friendly system to obtain complex chemical structures biosynthesized by rare or endangered plant species that resist domestication. For successful results, the biotechnological production of PSM requires an optimized system, for which elicitation has proved one of the most effective strategies. In plant cell cultures, an elicitor can be defined as a compound introduced in small concentrations to a living system to promote the biosynthesis of the target metabolite. Traditionally, elicitors have been classified in two types, abiotic or biotic, according to their chemical nature and exogenous or endogenous origin, and notably include yeast extract, methyl jasmonate, salicylic acid, vanadyl sulphate and chitosan. In this review, we summarize the enhancing effects of elicitors on the production of high-added value plant compounds such as taxanes, ginsenosides, aryltetralin lignans and other types of polyphenols, focusing particularly on the use of a new generation of elicitors such as coronatine and cyclodextrins.

Optimization of large-ring cyclodextrin production from starch by amylomaltase from Corynebacterium glutamicum and effect of organic solvent on product size.

AIMS: To increase yield of starch conversion to large-ring cyclodextrins (LR-CDs) by amylomaltase from Corynebacterium glutamicum (CgAM). METHODS AND RESULTS: In this work, LR-CDs produced from pea, tapioca, corn, potato, rice and glutinous-rice starch by the recombinant CgAM were analysed by High-Performance Anion-Exchange Chromatography Using Pulsed Amperometric Detection (HPAEC-PAD). Among these, pea starch gave the highest yield of LR-CDs. Pretreatment of pea starch with isoamylase prior to incubation with CgAM resulted in the increase in LR-CD products by 20%. Surprisingly, CgAM converted starch into LR-CDs within a wide pH range (pH 5·5-9·0). LR-CD syntheses at alkaline pH or at a long incubation time favoured low-degree of polymerization (DP) products (CD22-CD32). Addition of 5-15% dimethyl sulfoxide (DMSO) promoted the synthesis of medium-DP species (CD33-CD43) by 10-25%. CONCLUSIONS: Pretreatment of pea starch with isoamylase could enhance the yield of LR-CDs. The ratio of LR-CD products depends on pH, incubation time and addition of organic solvents such as DMSO. SIGNIFICANCE AND IMPACT OF THE STUDY: LR-CD yield can be increased by thorough optimization of starch types, starch concentrations, enzyme activities, pH and incubation times. This study is the first report of the effect of organic solvents on LR-CD production by amylomaltase.

Antifungal activity improved by coproduction of cyclodextrins and anabaenolysins in Cyanobacteria.

Cyclodextrins are cyclic oligosaccharides widely used in the pharmaceutical industry to improve drug delivery and to increase the solubility of hydrophobic compounds. Anabaenolysins are lipopeptides produced by cyanobacteria with potent lytic activity in cholesterol-containing membranes. Here, we identified the 23- to 24-kb gene clusters responsible for the production of the lipopeptide anabaenolysin. The hybrid nonribosomal peptide synthetase and polyketide synthase biosynthetic gene cluster is encoded in the genomes of three anabaenolysin-producing strains of Anabaena. We detected previously unidentified strains producing known anabaenolysins A and B and discovered the production of new variants of anabaenolysins C and D. Bioassays demonstrated that anabaenolysins have weak antifungal activity against Candida albicans. Surprisingly, addition of the hydrophilic fraction of the whole-cell extracts increased the antifungal activity of the hydrophobic anabaenolysins. The fraction contained compounds identified by NMR as α-, ß-, and γ-cyclodextrins, which undergo acetylation. Cyclodextrins have been used for decades to improve the solubility and bioavailability of many drugs including antifungal compounds. This study shows a natural example of cyclodextrins improving the solubility and efficacy of an antifungal compound in an ancient lineage of photosynthetic bacteria.

Ultrafiltration system for cyclodextrin production in repetitive batches by CGTase from Bacillus firmus 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 ß-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 ß-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.

Continuous production of cyclodextrins in an ultrafiltration membrane reactor, catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R.

Cyclodextrins (CDs) are cyclic oligosaccharides of wide industrial application, whose synthesis is catalyzed by Cyclodextrin glycosyltransferase (CGTase) from starch. Here, CDs were produced using CGTase from Bacillus circulans DF 9R in continuous process and an ultrafiltration membrane reactor. The batch process was conducted as a control. This method allowed increasing the yield from 40 to 55.6% and the productivity from 26.1 to 99.5 mg of CD per unit of enzyme. The method also allowed obtaining a high-purity product. The flow rate remained at 50% of its initial value after 24 h of process, improving the results described in the literature for starch hydrolysis processes. CGTase remained active throughout the process, which could be explained by the protective effect of the substrate and reaction products on CGTase stability. In addition, batch processes were developed using starches from different sources. We concluded that any of the starches studied could be used as substrate for CD production with similar yields and product specificity.

[Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production].

The aim of this study was to isolate a novel amylomaltase gene from community DNA of soil samples collected from Ban Nong Khrok hot spring in Thailand without bacterial cultivation. Using PCR, a 1.5 kb full-length gene was amplified and ligated with pGEM-T easy vector to transform into Escherichia coli DH5 alpha for sequencing. The obtained gene encoding an amylomaltase consisted of 1.503 bp that translated into 500 amino acids. Amino acid sequence deduced from this gene was highly homologous with that of amylomaltase from Thermus thermophillus ATCC 33923. In order to express the enzyme, the cloned gene was subcloned into plasmid pET-17b and introduced into E. coli BL21 (DE3). The maximum expression was observed when the cloned cells were cultured at 37 degrees C for 6 h with 0.5 mM IPTG induction. By 10% SDS-PAGE, the relative molecular mass of the purified amylomaltase was approximately 58 kDa. This enzyme was optimally active at 70 degrees C and pH 9.0. In addition, the enzyme could hydrolyze pea starch to yield the large-ring cyclodextrins with degrees of polymerization of 23 and higher. It is noted that CD29 was the product in the largest quantity under all tested conditions.

Cycloamylose production from amylomaize by isoamylase and Thermus aquaticus 4-α-glucanotransferase.

Amylomaize (AMM) was utilized as the substrate for cycloamylose (CA) production in this study. After debranching, AMM was incubated with 10 U/g of Thermus aquaticus 4-α-glucanotransferase (TA 4αGTase) for various reaction times in water or in DMSO reaction system. The maximum conversion yield of CA was greatly improved from 24.55% to 45.58% and from 27.40% to 47.25% after debranching in the water and DMSO reaction systems, respectively. Compared with the method that produced CA from commercial potato amylose, this method produced CA from a natural amylopectin containing starch with enhanced conversion yield after debranching. Meanwhile, we found that the minimum degree of polymerization (DP) of CA from TA 4αGTase treatment was 5, regardless of the reaction conditions. These results were different from those reported in the literature that stated the minimum DP of CA produced with a TA 4αGTase treatment was 22 regardless of the reaction conditions.

Molecular mutagenesis at Tyr-101 of the amylomaltase transcribed from a gene isolated from soil DNA.

The wild-type (WT) amylomaltase gene was directly isolated from soil DNA and cloned into a pET19b vector to express in E. coli BL21(DE3). The ORF of this gene consisted of 1,572 bp, encoding an enzyme of 523 amino acids. Though showing 99% sequence identity to amylomaltse from Thermus thermophilus ATCC 33923, this enzyme is unique in its alkaline optimum pH. In order to alter amylomaltase with less coupling or hydrolytic activity to enhance cycloamylose (CA) formation through cyclization reaction, site-directed mutagenesis of the second glucan binding site involving in CA production was performed at Tyr-101. The result revealed that the mutated Y101S enzyme showed a small increase in cyclization activity while significantly decreased disproportionation, coupling and hydrolytic activities. Mutation also resulted in the change in substrate specificity for disproportionation reaction. The WT enzyme preferred maltotriose, while the activity of mutated enzyme was the highest with maltopentaose substrate. Product analysis by HPAEC-PAD demonstrated that the main CAs of the WT amylomaltase were CA29-CA37. Y101S mutation did not change the product pattern, however, the amount of CAs formed by the mutated enzyme tended to increase especially at long incubation time.

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K.

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the k(cat)/K(M) value of CITase-598K was approximately two times higher (32.2s(-1)mM(-1)) than that of CITase-T3040 (17.8s(-1)mM(-1)). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.

Reaction conditions for maximal cyclodextrin production by cyclodextrin glucanotransferase from Bacillus megaterium.

The effect of the reaction conditions (substrate concentration, enzyme dosage, and pH) on cyclodextrin production by cyclodextrin glucanotransferase from Bacillus megaterium was investigated by applying mathematical modeling methods. Adequate models were developed and they were used for determination of the optimal conditions for maximal formation of beta-cyclodextrins at minimal concentrations of a- and gamma-cydclodextrins. The main factor affecting the ratio of the products was pH of the reaction mixture. At pH 9 the enzyme formed mainly beta- and y-cyclodextrins and the ratio a:beta:gamma was 2.6:83.5:13.9; at pH 5 the ratio changed to 8.6:84.6:6.8. Mathematical models were used for determination of the conditions for maximal conversion of the substrate into cyclodextrins. 45.88% conversion of starch was achieved at 5% substrate concentration, 3.5 U/g enzyme dosage, and pH 7.4.

Effects of substrates and reaction conditions on production of cyclodextrins using cyclodextrin glucanotransferase from newly isolated Bacillus agaradhaerens KSU-A11

The effects of reaction conditions on cyclodextrins (CDs) production by CGTase from newly isolated Bacillus agaradhaerens KSU-A11 is reported. Among six types of starch tested, potato starch gave highest starch conversion into CDs. In addition, CDs yield was about three fold higher when using gelatinized potato starch in comparison to raw starch. The total CDs production was increased with increasing pH, showing maximum starch conversion at pH 10. Furthermore, the proportion of gamma-CD was relatively higher under slightly acidic-neutral conditions than at alkaline pH with a maximum proportion of 35.6 percent at pH 7 compared to 7.6 percent at pH 10. Maximum starch conversion into CDs was seen at reaction temperature of 55ºC. Lower reaction temperature led to higher proportion of gamma-CD with maximum percentage at 35ºC. Cyclization reaction was significantly promoted in the presence CaCl2 (10 mM), while in the presence of ethyl alcohol there was significant decrease in CD production particularly at high concentration. beta-CD was the major product up to 1 hr reaction period with traces of alpha-CD and no detectable gamma-CD. However, as the reaction proceed, gamma-CD started to be synthesised and alpha-CD concentration increased up to 4 hrs, where the CDs ratios were 0.27:0.65:0.07 for alpha-CD:beta-CD:gamma-CD, respectively. In addition, optimum CGTase/starch ratio was obtained at 80 U/g starch, showing highest starch conversion into CDs. All the parameters involved have been shown to affect the products yield and/or specificity of B. agaradhaerens KSU-A11 CGTase.

Effect of temperature on cyclodextrin production and characterization of paracetamol/cyclodextrin complexes.

OBJECTIVE: To compare the effect of different reaction temperatures on the cyclization and coupling reactions of the Toruzyme CGTase influencing the yield of cyclodextrins (CDs) and to study the solubility of paracetamol with CDs. MATERIAL AND METHOD: Type and amount of CDs were analyzed by HPAEC-PAD. The stability constants for the inclusion complex formed between CDs and paracetamol were determined using the phase solubility method. The solubility of paracetamol with CDs was measured by UV-spectrophotometer at 240 nm. RESULTS: The result has shown that the reaction temperature has effect on the Toruzyme CGTase reactions in production of CDs. The CDs yield after 30 min of incubation was higher at 60 degrees C than at 80 degrees C. The catalytic efficiency (k(cat)/K(m)) of this enzyme indicated the higher value of the cyclization reaction at 60 degrees C compared to 80 degrees C while the opposite was found for the coupling reaction. Paracetamol is used as an analgesic and antipyretic but it is poorly water-soluble drug. To improve the solubility of paracetamol CDs obtained were used to study for paracetamol/CDs complexes. The phase-solubility diagrams of paracetamol with alpha-, beta- and gamma-CD were A(N) type while that of paracetamol with maltosyl-beta-CD (G2-beta-CD) complex was A(L) type. The stability constants (K(c)) for the inclusion complex of paracetamol with alpha-, beta-, gamma-CD and G2-beta-CD were 5.69, 16.75, 4.73 and 2,223.25 M(-1), respectively. CONCLUSION: The optimum temperature for CDs production was at 60 degrees C and the low solubility of paracetamol was significantly improved by complexation with CDs, where the enhancing effect was in the order of G2-beta-CD > beta-CD > alpha-CD > gamma-CD.

Contribution of AmyA, an extracellular alpha-glucan degrading enzyme, to group A streptococcal host-pathogen interaction.

alpha-Glucans such as starch and glycogen are abundant in the human oropharynx, the main site of group A Streptococcus (GAS) infection. However, the role in pathogenesis of GAS extracellular alpha-glucan binding and degrading enzymes is unknown. The serotype M1 GAS genome encodes two extracellular proteins putatively involved in alpha-glucan binding and degradation; pulA encodes a cell wall anchored pullulanase and amyA encodes a freely secreted putative cyclomaltodextrin alpha-glucanotransferase. Genetic inactivation of amyA, but not pulA, abolished GAS alpha-glucan degradation. The DeltaamyA strain had a slower rate of translocation across human pharyngeal epithelial cells. Consistent with this finding, the DeltaamyA strain was less virulent following mouse mucosal challenge. Recombinant AmyA degraded alpha-glucans into beta-cyclomaltodextrins that reduced pharyngeal cell transepithelial resistance, providing a physiologic explanation for the observed transepithelial migration phenotype. Higher amyA transcript levels were present in serotype M1 GAS strains causing invasive infection compared with strains causing pharyngitis. GAS proliferation in a defined alpha-glucan-containing medium was dependent on the presence of human salivary alpha-amylase. These data delineate the molecular mechanisms by which alpha-glucan degradation contributes to GAS host-pathogen interaction, including how GAS uses human salivary alpha-amylase for its own metabolic benefit.

Characterization of 4-alpha-glucanotransferase from Synechocystis sp. PCC 6803 and its application to various corn starches.

A putative 4-alpha-glucanotransferase (alphaGTase) gene from Synechocystis sp. PCC 6803 was identified being composed of 1505 nucleotides, and the overexpressed protein was purified with an affinity chromatography. The recombinant alphaGTase had about 57kDa of molecular mass when judged by SDS-PAGE analysis. The optimum reaction condition of the alphaGTase was shown to be pH 7 at 45 degrees C in 50mm phosphate buffer. This enzyme displayed transglycosylating activity on various maltooligosaccharides, of which the smallest donor and acceptor molecules were determined to be maltose and glucose, respectively. Various corn starches consisting of different proportions of amylopectin and amylose were incubated with the recombinant alphaGTase. The change in molecular weight distribution of alphaGTase-modified starch was analyzed by HPSEC. The reaction pattern of alphaGTase showed substantial decrease in amylopectin and increase in the peak corresponding to cycloamylose (CA). The production yield of CA tended to increase from 5 to 30% along with the increase in the apparent amylose content in corn starch, which suggested that linear amylose chain would be preferred to produce CA in the alphaGTase treatment. The detectable minimum degree of polymerization (DP) of CA was shown to be 22 by MALDI-TOF-MS analysis. As another action mode of alphaGTase, the rearrangement of amylopectin branch-chain distribution occurred without hydrolysis to small oligosaccharides. After isoamylolysis, alphaGTase-treated starch displayed the increase in DP 4-9 and longer than DP 21 when the relative proportion of branch chains in amylopectin was determined by HPAEC.

Isolation of Bacillus and Paenibacillus bacterial strains that produce large molecules of cyclic isomaltooligosaccharides.

Cyclic isomaltooligosaccharides (CIs) usually consist of 7 to 12 glucose units, although only CI-10 has strong inclusion complex-forming ability. Four Bacillus strains and two Paenibacillus strains were isolated as novel CI-producing bacteria. Among these, five strains produced small amounts of CI-7 to CI-9, but mainly produced CI-10 to CI-12. Larger CIs, up to CI-17, were also identified.