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.     

Modulation of the multisubstrate specificity of Thermus maltogenic amylase by truncation of the N-terminal domain and by a salt-induced shift of the monomer/dimer equilibrium.

The relation between the quaternary structure and the substrate specificity of Thermus maltogenic amylase (ThMA) has been investigated. Sedimentation diffusion equilibrium ultracentrifugation and gel filtration analyses, in combination with the crystal structure determined recently, have demonstrated that ThMA existed in a monomer/dimer equilibrium. The truncation of ThMA by removing the N-terminal domain, which is composed of 124 amino acid residues, resulted in the complete monomerization of the enzyme (ThMADelta124) accompanied by a drastic decrease in the activity for beta-cyclodextrin (beta-CD) and a relatively smaller reduction of the activity for starch. Despite the overall low activity of ThMADelta124, the activity was higher toward starch than beta-CD, and the ratio of the specific activities toward these substrates was approximately 100 fold higher than that of wild-type ThMA. Furthermore, the addition of KCl to wild-type ThMA shifted the monomer/dimer equilibrium toward the monomer. In the presence of 1.0 M KCl, the relative activity of ThMA toward beta-CD decreased to 74%, while that for soluble starch increased to 194% compared to the activities in the absence of KCl. Thus, the ThMA monomer and dimer are both inferred to be enzymatically active but with a somewhat different substrate preference. Kinetic parameters of the wild-type and truncated enzymes also are in accordance with the changes in their specific activities. We thus provide evidence in support of a model, which shows that the relative multisubstrate specificity of ThMA is influenced by the monomer/dimer equilibrium of the enzyme.     

Comparative characteristics of cyclodextrin glycosyltransferases from various groups of microorganisms]

Cyclodextrin glycosyltransferases (CGT-ase, 1.4-alpha-glucanotransferase, cyclizing, EC 2.4.1.19) produced by some thermophilic, alkalophilic and mesophilic bacterial strains, were isolated and characterized. It was shown that thermophilic and mesophilic CGT-ases represent a mixture of alpha-, beta- and gamma-cyclodextrins (CD), alpha-cyclodextrin being the predominant component. Alkalophilic enzymes produce only beta-CD and are able to produce CD not only from starch but also from maltose, melibiose, maltotriose, amylose and amylopectin. The optimal conditions for the catalytic activity of the enzymes were determined. It was found that calcium, magnesium and zinc ions have a beneficial effect on the specific activity of these enzymes. The amino acid composition of the enzymes was studied.     

Enzymatic degradation of and bovine serum albumin release from starch-acetate films

The effect of acetylation of potato starch on swelling, enzymatic degradation, and bovine serum albumin (BSA, molecular mass 68 kDa) release rate from polymer films was studied. Potato starch and potato starch acetates (SA), having a degree of substitution of 1.9 or 2.6, were investigated. Polymer films were incubated in phosphate buffer solution pH 7.4 in the absence and presence of enzymes (alpha-amylase, amyloglucosidase, esterase) or in human serum. The acetylation of potato starch decreased its swelling considerably. Increased acetylation of starch also considerably retarded its enzymatic degradation. Due to the decreased swelling and degradation of SA films, BSA was released much slower from SA films than from potato starch films, both in the presence and absence of enzymes.     

Cyclodextrin production by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrins (CD) are cyclic oligosaccharides with multiple applications in the food, pharmaceutical, cosmetic, agricultural and chemical industries. In this work, the conditions used to produce CD with cyclodextrin glycosyltransferase from Bacillus circulans DF 9R were optimized using experimental designs. The developed method allowed the partial purification and concentration of the enzyme from the cultural broth and, subsequently, the CD production, using the same cassava starch as enzyme adsorbent and as substrate. Heat-treatment of raw starch at 70 degrees C for 15 min in the presence of adsorbed cyclodextrin glycosyltransferase allowed the starch liquefaction without enzyme inactivation. The optimum conditions for CD production were: 5% (w/v) cassava starch, 15 U of enzyme per gram of substrate, reaction temperature of 56 degrees C and pH 6.4. After 4h, the proportion of starch converted to CD reached 66% (w/w) and the weight ratio of alpha-CD:beta-CD:gamma-CD was 1.00:0.70:0.16.     

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.     

Insight into the chiral recognition of warfarin enantiomers by epichlorhydrin/beta-cyclodextrin polymer-based supports: determination of stoichiometry and stability of warfarin/beta-cyclodextrin polymer complexes

The complexation of warfarin (W) enantiomers by a hydrosoluble high-molecular-weight beta-cyclodextrin/epichlohydrin polymer (EP/beta-CD polymer) was studied using HPLC with a mobile phase of methanol/0.1 M Na acetate/acetic acid (pH 4) at 22 degrees C. It was found that the complexes (W/beta-CD unit) have a 1:1 stoichiometry. The stability constants of the complex involving each enantiomer and the polymer beta-CD units were determined in the mobile phase, and the highest stability of the complex (S-warfarin/beta-CD unit) was observed. From the chromatographic separations of warfarin enantiomers on different beta-CD or its derivative supports, we have deduced the role of the simultaneous presence of several glyceryl (-O-CH(2)-CHOH-CH(2)-O-) and dihydroxypropyl (-O-CH(2)-CHOH-CH(2)OH) groups on one beta-CD ring in promoting the chiral recognition of warfarin enantiomers.     

Effects of beta-cyclodextrin addition and temperature on the modulation of hydrophobic interactions in aqueous solutions of an associative alginate

Novel information about the effects of beta-cyclodextrin (beta-CD) addition and temperature on structural and rheological features of semidilute solutions of alginate and its hydrophobically modified analogue (HM-alginate) is given. Enhanced turbidity is observed for the HM-alginate solutions at high levels of beta-CD addition and low temperatures. The viscosity results revealed cross-linking of the alginate chains at high beta-CD concentrations and low temperatures. Rheological results for the HM-alginate solutions demonstrated that high levels of beta-CD addition and elevated temperatures promoted decoupling of the hydrophobic polymer-polymer associations via inclusion complex formation between beta-CD cavities and the hydrophobic side chains of the polymer. Analysis of small-angle neutron scattering (SANS) results from HM-alginate solutions in the presence of beta-CD suggested that the polymer chains are locally stretched at all of the considered levels of beta-CD and temperatures. The SANS results revealed association structures. The general picture that emerges is that beta-CD addition and temperature can be combined to tune the intensity of the hydrophobic interactions and to cross-link the unmodified alginate.     

Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other

Over 20 enzymes denoted as cyclomaltodextrinase, maltogenic amylase, or neopullulanase that share 40-86% sequence identity with each other are found in public data bases. These enzymes are distinguished from typical alpha-amylases by containing a novel N-terminal domain and exhibiting preferential substrate specificities for cyclomaltodextrins (CDs) over starch. In this research field, a great deal of confusion exists regarding the features distinguishing the three groups of enzymes from one another. Although a different enzyme code has been assigned to each of the three different enzyme names, even a single differentiating enzymatic property has not been documented in the literature. On the other hand, an outstanding question related to this issue concerns the structural basis for the preference of these enzymes for CDs. To clarify the confusion and to address this question, we have determined the structures of two enzymes, one from alkalophilic Bacillus sp. I-5 and named cyclomaltodextrinase and the other from a Thermus species and named maltogenic amylase. The structure of the Bacillus enzyme reveals a dodecameric assembly composed of six copies of the dimer, which is the structural and functional unit of the Thermus enzyme and an enzyme named neopullulanase. The structure of the Thermus enzyme in complex with beta-CD led to the conclusion that Trp47, a well conserved N-terminal domain residue, contributes greatly to the preference for beta-CD. The common dimer formation through the novel N-terminal domain, which contributes to the preference for CDs by lining the active-site cavity, convincingly indicates that the three groups of enzymes are not different enough to preserve the different names and enzyme codes.     

Molecular cloning and biochemical characterization of the first archaeal maltogenic amylase from the hyperthermophilic archaeon Thermoplasma volcanium GSS1

Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes belonging to glycoside hydrolase family 13, have been studied extensively, but their physiological roles in microbes and evolutionary relationships with other amylolytic enzymes remain unclear. Here, we report the biochemical properties of a thermostable archaeal MAase from Thermoplasma volcanium GSS1 (TpMA) for the first time. The primary structure and catalytic properties of TpMA were similar to those of MAases, such as possession of an extra domain at its N-terminal and preference for CD over starch. TpMA showed high thermostability and optimal activity at 75 degrees C and 80 degrees C for beta-CD and soluble starch, respectively. The recombinant TpMA exists as a high oligomer in a solution and the oligomeric TpMA was dissociated into dimer and monomer mixture by a high concentration of NaCl. The substrate preference and thermostability of TpMA were significantly dependent on the oligomeric state of the enzyme. However, TpMA exhibited distinguishable characteristics from those of bacterial MAases. The transglycosylation pattern of TpMA was opposite to that of bacterial MAases. TpMA formed more alpha-1,4-glycosidic linked transfer product than alpha-1,6-linked products. Like as alpha-amylases, notably, TpMA has a longer subsite structure than those of other CD-degrading enzymes. Our findings in this study suggest that TpMA, the archaeal MAase, shares characteristics of both bacterial MAases and alpha-amylases, and locates in the middle of the evolutionary process between alpha-amylases and bacterial MAases.     

Different behavior towards raw starch of three forms of glucoamylase from a Rhizopus sp

Three forms of glucoamylase [EC 3.2.1.3] of a Rhizopus sp., Gluc1 (M.W. 74,000), Gluc2 (M.W. 58,600), and Gluc3 (M.W. 61,400), which have similar pH optima and specific activities towards soluble starch were studied as to their behavior towards raw starch. The pH optima for raw starch digestion were different, that is, 4.5 for Gluc1 and 5.0 for both Gluc2 and Gluc3. All the enzymes digested raw starch almost completely but at quite different rates; Gluc2 and Gluc3, which lack the N-terminal portions of Gluc1, were 22 and 25 times less effective, respectively, for raw starch digestion than Gluc1. Of the three enzymes, only Gluc1 tightly bound to raw starch. Binding of Gluc1 to raw starch occurred pH-dependently with a broad pH optimum of 4.5-5.5, but temperature and ionic strength affected it only slightly and little, respectively. The binding constant of Gluc1 for raw starch at pH 5.0 and 4 degrees C was estimated to be 1.2 X 10(5) M-1. Fragment H (M.W. 16,700), presumably released from the N-terminal part of Gluc1, not only bound to raw starch itself but also inhibited the binding of Gluc1 to raw starch. pap-Gluc (M.W. 57,000) and chymo-Gluc (M.W. 64,000), which are papain- and chymotrypsin-modified Gluc1, respectively, and lack the N-terminal portions of Gluc1, resembled Gluc2 and Gluc3 in raw starch binding as well as digestion. All these results indicate that Gluc1 has a raw starch-binding site, different from the active center, in the N-terminal region. Various substrates and analogs inhibited the binding of Gluc1 to raw starch, presumably due to steric hindrance.     

Selective measurement of starch synthesizing enzymes in permeabilized potato tuber slices

Osmotically permeabilized potato (Solanum tuberosum L.) tuber slices were used to study the biosynthesis of starch under semi in vivo conditions. Criteria to distinguish the various enzymes involved in starch biosynthesis were developed based on the characteristics of the enzymes in in vitro experiments. Branching enzyme activity was inhibited at pH 8.5 or higher, while the starch synthases functioned optimally between pH 8.8 and 9.1. Unprimed soluble starch synthase activity was only apparent in the presence of sodium citrate (0.4 molar or higher). Granulebound and primed soluble starch synthase were active in the absence of sodium citrate. Primed soluble starch synthase activity was susceptible to inhibition by 10 millimolar zinc sulfate, while granule-bound starch synthase activity was not. The incorporation of the Glc moiety of ADP-Glc into starch in tissue slices by the various starch synthases was consistent with in vitro data with respect to the affinity of the enzymes for substrate, the pH profile, the stimulation by citrate, and the inhibition by zinc sulfate. These data were used to determine the activity of each of the starch synthases in tissue slices: granule-bound and soluble starch synthase transferred 37 and 55 picomoles ADP-Glc per hour per milligram fresh weight into starch of permeabilized tissue slices at 30°C and pH 9.1. In the presence of 0.5 molar sodium citrate, at least 40 picomoles ADP-Glc per hour per milligram fresh weight as transferred into starch by unprimed soluble starch synthase activity.     

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.     

Biochemical and structural features of a novel cyclodextrinase from cow rumen metagenome

A novel enzyme, RA.04, belonging to the alpha-amylase family was obtained after expression of metagenomic DNA from rumen fluid (Ferrer et al.: Environ. Microbiol. 2005, 7, 1996-2010). The purified RA.04 has a tetrameric structure (280 kDa) and exhibited maximum activity (5000 U/mg protein) at 70 degrees C and was active within an unusually broad pH range from 5.5 to 9.0. It maintained 80% activity at pH 5.0 and 9.5 and 75 degrees C. The enzyme hydrolyzed alpha-D-(1,4) bonds 13-fold faster than alpha-D-(1,6) bonds to yield maltose and glucose as the main products, and it exhibited transglycosylation activity. Its preferred substrates, in the descending order, were maltooligosaccharides (C3-C7), cyclomaltoheptaose (beta-CD), cyclomaltohexaose (alpha-CD), cyclomaltooctaose (gamma-CD), soluble starch, amylose, pullulan and amylopectin. The biochemical properties and amino acid sequence alignments suggested that this enzyme is a cyclomaltodextrinase. However, despite the similarity in the catalytic module (with Glu359 and Asp331 being the catalytic nucleophile and substrate-binding residues, respectively), the enzyme bears a shorter N-terminal domain that may keep the active site more accessible for both starch and pullulan, compared to the other known CDases. Moreover, RA.04 lacks the well-conserved N-terminal Trp responsible for the substrate preference typical of CDases/MAases/PNases, suggesting a new residue is implicated in the preference for cyclic maltooligosaccharides. This study has demonstrated the usefulness of a metagenomic approach to gain novel debranching enzymes, important for the bread/food industries, from microbial environments with a high rate of plant polymer turnover, exemplified by the cow rumen.     

Studies on α-Glucosidase in Rice

Two kinds of αglucosidase which were homogeneous in disc electrophoretic and ultra-centrifugal analysis were isolated from rice seeds by means of ammonium sulfate fractionation and CM-cellulose, Sephadex G–100 and DEAE-cellulose column chromatography and designated as α-glucosidase I and α-glucosidase II.

Both α-glucosidases hydrolyzed maltose and soluble starch to glucose and showed same optimal pH (4.0) on the both substrates. In addition, both enzymes acted on various α-linked gluco-oligosaccharides and soluble starch but little or not on α-linked hetero-glucosides and α-l,6-glucan (dextran).

Activity of the enzymes on maltose and soluble starch was inhibited by Tris and erythritol. α-Glucosidase II was more sensitive to the inhibitors than α-glucosidase I.

Km value for maltose was 1.1 mM for α-glucosidase I and 2.0 mM for α-glucosidase II.     

Expression of the promoter for the maltogenic amylase gene in Bacillus subtilis 168

An additional amylase, besides the typical alpha-amylase, was detected for the first time in the cytoplasm of B. subtilis SUH4-2, an isolate from Korean soil. The corresponding gene (bbmA) encoded a maltogenic amylase (MAase) and its sequence was almost identical to the yvdF gene of B. subtilis 168, whose function was unknown. Southern blot analysis using bbmA as the probe indicated that this gene was ubiquitous among various B. subtilis strains. In an effort to understand the physiological function of the bbmA gene in B. subtilis, the expression pattern of the gene was monitored by measuring the beta-galactosidase activity produced from the bbmA promoter fused to the amino terminus of the lacZ structural gene, which was then integrated into the amyE locus on the B. subtilis 168 chromosome. The promoter was induced during the mid-log phase and fully expressed at the early stationary phase in defined media containing beta-cyclodextrin (beta-CD), maltose, or starch. On the other hand, it was kept repressed in the presence of glucose, fructose, sucrose, or glycerol, suggesting that catabolite repression might be involved in the expression of the gene. Production of the beta-CD hydrolyzing activity was impaired by the spo0A mutation in B. subtilis 168, indicating the involvement of an additional regulatory system exerting control on the promoter. Inactivation of yvdF resulted in a significant decrease of the beta-CD hydrolyzing activity, if not all. This result implied the presence of an additional enzyme(s) that is capable of hydrolyzing beta-CD in B. subtilis 168. Based on the results, MAase encoded by bbmA is likely to be involved in maltose and beta-CD utilization when other sugars, which are readily usable as an energy source, are not available during the stationary phase.     

Immobilized cyclomaltodextrin glucanotransferase of an alkalophilic Bacillus sp. No. 38-2

Cyclomaltodextrin glucanotransferase [1,4-alpha-D-glucan-4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing), E.C.-2.4.1.19] of an alkalophilic Bacillus sp. No. 38-2 (ATCC 21783), which contains three types of enzymes (acid, neutral, and alkaline enzymes), was immobilized on synthetic adsorption resin. No distinguishing changes in pH or thermal stabilities of enzyme were observed due to the immobilization. Since acid-enzyme activity had disappeared, the optimum pH of immobilized enzyme was 9.0. Optimum temperature for the enzyme activity changed from 50 to 55 degrees C. The enzyme converted starch to cyclodextrins without significant loss of activity under the conditions of continuous reaction for about two weeks by using the column system (60 degrees C at pH 8.0). About 63% of soluble starch solution [4% (w/v)] was changed to cyclodextrins, as tested so far.     

Complex structures of Thermoactinomyces vulgaris R-47 alpha-amylase 2 with acarbose and cyclodextrins demonstrate the multiple substrate recognition mechanism

Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII) has the unique ability to hydrolyze cyclodextrins (CDs), with various sized cavities, as well as starch. To understand the relationship between structure and substrate specificity, x-ray structures of a TVAII-acarbose complex and inactive mutant TVAII (D325N/D421N)/alpha-, beta- and gamma-CDs complexes were determined at resolutions of 2.9, 2.9, 2.8, and 3.1 A, respectively. In all complexes, the interactions between ligands and enzymes at subsites -1, -2, and -3 were almost the same, but striking differences in the catalytic site structure were found at subsites +1 and +2, where Trp(356) and Tyr(374) changed the conformation of the side chain depending on the structure and size of the ligands. Trp(356) and Tyr(374) are thought to be responsible for the multiple substrate-recognition mechanism of TVAII, providing the unique substrate specificity. In the beta-CD complex, the beta-CD maintains a regular conical structure, making it difficult for Glu(354) to protonate the O-4 atom at the hydrolyzing site as a previously proposed hydrolyzing mechanism of alpha-amylase. From the x-ray structures, it is suggested that the protonation of the O-4 atom is possibly carried out via a hydrogen atom of the inter-glucose hydrogen bond at the hydrolyzing site.     

The Role of Pea Chloroplast [alpha]-Glucosidase in Transitory Starch Degradation

Pea chloroplastic [alpha]-glucosidase (EC 3.2.1.20) involved in transitory starch degradation was purified to apparent homogeneity by ion exchange, reactive dye, hydroxylapatite, hydrophobic interaction, and gel filtration column chromatography. The native molecular mass and the subunit molecular mass were about 49.1 and 24.4 kD, respectively, suggesting that the enzyme is a homodimer. The enzyme had a Km of 7.18 mM for maltose. The enzyme's maximal activity at pH 7.0 and stability at pH 6.5 are compatible with the diurnal oscillations of the chloroplastic stromal pH and transitory starch accumulation. This pH modulation of the [alpha]-glucosidase's activity and stability is the only mechanism known to regulate starch degradative enzymes in leaves. Although the enzyme was specific for the [alpha]-D-glucose in the nonreducing end as the glycon, the aglycon moieties could be composed of a variety of groups. However, the hydrolysis rate was greatly influenced by the aglycon residues. Also, the enzyme could hydrolyze glucans in which carbon 1 of the glycon was linked to different carbon positions of the penultimate glucose residue. The ability of the [alpha]-glucosidase to hydrolyze [alpha]-1,2- and [alpha]-1,3-glucosidic bonds may be vital if these bonds exist in starch granules because they would be barriers to other starch degradative enzymes. This purified pea chloroplastic [alpha]-glucosidase was demonstrated to initiate attacks on native transitory chloroplastic starch granules.