Comparison of aesthetic properties of tooth-colored restorative materials

A capillary electrophoresis system that integrated an enzymatic reaction and capillary polymer sieving electrophoresis was used to check the enzymatic stability of oligonucleotides. Phosphodiesterase I was employed to assess the susceptibility to 3'-exonucleolytic breakdown of some unmodified and modified oligonucleotides. Before degradation, the purity of the synthetic oligodeoxynucleotides was checked by capillary electrophoresis with a replaceable hydroxyethyl cellulose polymer solution. Enzymatic breakdown was achieved inside the capillary by merging substrate and enzyme zones based on their difference in electrophoretic mobility. After reaction, oligonucleotide fragments were swept to the detector, where they were individually detected and the remaining substrate was quantified. The results from the in-capillary degradation were compared to an off-line incubation and separation.     

Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations

To stabilize Aspergillus awamori glucoamylase (GA), three proline substitution mutations were constructed. When expressed in Saccharomyces cerevisiae, Ser30-->Pro (S30P) stabilized the enzyme without decreased activity, whereas Asp345-->Pro (D345P) did not significantly alter and Glu408-->Pro (E408P) greatly decreased enzyme thermostability. The S30P mutation was combined with two previously identified stabilizing mutations: Gly137-->Ala, and Asn20-->Cys/Ala27-->Cys (which creates a disulfide bond between positions 20 and 27). The combined mutants demonstrated cumulative stabilization as shown by decreased irreversible thermoinactivation rates between 65 and 80 degrees C. Additionally, two of the combined mutants outperformed wild-type GA in high-temperature (65 degrees C) saccharifications of DE 10 maltodextrin and were more active than the wild-type enzyme when assayed using maltose as substrate.     

Molecular mechanism in alpha-glucosidase and glucoamylase

The hydrolysis of glucosidic linkage catalyzed by every carbohydrate-hydrolase is a reaction in which the product retains (alpha-->alpha or beta-->beta) or inverts (alpha-->beta or beta-->alpha) the anomeric configuration of the substrate. alpha-Glucosidase and glucoamylase are essentially distinguished by releasing alpha-glucose and beta-glucose, respectively, from the common substrates having alpha-glucosidic linkage. The distinction in the substrate specificities of the two enzymes was explained by the subsite affinities in their active sites. The amino acid sequences of the regions containing the catalytic sites were compared in alpha-glucosidases and glucoamylases from various sources. alpha-Glucosidases were suggested to be grouped into two families by their primary structures. The catalytic reaction mechanisms of carbohydrate-hydrolases were discussed in the two significant models of a nucleophilic displacement mechanism and an oxocarbenium ion intermediate mechanism.     

Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.     

Separation and purification of glucoamylase in aqueous two-phase systems by a two-step extraction

Extraction in two steps of glucoamylase was studied in poly(ethylene glycol) (PEG) and potassium phosphate systems at pH values of 6, 7 and 9. Ten different conditions using PEG 300, 600, 1500, 4000 and 6000 were studied. The bottom phase of the first extraction step, with the enzyme, was reused in an appropriate concentration of PEG to form the second extraction step. The optimal partitioning conditions for glucoamylase separation were obtained in PEG 4000 (first step), PEG 1500 (second step) at pH 7 and resulted in a three-fold increase in glucoamylase purification.     

Enzymatic, fluorometric assay of alpha-amylase in serum

An enzymatic, fluorometric method for kinetic assay of serum alpha-amylase (1,4alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1) is described. A soluble starch is used as substrate, in tris(hydroxymethyl)methylamine buffer. All measurements are made in Pyrex cuvettes at 37 degrees C, with a reaction volume of 1.16 ml. The assay is based on the following reaction sequence: (see article) The rate of appearance of fluorescence of NADH (lambdaex = 365 nm, lambdaem = 460 nm), developed in the indicator reaction (4), is measured and equated to the activity of alpha-amylase in serum. A calibration plot of the change of fluorescence per min vs. enzyme concentration shows a good proportionality in the range of 0.50-5.0 kU/liter.     

Enhanced recovery and purification of Aspergillus glucoamylase from Saccharomyces cerevisiae by the addition of poly(aspartic acid) tails

Poly(aspartic acid) tails of different lengths were fused to the glucoamylase (GA) of Aspergillus awamori by genetic engineering techniques. Tails consisting of 5, 7, and 10 aspartate residues were fused to the N-terminus of the full-length mature GA (aa 1-616) downstream from the intact leader peptide to produce fusion proteins designated GAND5, GAND7, and GAND10, respectively. Three fusion proteins with C-terminal tails were also constructed, designated GACD0, GACD5, and GACD10 (0, 5, and 10 aspartate residues, respectively). For the C-terminal fusion proteins, the tails were fused to a catalytically active but truncated form of GA (aa 1-484). All of the charged tails had the general sequence Met-Ala-Aspn-Tyr, where n = 0, 5, 7, or 10. The modified genes were expressed in the yeast Saccharomyces cerevisiae and the proteins secreted into the culture medium. The enzymes were subsequently purified by affinity chromatography. The specific activity of each purified enzyme was found to be comparable to the wild-type enzyme. The C-terminal tails did not interfere with expression, whereas decreased extracellular glucoamylase activities corresponding to increased tail length were found for the N-terminal fusion proteins. Amino-terminal amino acid sequence analysis of the purified GAND proteins confirmed the authenticity of the amino termini of the modified proteins and showed that both the leader peptidase and KEX2 protease cleavages had occurred faithfully. The increased net negative charge of the GAND and GACD proteins was indicated by both nondenaturing PAGE and isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Automated docking of glucosyl disaccharides in the glucoamylase active site

To better understand the molecular basis of glucomylase selectivity, low-energy conformers of glucosyl disaccharides obtained from relaxed-residue conformational mapping were flexibly docked into the glucoamylase active site using AutoDock 2.2. This procedure ensures that significant conformational space is searched and can produce bound structures comparable to those obtained by protein crystallography. alpha-Linked glucosyl disaccharides except alpha,alpha-trehalose dock easily into the active site while exclusively beta-linked disaccharides do not, explaining why only the former are glucoamylase substrates. The optimized docking modes are similar at the nonreducing end of the different substrates. Individual atomic energies of intermolecular interaction allow the definite identification of key hydroxyl groups for each substrate. This approach confirmed the versatility of the second subsite of the glucoamylase active site in binding different substrates.     

Physicochemical and serological characterization of rice alpha-amylase isoforms and identification of their corresponding genes

We have identified, purified, and characterized 10 alpha-amylase isoforms from suspension-cultured rice (Oryza sativa L.) cells having different isoelectric point values. They had distinguishable optimum temperatures for enzymatic activity and molecular sizes. The results of immunoblotting indicated that polyclonal anti-A + B antibodies bound well to isoforms A, B, Y, and Z but weakly or not at all to E, F, G, H, I, and J. However, the anti-A + B antibodies inhibited the enzyme activities of only isoforms A and B. Polyclonal anti-H antibodies strongly bound to isoforms F, G, H, I, and J, whereas polyclonal anti-E antibodies preferentially recognized isoform E. A monoclonal antibody against isoform H (H-G49) inhibited the activities of isoforms E, G, H, I, and J, whereas it did not inhibit those of isoforms A, B, Y, and Z. Judging from their physicochemical and serological properties, we classified the rice alpha-amylase isoforms into two major classes, class I (A, B, Y, and Z) and class II (E, F, G, H, I, and J), and into four subgroups, group 1 (A and B), group 2 (Y and Z), group 3 (E), and group 4 (F, G, H, I, and J). Partial amino acid sequences for isoforms A, E, G, and H were also determined. In addition, the recombinant alpha-amylases expressed by plasmid pEno/103 containing the rice alpha-amylase gene RAmy1A in yeast were identified as both isoforms A and B. These analyses indicated that isoforms A and B were encoded by the gene RAmy1A, isoforms G and H were encoded by the gene RAmy3D, and isoform E was encoded by RAmy3E. The results strongly suggest that some isoforms within subgroups are formed by posttranslational modifications.     

Enhanced expression of GTP-binding proteins in differentiated U937 monocytic cells: possible involvement of tyrosine kinase and protein kinase C

Seven Arxula adeninivorans strains originating from The Netherlands (CSIR 1136, CSIR 1138 and CBS 8244T), South Africa (CSIR 1147, CSIR 1148 and CSIR 1149) and Siberia (Ls3) were compared concerning their secretory glucoamylase. Within the spectrum of secretory polypeptides obtained by SDS-polyacrylamide gel electrophoresis, the glucoamylase corresponding polypeptide, could be identified by means of specific antibodies directed against the glucoamylase of strain Ls3. The molecular masses of the glucoamylases vary from 84 to 95 kDa. After endoglycosidase F treatment of the secretory proteins, the N-linked carbohydrate content for each glucoamylase could be quantified at about 25%. Glucoamylase activity staining of secretory proteins separated under nondenaturing conditions revealed one glucoamylase active protein for the Dutch strains and two active forms for the strains originating from South Africa and Siberia. Highest activities of glucoamylase can be measured at the end of the exponential growth phase for each strain. The Dutch strain CSIR 1138 secretes the highest activity. Optimum values for temperature and pH were determined and compared. By means of pulsed field gel electrophoresis and Southern analysis, the glucoamylase corresponding gene could be localized on the second of the four chromosomes of each yeast strain.     

Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate

Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.     

Comparison of o-phthalaldehyde modification of alpha-amylases from porcine pancreas and Bacillus subtilis with Taka-amylase A

A fluorescent reagent, o-phthalaldehyde (OPA), competitively inhibited porcine pancreatic alpha-amylase (PPA) with Ki values of 0.7-0.9 mM, while alpha-amylase from Bacillus subtilis (BS) was uncompetitively inhibited, with Ki values of 5.8-7.6 mM. In both cases, OPA gave a time-dependent irreversible inactivation, where the amylase activity was lost faster than the maltosidase activity. Zymograms of the course of OPA modification showed that PPA was converted into at least six, faster moving components and BS gave two components. The OPA modification was retarded by the addition of the substrate analog, cyclodextrins, and the OPA modified enzymes decreased in affinity for the substrate soluble starch. Stoichiometric measurement showed that both PPA and BS was inactivated by the incorporation of 1 mol of OPA per mol of enzyme. The role of OPA modification of alpha-amylases was discussed in relation to the regulation of catalytic activity of enzymes.     

Synthesis of novel oligosaccharides from leucrose by an alpha-glucosidase

An alpha-glucosidase was purified in an electrophoretically pure state from an extract of koji culture of Aspergillus sp. KT-11. This enzyme was found to have a transferring activity when the reaction was done in a high concentration of leucrose at pH 4.5. Two kinds of transfer products, fractions I and II, were obtained from leucrose by the enzyme and they were identified as [(alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-alpha-D- glucopyranosyl-(1-->5)-D-fructopyranose] and [alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->5)-D- fructopyranose], respectively. These are considered to be novel oligosaccharides.     

Effects of mutations in the starch-binding domain of Bacillus macerans cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) is an industrially important enzyme that produces cyclodextrins (CD) from starch by intramolecular transglycosylation. CGTase consists of five globular domains labeled A through E. To better understand the role of domain E in CGTase catalysis, we have constructed several mutants of Bacillus macerans CGTase. Removing the entire E domain resulted in an inactive enzyme. Adding six amino acids between domains D and E caused a decrease in activity and thermostability. Replacing domain E with the similar starch-binding domain from Aspergillus awamori glucoamylase I caused a drastic decrease in activity, indicating the necessity of correct alignment of bound substrate. Substituting tyrosine residue 634 (Tyr634) with phenylalanine had very little effect on activity or thermostability. Substituting Tyr634 with glycine resulted in a 25% increase of specific cyclization and starch-hydrolyzing activities compared with that of the wild-type enzyme. The latter mutant was less thermostable. The results of this study indicate that domain E is important for the stability and integrity of B. macerans CGTase.     

Sugar sensing and alpha-amylase gene repression in rice embryos

We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) alpha-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the alpha-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the alpha-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization.     

Thermal unfolding of the starch binding domain of Aspergillus niger glucoamylase

A fragment of the starch-binding domain (SBDF) of Aspergillus niger glucoamylase was prepared using recombinant DNA techniques, and its thermal unfolding was investigated by high-sensitivity differential scanning calorimetry (DSC). Thermal unfolding of SBDF was found to be reversible at pH 7 as expected from a DSC study of the whole enzyme molecule [Tanaka A. et al., J. Biochem., 117, 1024-1028 (1995)] but not reversible at acidic region. Numerical analysis of the DSC curves showed that the denaturation was two-state, and some of the SBDF molecules were oligomeric (average degree of oligomerization was 1.2) at pH 7. It was suggested that the denaturation temperature of SBDF was lower than that of the starch-binding domain in the whole enzyme molecule by about 4.5 degrees (decrease in the Gibbs energy change was 5.3 kJ mol-1) indicating a possibility that the starch-binding domain is stabilized by glycosylation of the domain itself, or by the highly glycosylated linker region.     

Integration of glucoamylase gene from Aspergillus niger into Saccharomyces cerevisiae genome and its stable expression

The proteolitic enzyme pepsin (EC 3.4.23.1) was purified from chicken stomach by a modification of the method of Bohak (1970): after homogenazing the raw material, the zymogen was extracted with NaCl and NaHCO3, activated with 3N HCl and precipitated with NaCl (28% final concentration). The precipitate was lyophilised; fractions of it were suspended in 0.02N HCl. The solution was filtered through a column (2.6 x 80 cm) of Sephadex G-100 at an elution rate of 8-10 ml x cm-2 x h-1. Two protein peaks were obtained, the first one corresponding to the pepsin (2.0 mg/ml in pooled fractions). The milk clotting activity of the enzyme was determined on skimmed milk as a substrate (Berridge, 1955). Its proteolitic activity on the artificial substrate N-acetyl-L-phenylalanyl-L-3, 5-diiodo tyrosin (APD) also was determined (Rick-Fritsch, 1974). Mean clotting activity value was 5.52 UC, higher (P < 0.01) than that of the reference chymosin (0.64 UC). The activity with APD was unsatisfactory, due to very high absorbance values of the blanks. It is concluded, that the purification steps followed in this trial are simple and rapid, conferring a strong stimulus to using chicken pepsin as a clotting agent for the industrial production of pasteurized white cheese.     

Antibody responses of cows during an outbreak of neosporosis evaluated by indirect fluorescent antibody test and different enzyme-linked immunosorbent assays

Hairpin ribozymes with high cleavage activities were designed. An extra sequence was introduced at the 3'-end of the hairpin ribozyme to increase the binding to the substrate RNA, as compared to the wild-type hairpin ribozyme. A three-way junction (TWJ) was formed between the newly designed ribozyme and the substrate RNA. The complex with a solid TWJ showed less RNA cleavage activity than the wild-type hairpin ribozyme. However, the ribozyme with a TWJ with five unpaired bases or propandiol phosphate linkers had higher cleavage activity than the parent ribozyme without the TWJ. When a cis-cleavage system, in which the 5'-end of the substrate RNA was conjugated to the 3'-end of the ribozyme, was employed, the complex with the TWJ containing unpaired bases was also cleaved faster than the complex with the solid TWJ. This suggested that these differences in the cleavage activities were derived from the confirmation, and this was proven by nondenaturing gel electrophoresis. The TWJ hairpin ribozyme containing unpaired bases is able to bind strongly with substrate RNAs and to cleave them efficiently. Since the three-way ribozyme presented here is more active than the wild-type ribozyme, this type of ribozyme can serve as a more efficient tool to control RNA activities in vitro and in vivo.     

Metabolic regulation of alpha-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the alpha-amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice alpha-amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.