Isolation and structural analyses of positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) and 6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose.

6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose (beta CD) and three positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) in a mixture of products from beta CD and N-acetylglucosamine by the reversed reaction of beta-N-acetylhexosaminidase from jack bean were isolated and purified by HPLC. The structures of four isomers of di-N-acetylglucosaminyl-beta CDs were determined by FABMS and NMR spectroscopy. The degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying alpha-amylase (BSA) was established by LC-MS methods.     

New chromogenic substrates for the assay of alpha-amylase and (1 leads to 4)-beta-D-glucanase

New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1 leads to 4)-beta-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1 leads to 4)-beta-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.     

Microfluidic analysis of antibody specificity in a compact disk format

A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His6-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three-dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.     

An ESR assay for alpha-amylase activity toward succinylated starch, amylose and amylopectin

The esterification of the three polysaccharides, starch, amylose and amylopectin was carried out in pyridine-DMSO by succinic anhydride. The carboxylic groups in the succinylated polysaccharides were measured by FT-IR spectroscopy. The succinic derivatives were tested as alpha-amylase (1,4-alpha-D-glucan glucano hydrolase, E.C. 3.2.1.1) substrates. A colorimetric assay of the alpha-amylase activity indicated that this enzyme is active on succinic esters of starch and amylose and that the activity shows a linear decrease with the number of succinic units introduced into the polysaccharide. Since the colorimetric test was not suitable for the detection of the alpha-amylase activity when succinylated amylopectin was the substrate, we set-up an assay based on the labeling by a paramagnetic probe of the free carboxylic groups of succinylated polysaccharides. The kinetics of the alpha-amylase reaction were monitored by ESR spectroscopy through the increase of the mobility of the paramagnetic probe. The spin label used was the commercially available 4-amino-tempo. By this method we demonstrated that alpha-amylase is active on succinylated amylopectin. The utility of the assay for monitoring alpha-amylase activity when other methods (i.e. colorimetric tests) fail, is discussed.     

A simple and reliable turbidimetric and kinetic assay for alpha-amylase that is readily applied to culture supernatants and cell extracts

Summary A simple, reliable and sensitive assay for alpha-amylase activity is reported, together with its theoretical derivation, that overcomes many of the problems encountered with other assays, especially when attempting to assay alpha-amylase activity in crude cell extracts or culture supernatants. The method relies on the reduction in turbidity that occurs upon digestion of a starch suspension with alpha-amylase. The initial rate of decrease in turbidity is shown to be proportional to a wide range of enzyme concentrations, permitting a rapid spectrophotometric and kinetic determination of alpha-amylase activity.     

Enzyme‐triggered fluorescence turn‐off/turn‐on of carbon dots for monitoring β‐glucosidase and its inhibitor in living cells

Energy transfer engineering based on fluorescent probes for directly sensing enzyme activities are in great demand as enzyme‐mediated transformations, which are central to all biological processes. Here, a fluorescence carbon dot (CD)‐based assay exhibiting selective responses to the quantitation of β‐glucosidase and the effect of its inhibitor was developed. The most common substrate, para‐nitrophenyl‐β‐d ‐glucopyranoside (pNPG) was hydrolyzed by β‐glucosidase to release p‐nitrophenol (pNP), which can efficiently quench fluorescence of CDs via an inner filter effect and electron transfer. However, in the presence of inhibitors of β‐glucosidase, the fluorescence intensity gradually recovered as the concentration of inhibitors increased. Therefore, the enzyme‐triggered fluorescence turn‐off/turn‐on of specific CDs successfully achieved sensitive detection of β‐glucosidase and monitored the effect of its inhibitors. This new strategy was applied to detect β‐glucosidase and monitor β‐glucosidase inhibitor in hepatoma cells using cell imaging. All results suggest that the new method is sensitive and promising for use in cancer diagnosis and treatment.     

Preparation of non-reducing-end substituted p-nitrophenyl alpha-maltopentaoside (FG5P) as a substrate for a coupled enzymatic assay for alpha-amylases

p-Nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D - glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside, FG5P, was prepared, taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase on a mixture of O-6-deoxy-6-[(2-pyridyl)-amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose and p-nitrophenyl alpha-glucoside. The maltopentaose derivative is resistant to alpha-glucosidase and is suitable as a substrate for the alpha-amylase assay coupled with alpha-glucosidase in which the activity of alpha-amylase is determined by measuring the amount of p-nitrophenol liberated by alpha-glucosidase from p-nitrophenyl alpha-glucoside and p-nitrophenyl alpha-maltoside produced by the action of alpha-amylase. This alpha-amylase assay method was applied for determination of alpha-amylases in human serum.     

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. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:695–699, 2015     

Specific inhibition by cyclodextrins of raw starch digestion by fungal glucoamylase.

alpha-, beta-, and gamma-cyclodextrins (CDs) completely inhibited raw starch digestion by glucoamylase I (GA I, MW 90,000) from Aspergillus awamori var. kawachi, and inhibited by 85% the raw starch adsorption of GA I at the CD concentrations of 1-5 mM. CDs at 1-5 mM did not inhibit gelatinized starch hydrolysis by GA I, but at the concentration of 50 mM, they inhibited such hydrolysis slightly. GA I was specifically adsorbed onto CD-Sepharose 6B, but glucoamylase I' (GA I', MW 73,000), which does not adsorb onto or digest raw starch, from the same strain was not adsorbed onto that gel. The adsorption of the glucoamylases onto raw starch and CD-Sepharose 6B was correlated to their digestion of raw starch. The hydrophobic adsorption of GA I onto CDs and raw starch occurred competitively at the Cp region, which is on the C-terminal side of Gp-I in the site for raw starch affinity of GA I, and inclusion complexes were formed.     

Production of cyclodextrins in ultrafiltration membrane reactor containing cyclodextrin glycosyltransferase from Bacillus macerans

An enzyme reactor installed with ultrafiltration membrane was developed to produce alpha-, beta-, and gamma-cyclodextrins (CDs) from soluble starch by Bacillus macerans cyclodextrin glycosyltransferase (CGTase) tagged with 10 lysines at its C-terminus (CGTK10ase). Ultrafiltration membrane YM10 with 10,000 of molecular cutoff was chosen for membrane modification and CD production. A repeated-batch type of the enzyme reaction with free CGTK10ase resulted in a alpha-CD yield of 24.0 (+/-1.5)% and a productivity of 4.68 (+/-0.88) g/l-h, which were 7 times higher that those for CGTK10ase immobilized on modified YM10 membrane. Addition of 1- nonanol increased CD yields by 30% relative to the control, which might be due to prevention of the reversible hydrolysis of CDs.     

利用高效阴离子色谱快速检测筛选环糊精糖基转移酶产生菌>

利用高效阴离子色谱快速直接地检测微生物发酵液中的环糊精成分,尤其是大环环糊精的组成,进而创造了一种能快速准确地从土壤中筛选产环糊精糖基转移酶菌种的方法。共分离了149个产胞外淀粉水解酶的微生物菌株,利用高效阴离子交换色谱共检测了其中11株菌,其中6株主要产CD6 ,5株主要产CD7,主要产CD8的没有。在直接鉴定产生环糊精糖基转移酶菌株的过程中,也可以定量检测各种环糊精包括大环糊精（CD大于8）的含量。     

Enzymatic hydrolysis of soluble starch with an alpha-amylase from Bacillus licheniformis

The enzymatic hydrolysis of soluble starch with an alpha-amylase from Bacillus licheniformis (commercial enzyme Termamyl 300 L Type DX) have been experimentally studied at pH 7.5, within the temperature range of 37-75 degrees C, at initial substrate concentrations of between 0.25 and 2.00 g/L, and enzyme concentrations of between 0.575 x 10(-4) and 13.8 x 10(-4) g/L. To follow the reaction a procedure based on the iodometric method for measuring alpha-amylase activity was used. The kinetics of the enzymatic hydrolysis was fitted to the Michaelis-Menten equation using the integral method, taking into account that the thermal deactivation of the enzyme follows a second-order kinetic. These parameters were fitted to the Arrhenius equation obtaining activation energies of 24.4 and 41.7 kJ/mol and preexponential factors of 734.9 g/L and 1.74 x 10(8) min(-1) for K(M) and k, respectively.     

Properties and gene structure of the Thermotoga maritima alpha-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium.

Thermotoga maritima MSB8 has a chromosomal alpha-amylase gene, designated amyA, that is predicted to code for a 553-amino-acid preprotein with significant amino acid sequence similarity to the 4-alpha-glucanotransferase of the same strain and to alpha-amylase primary structures of other organisms. Upstream of the amylase gene, a divergently oriented open reading frame which can be translated into a polypeptide with similarity to the maltose-binding protein MalE of Escherichia coli was found. The T. maritima alpha-amylase appears to be the first known example of a lipoprotein alpha-amylase. This is in agreement with observations pointing to the membrane localization of this enzyme in T. maritima. Following the signal peptide, a 25-residue putative linker sequence rich in serine and threonine was found. The amylase gene was expressed in E. coli, and the recombinant enzyme was purified and characterized. The molecular mass of the recombinant enzyme was estimated at 61 kDa by denaturing gel electrophoresis (63 kDa by gel permeation chromatography). In a 10-min assay at the optimum pH of 7.0, the optimum temperature of amylase activity was 85 to 90 degrees C. Like the alpha-amylases of many other organisms, the activity of the T. maritima alpha-amylase was dependent on Ca2+. The final products of hydrolysis of soluble starch and amylose were mainly glucose and maltose. The extraordinarily high specific activity of the T. maritima alpha-amylase (about 5.6 x 10(3) U/mg of protein at 80 degrees C, pH 7, with amylose as the substrate) together with its extreme thermal stability makes this enzyme an interesting candidate for biotechnological applications in the starch processing industry.     

Gene cloning and enzymatic characteristics of a novel gamma-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364

A new gene, cda, was found in the downstream region of the cgt gene encoding cyclodextrin (CD) glucanotransferase from Bacillus clarkii 7364. Cda encoded by the cda was a cyclodextrinase that has extremely high specificity for gamma-CD. The rates of hydrolysis toward alpha- and beta-CD, maltooctaose and polysaccharides were less than 4% of that toward gamma-CD. Cda also has a transglycosylation activity, by which the maltotriose moiety was transferred from maltohexaose and maltopentaose. The comparison of the amino acid sequences between Cda and CD-degrading enzymes revealed the sequence of Cda has unique features. One of them is Gly247 next to the catalytic nucleophile Asp246. Most enzymes in GH family 13 have more bulky amino acids at this position. Other features in Cda are the lack of the N-domain in CD-degrading enzymes involving in the dimerization contributing to the preference of CDs and the existence of a long extra sequence in the C-terminus. Despite the lack of N-domain, Cda showed a dodecameric structure. The long extra sequence in the C-terminus might contribute to the oligomerization of Cda through a new mechanism. These unique features indicate that Cda is a novel type of CD-degrading enzyme.     

alpha-Amylase immobilized on bulk acoustic-wave sensor by UV-curing coating

A new method for immobilization of alpha-amylase by UV-curing coating is proposed in this paper. The immobilization procedure of UV-curing coating on piezoelectric quartz crystal is simple and convenient, and causes less loss of enzymatic activity. The activity of the immobilized alpha-amylase is monitored by a technique based on bulk acoustic-wave (BAW) sensor. The frequency shift of BAW sensor can reflect the degree of hydrolysis of starch by the immobilized alpha-amylase. It is appropriate for the immobilized alpha-amylase to hydrolyze the soluble starch under pH 7.0 condition, which is similar to that of the free alpha-amylase. Kinetic parameters (the Michaelis constant, K(m), and the maximum initial rate V(max)) of the enzymatic hydrolysis of starch by the immobilized alpha-amylase are estimated by using a linear method of Lineweaver-Burk plot. K(m)=12.7mgml(-1) and V(max)=15.9Hzmin(-1). And the experimental results show that the immobilized alpha-amylase entrapped by the UV-curing coating retains adequate enzymatic activity and can be reused more than 50 times under certain experimental conditions.     

Preparation of a new fluorogenic substrate of alpha-amylases and a simple alpha-amylase assay by HPLC

A new substrate of alpha-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, was prepared using dextrin as a starting material. Compared with other substrates so far reported, the fluorogenic substrate is unique in that it is resistant to exo-alpha-glucosidases due to the blocking group introduced into the non-reducing end glucose residue. The product of alpha-amylase digestion was rapidly separated from the substrate and was detected very sensitively by HPLC and a fluorescence detector. This method for alpha-amylase assay was also applied for determination of alpha-amylase in human serum.     

Fluorometric rate assay of alpha-amylase using an intramolecularly-quenched fluorescent substrate (FG5P)

The complete hydrolysis of a fluorogenic derivative of rho-nitrophenyl alpha-maltopentaoside, FG5P, by human salivary alpha-amylase, resulted in a 5-fold increase in fluorescence. This is due to disruption of the intramolecular quenching of the fluorescence of the 2-pyridylamino residue by the rho-nitrophenyl residue by separation of the two residues. This change of fluorescence accompanying the cleavage of the glucosidic bond was exploited to develop a fluorometric rate assay of alpha-amylase in human serum.     

Study on peptide hydrolysis by aminopeptidases from Streptomyces griseus, Streptomyces septatus and Aeromonas proteolytica

We developed a spectrophotometric assay for peptide hydrolysis by aminopeptidases (APs). The assay enables the measurement of free amino acids liberated by AP-catalyzed peptide hydrolysis using 4-aminoantipyrine, phenol, peroxidase, and l-amino acid oxidase. We investigated the specificity of bacterial APs [enzymes from Streptomyces griseus (SGAP), Streptomyces septatus (SSAP), and Aeromonas proteolytica (AAP)] toward peptide substrates using this assay method. Although these enzymes most efficiently cleave leucyl derivatives among 20 aminoacyl derivatives, in peptide hydrolysis, the catalytic efficiencies of Phe-Phe hydrolysis by SGAP and SSAP exceed that of Leu-Phe hydrolysis. Furthermore, all enzymes showed the maximum catalytic efficiencies for Phe-Phe-Phe hydrolysis. These results indicate that the hydrolytic activities of bacterial APs are affected by the nature of the penultimate residue or flanking moiety and the length of the peptide substrate.     

Gene cloning and enzymatic characteristics of a novel γ-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364

A new gene, cda, was found in the downstream region of the cgt gene encoding cyclodextrin (CD) glucanotransferase from Bacillus clarkii 7364. Cda encoded by the cda was a cyclodextrinase that has extremely high specificity for γ-CD. The rates of hydrolysis toward α- and β-CD, maltooctaose and polysaccharides were less than 4% of that toward γ-CD. Cda also has a transglycosylation activity, by which the maltotriose moiety was transferred from maltohexaose and maltopentaose. The comparison of the amino acid sequences between Cda and CD-degrading enzymes revealed the sequence of Cda has unique features. One of them is Gly247 next to the catalytic nucleophile Asp246. Most enzymes in GH family 13 have more bulky amino acids at this position. Other features in Cda are the lack of the N-domain in CD-degrading enzymes involving in the dimerization contributing to the preference of CDs and the existence of a long extra sequence in the C-terminus. Despite the lack of N-domain, Cda showed a dodecameric structure. The long extra sequence in the C-terminus might contribute to the oligomerization of Cda through a new mechanism. These unique features indicate that Cda is a novel type of CD-degrading enzyme.