Purification and Characterization of Thermostable Pullulanase from Bacillus stearothermophilus and Molecular Cloning and Expression of the Gene in Bacillus subtilis

A thermostable pullulanase (alpha-dextrin 6-glucanohydrolase [EC 3.2.1.41]) from a newly isolated Bacillus stearothermophilus strain (TRS128) was purified and characterized. The enzyme hydrolyzed (1-->6)-alpha-d-glucosidic linkages of pullulan to produce maltotriose, and the optimum temperature was 65 degrees C. About 90% of the enzyme activity was retained after treatment at 65 degrees C for 60 min. By using pTB522 as a vector plasmid, the pullulanase gene was cloned and expressed in Bacillus subtilis.     

Production of Schardinger beta-dextrin by soluble and immobilized cyclodextrin glycosyltransferase of an alkalophilic Bacillus sp.

Succinylated cyclodextrin glycosyltransferase (EC 3.2.1.19) of an alkalophilic Bacillus sp. was adsorbed on a vinylpyridine copolymer. The enzyme had about 25% of the activity of soluble enzyme added. No increase of pH or thermal stability of the enzyme was observed by the adsorption, whereas optimum temperature for the enzyme action was shifted from 50 to 55 degrees C. The enzyme converted starch to cyclodextrine without significant loss of activity under the conditions of 4 times reusing of 6 hr conversion by the batch system or 2 weeks continuous reaction by the column system at 55 degrees C and pH 8.0. About 46% of the potato starch solution [15% (w/v)] was converted to cyclodextrins by the enzyme, and 52% was converted by the simultaneous action of the enzyme and alkaline pullulanase of alkalophilic Bacillus sp. (No. 202-1). These values were almost the same as those obtained by the soluble enzyme or enzymes system.     

Kinetic analysis about the effects of neutral salts on the thermal stability of yeast alcohol dehydrogenase

The effects of salts on the rate constants of inactivation by heat of yeast alcohol dehydrogenase (YADH) at 60.0 degrees C were measured. Different effects were observed at low and high salt concentrations. At high concentrations, some salts had stabilizing effects, while others were destabilizing. The effects of salts in the high concentration range examined can be described as follows: (decreased thermal stability) NaClO(4) < NaI = (C(2)H(5))(4)NBr < NH(4)Br < NaBr = KBr = CsBr = (no addition) < (CH(3))(4)NBr < KCl < KF < Na(2)SO(4) (increased thermal stability). The decreasing effect of NaClO(4) on YADH controlled the thermal stability of the enzyme absolutely and was not compensated by the addition of Na(2)SO(4), a salt which stabilized the enzyme. However, Na(2)SO(4) compensation did occur in response to the decrease in thermal stability caused by (C(2)H(5))(4)NBr. The rate constants of inactivation by heat (k (in)) of the enzyme were measured at various temperatures. Effective values of the thermodynamic activation parameters of thermal inactivation, activation of free energy (DeltaG (double dagger)), activation enthalpy (DeltaH (double dagger)), and activation entropy (DeltaS (double dagger)), were determined. The thermal stability of YADH in 0.8 M Na(2)SO(4) increased more than that of pyruvate kinase from Bacillus stearothermophilus, a moderate thermophile. The changes in the values of DeltaH (double dagger) and DeltaS (double dagger) were great and showed a general compensatory tendency, with the exception of in the case of NaClO(4). The temperature for the general compensation effect (T (c)) was approximately 123 degrees C. With Na(2)SO(4), the thermal stability of YADH at a temperature below T (c) was greater than that in the absence of salt due to the higher values of DeltaH (double dagger) and DeltaS (double dagger), respectively, and thus was an example of low-temperature enzymatic stabilization. With (C(2)H(5))(4)NBr, the thermal stability of YADH at a temperature below T (c) was lower than that in the absence of salt due to the lower values of DeltaH (double dagger) and DeltaS (double dagger), respectively, and thus was an example of low-temperature enzymatic destabilization. But with NaClO(4), the changes in the values of DeltaH (double dagger) and DeltaS (double dagger) were small and the thermal stability of YADH was thus an example of high-temperature enzymatic destabilization.     

Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment

High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.     

Enhancement of starch conversion efficiency with free and immobilized pullulanase and alpha-1,4-glucosidase

Glucoamylase and pullulanase were immobilized on reconstituted bovine-hide collagen membranes using the covalent azide linkage method. A pretanning step was incorporated into the immobilization procedure to enable the support matrix to resist proteolytic activity while accommodating an operating temperature of 50 degrees C. The immobilized glucoamylase and pullulanase activities were 0.91 and 0.022 mg dextrose equivalent (DE) min(-1) cm(-2) of membrane, respectively. Immobilized glucoamylase had a half-life of 50 days while the immobilized pullulanase had a half-life of 7 days. This is a considerably improved stability over that reported by other researchers. The enzymes were studied in their free and immobilized forms on a variety of starch substrates including waxy maize, a material which contains 80% alpha-1-6-glucosidic linkages. Substrate concentrations ranged from 1% to a typical commercial concentration of 30%. Conversion efficiencies of 90-92% DE were obtained with free and immobilized glucoamylase preparations. Conversion enhancements of 4-5 mg of DE above this level were obtained by the use of pullulanase in its free or immobilized forms. Close examination of free pullulanase stability as a function of pH indicated improved thermal stability at higher pH values. At 50 degrees C and pH 5.0, the free enzyme was inactivated after 24 h. At pH 7.0, the enzyme still possessed one-half its activity after 72 h. Studies were conducted in both batch and continuous total recycle reactors. All experiments were conducted at 50 degrees C. Experiments conducted with coimmobilized enzymes proved quite promising. Levels of conversion equivalent to those obtained with the individually immobilized enzymes were realized.     

Three domains comprised in thermostable molecular weight 54,000 pullulanase of type I from Bacillus flavocaldarius KP1228

The gene that coded for a cellular pullulanase of type I (alpha-dextrin 6-glucanohydrolase, EC 3.2.1.41) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51 to 82 degrees C was expressed in Escherichia coli MV1184. The enzyme had a half-life of 10 min at 107 degrees C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 475 residues with a molecular weight of 53,856 deduced from the gene was 15-21% and 43% identical to the corresponding C-terminal regions in the sequences of 2 plant and 6 bacterial pullulanases of type I, and of Bacillus stearothermophilus TRS40 neoplullulanase, respectively. Sequence analysis showed that B. flavocaldarius pullulanase comprised 3 domains, i.e., one catalytic (beta/alpha)8-barrel domain, one domain made of the region protruding from the barrel between the third beta-strand and the third alpha-helix, and one beta-stranded domain attached to the C-end of the barrel domain, but that the pullulanase lacked the beta-stranded domain commonly found in addition to the 3 domains in the neopullulanase and all other pullulanases, and attached to the N-end of the barrel domain.     

Inactivation of bacillus spores in dry systems at low and high temperatures.

A plot of the thermal resistance of Bacillus subtilis var. niger spores (log D value) against temperature was linear between 37 and 190 degrees C (z = 23 degrees C), provided that the relative humidity of the spore environment was kept below a certain critical level. The corresponding plot for Bacillus stearothermophilus spores was linear in the range 150 to 180 degrees C (z = 29 degrees C) but departed from linearity at lower temperatures (decreasing z value). However, the z value of 29 degrees C was decreased to 23 degrees C if spores were dried before heat treatment. The straight line corresponding to this new z value was consistent with the inactivation rate at a lower temperature (60 degrees C). The data indicate that bacterial spores which are treated in dry heat at an environmental relative humidity near zero are inactivated mainly by a drying process. By extrapolation of the thermal resistance plot obtained under these conditions for B. subtilis var. niger spores, the D value at 0 degrees C would be about 4 years.     

Microbial reclamation of shellfish wastes for the production of chitinases

Shrimp and crab shell powder (SCSP), prepared by treating shellfish processing waste with boiling and crushing, was used as a substrate for isolating chitinolytic microorganisms. Three potential strains (E1, J1, and J1-1) were isolated and identified as Bacillus cereus, B. alvei, and B. sphaericus, respectively. Three extracellular chitinases (FB1, FB2, and FB3) were purified from the culture supernatants of Bacillus cereus E1, B. alvei J1, and B. sphaericus J1-1, respectively. The molecular weights of FB1, FB2, and FB3 were 71,000, 71,000, and 65,000, respectively, by SDS-PAGE. The pIs for FB1, FB2, and FB3 were 7.1, 7.2, and 7.4, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FB1 were pH 9, 50 degrees C, pH 7 to 10, and 70 degrees C; those of FB2 were pH 9, 60 degrees C, pH 5 to 9, and 70 degrees C; and those of FB3 were pH 7, 50 degrees C, pH 5 to 9, and 60 degrees C. The activities of all enzymes were strongly inhibited by Hg(2+) and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol.     

Truncation of the cellulose binding domain improved thermal stability of endo-beta-1,4-glucanase from Bacillus subtilis JA18

The C-terminus region of endo-beta-glucanase Egl499 from Bacillus subtilis JA18 was suggested to be a putative family 3 cellulose-binding domain (CBD) by computer analysis. To prove this proposal, C-terminus truncation mutant Egl330 was constructed and expressed. Compared with Egl499, Egl330 lost the cellulose binding capability at 4 degrees C, confirming the C-terminus region was a CBD. Binding of the CBD to Avicel was inhibited by carboxymethylcellulose (CMC), but not by barley beta-glucan and glucose at concentration of 0.1% and 0.5%. Kinetic analysis showed both the turnover rate (k(cat)) and the catalytic efficiency (k(cat)/K(m)) of Egl330 increased for the substrate CMC compared to Egl499. A great improvement in thermal stability was observed in Egl330. The half life of Egl330 at 65 degrees C increased to three folds that of Egl499, from 10 to 29 min. After treated at 80 degrees C for 10 min, Egl330 could recover more than 60% of its original activity while Egl499 only recovered 12% activity. UV spectrometry analysis showed Egl330 and Egl499 differed in refolding efficiency after heat treatment.     

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.     

Stabilizing effects of caprylate and acetyltryptophanate on heat-induced aggregation of bovine serum albumin

Acetyltryptophanate (AT) and caprylate (Cap) have been used to stabilize serum albumin against heat treatment. However, the mechanism of stabilization by these additives has never been fully elucidated. Here we used thermal melting to determine the effects of these additives on the melting temperature of bovine serum albumin (BSA) and heat stress at 60 degrees C to follow degradation of the protein in the presence of varying concentrations of AT or Cap. Native polyacrylamide gel electrophoresis was used to examine degradation products generated by heat treatment. Both additives increased the melting temperature of BSA, resulting in an increase by 12 degrees C at 5 mM AT and 3 degrees C at 1 mM Cap. They also conferred stability to BSA against heat stress at 60 degrees C. Complete protection was observed at 5 mM AT and 1 mM Cap. Comparison of AT and Cap in their effects on melting temperature and heat stress-induced degradation showed that a greater protection occurs with Cap which has a weaker effect on melting temperature. Based on this observation it was concluded that the observed protection by AT may be explained by its effects on melting temperature while that of Cap should be ascribed to other mechanisms.     

Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site

The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.     

Purification and Characterization of Protease and Chitinase from Bacillus cereus TKU006 and Conversion of Marine Wastes by These Enzymes

A chitinase- and protease-producing bacterium was isolated and identified as Bacillus cereus TKU006. The better condition on our tests for protease and chitinase production was found when the culture was shaken at 25 degrees C for 2 days in 25 mL of medium containing 2% shrimp shell powder (w/v), 0.1% K(2)HPO(4), and 0.05% MgSO(4).7H(2)O. The molecular masses of TKU006 protease and chitinase determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were approximately 39 and 35 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU006 protease and chitinase were 9, 50 degrees C, 3-11, 50 degrees C and 5, 40 degrees C, 3-11, 60 degrees C, respectively. TKU006 protease was inhibited completely by EDTA, indicating that the TKU006 protease was a metalloprotease. The TKU006 protease and chitinase retained 61%, 60%, 73%, and 100% and 60%, 60%, 71%, and 96% of its original activity in the presence of 2% Tween 20, 2% Tween 40, 2% Triton X-100, and 1 mM SDS, respectively. The antioxidant activity of TKU006 culture supernatant was determined through the scavenging ability on DPPH with 70% per milliliter. In conclusion, the novelties of the TKU006 protease and chitinase include its high stability to the surfactants and pH. Besides, with this method, we have shown that marine wastes can be utilized to generate a high-value-added product and have revealed its hidden potential in the production of functional foods.     

Heat-stable pullulanase from Bacillus acidopullulyticus: characterization and refolding after guanidinium chloride-induced unfolding

Heat-stable pullulanase from Bacillus acidopullulyticus was characterized with respect to its stability against thermal and chemical denaturation and its reactivation after complete chemical unfolding. The enzyme was quite thermostable and retained 55% of activity after heating at 60°C for 30 min at pH 5.5. At pH 6.0, only 9% residual activity was observed. The addition of sucrose, polyols, and Na₂SO₄ strongly stabilized the enzyme against thermal inactivation. The processes of chemical unfolding by guanidinium chloride (GdmCl) and refolding were studied by enzymological and spectroscopic criteria. B. acidopullulyticus pullulanase was very sensitive to GdmCl denaturation and had a transition midpoint at 1.2 M GdmCl. Reactivation after complete unfolding in 5 M GdmCl was initiated by dilution of the unfolding mixture; 67% reactivation was observed under standard conditions. The influence of some chemical and physical parameters (pH, chemical agents, temperature, and unfolding and refolding time) on refolding was investigated. Of the additives tested to assist reactivation, only bovine serum albumin (BSA) increased the yield of activity to 80%. The full regain of structure and activity was proven by comparing the enzymological, physicochemical, and spectroscopic properties of the native and refolded pullulanase. Received: June 22, 1998 / Accepted: December 11, 1998     

Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM 13/ATCC14580) was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. These were selected by using a consensus approach together with assessing protein stability changes (PoPMuSiC) and B-factor iterative test (B-FIT). The second-generation mutants involved combinations of two to seven individually favorable single mutations. Thermal stability was examined as half-life at 60 °C and by recording of thermal transitions by circular dichroism. Surprisingly, the biggest increment in thermal stability was achieved by producing the wild-type RGI lyase in Bacillus subtilis as opposed to in Pichia pastoris; this effect is suggested to be a negative result of glycosylation of the P. pastoris expressed enzyme. A ~ twofold improvement in thermal stability at 60 °C, accompanied by less significant increases in T _m of the enzyme mutants, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino acids to hydrophobic ones in surface-exposed loops produced favorable thermal stability effects.     

Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 1. Thermal stability of nicotinamide adenine dinucleotide dependent dehydrogenases.

The technique of differential scanning calorimetry (DSC) has been applied to the study of temperature-induced irreversible denturation and thus to the heat stability of soluble and Sepharose-bound liver alcohol dehydrogenase (LADH, EC 1.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) in the presence of various coenzymes or coenzyme fragments. The transition temperature (Ttr) of 82.5 degrees C obtained for soluble LADH was increased by 12.5 degrees C in the presence of a saturating concentration of NACH. In the presence of NAD+, Ttr increased by 8.5 degrees C, whereas ADP-ribose and AMP caused an increase in Ttr of only 2 and 1 degree C, respectively. The Ttr of 85.5 degrees C obtained for Sepharose-bound LADH was increased by about 12 degrees C after the addition of free NADH. However, when the enzyme was immobilized simultaneously with a NADH analogue (which also binds to the matrix), a broad endotherm with a Ttr of 91.5 degrees C was obtained, indicating the presence of immobilized enzyme molecules both with, and without, associated NADH. Corresponding increases in heat stability were observed for LDH in solution in the presence of NADH, NAD+, and AMP, leading to increases in Ttr from 72 to 79.5 and 74 and 73 degrees C, respectively. The addition of pyruvate and NAD+ to the enzyme to form an abortive ternary complex led to the same stabilization as that observed with NADH, attendant with a large increase in the enthalpy of transition, deltaHtr. In these studies the technique of DSC was utilized because it is applicable both to soluble and immobilized enzymes and (1) provides rapid information about Ttr and thus thermal stability of enzymes, (2) different energetic states of an enzyme molecule can be identified, and (3) an overall picture of the thermal process is rapidly obtained.     

The inactivation of foot and mouth disease, Aujeszky's disease and classical swine fever viruses in pig slurry

The aim of the study was to investigate the decontamination of pig slurry containing exotic viruses of pigs, foot AND mouth disease virus (FMDV), Aujeszky's disease virus (ADV) AND classical swine fever virus (CSFV). Laboratory-scale decontamination experiments showed that FMDV, ADV and CSFV were heat inactivated in slurry within 3 min at 67 degrees C, 3 min at 62 degrees C and 3 min at 60 degrees C and in Glasgow Eagles medium within 5 min at 67 degrees C, 4 min at 65 degrees C and 2 min at 65 degrees C, respectively. At pilot scale, FMDV was heat inactivated at 66 degrees C in water and 61 degrees C in slurry, ADV at 61 degrees C in water or slurry and CSFV at 62 degrees C in water and 50 degrees C in slurry. Treatment of pig slurry for the inactivation of exotic viruses may be achieved through the use of a thermal pilot plant operating in continuous mode. The work demonstrates the suitability of thermal treatment in ensuring the safety of pig slurry following a disease outbreak.     

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.     

Extracellular Maltase of Bacillus brevis

Bacillus brevis NRRL B-4389 produced extracellular maltase (alpha-glucosidase; EC 3.2.1.20) only in the presence of short alpha-1,4-glucosidic polymers, such as maltose and maltotriose. An optimum medium was developed; it contained 2.5% maltose, 0.5% nonfat dry milk, 0.4% yeast extract, and 0.01% CaCl(2). The enzyme was produced extracellularly during the logarithmic phase of growth; no cell-bound activity was detected at any time. Partial purification of the maltase was accomplished by using diethylaminoethyl cellulose batch adsorption, ammonium sulfate precipitation, and Sephadex G-200 gel filtration. Maltase, isomaltase (oligo-1,6-glucosidase), and glucosyltransferase activities were purified 20.0-, 19.1-, and 11.5-fold, respectively. Some properties of the partially purified maltase were determined: optimum pH, 6.5; optimum temperature, 48 to 50 degrees C; pH stability range, 5.0 to 7.0; temperature stability range, 0 to 50 degrees C; isoelectric point, pH 5.2; and molecular weight, 52,000. The relative rates of hydrolysis of maltose (G(2)), maltotriose (G(3)), G(4), methyl-alpha-d-maltoside, G(40), dextrin, and isomaltose were 100, 22, 12, 10, 10, 8, and 5%, respectively; the K(m) on maltose was 5.8 mM; d-glucose, p-nitrophenyl-alpha-d-glucoside, and tris (hydroxymethyl) aminomethane were competitive inhibitors; transglucosylase activity of the enzyme on maltose resulted in the synthesis of isomaltose, isomaltotroise, and larger oligosaccharides.     

Validation of a sensitive and automated 96-well solid-phase extraction liquid chromatography-tandem mass spectrometry method for the determination of desloratadine and 3-hydroxydesloratadine in human plasma

To support clinical development, a liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed and validated for the determination of desloratadine (descarboethoxyloratadine) and 3-OH desloratadine (3-hydroxydescarboethoxyloratadine) concentrations in human plasma. The method consisted of automated 96-well solid-phase extraction for sample preparation and liquid chromatography/turbo ionspray tandem mass spectrometry for analysis. [2H(4)]Desloratadine and [2H(4)]3-OH desloratadine were used as internal standards (I.S.). A quadratic regression (weighted 1/concentration(2)) gave the best fit for calibration curves over the concentration range of 25-10000 pg/ml for both desloratadine and 3-OH desloratadine. There was no interference from endogenous components in the blank plasma tested. The accuracy (%bias) at the lower limit of quantitation (LLOQ) was -12.8 and +3.4% for desloratadine and 3-OH desloratadine, respectively. The precision (%CV) for samples at the LLOQ was 15.1 and 10.9% for desloratadine and 3-OH desloratadine, respectively. For quality control samples at 75, 1000 and 7500 pg/ml, the between run %CV was 相似文献