Purification and some properties of three forms of glucoamylase from a Rhizopus species.

1. Three forms of glucoamylase [EC 3.2.1.3] were simultaneously purified from a Rhizopus species by (NH4)2SO4 fractionation and successive chromatographies on Sephadex G-75, DEAE-Sephadex, and CM-Sephadex, and were finally separated from each other by means of recycling chromatography on Bio-Gel P-150. The purification achieved was 3--4 fold from crude extract with respect to each glucoamylase; the yields of the three glucoamylases, designated as Gluc1, Gluc2, and Gluc3 in order of content, were 39, 7, and 0.4%, respectively. All the purified enzymes were homogeneous in polyacrylamide gel electrophoresis, isoelectric focusing, and ultracentrifugation. 2. The three glucoamylases were glycoproteins differing in both amino acid composition and carbohydrate content, but showed a common antigenicity in immunodiffusion. The molecular weights of Gluc1, Gluc2, and Gluc3 were estimated to be 74,000, 58,600, and 61,400, respectively, by sedimentation equilibrium and these values were verified by SDS-polyacrylamide gel electrophoresis. The specific activities of the three enzymes toward starch were in the opposite order to their molecular weights. 3. The three glucoamylases had the same broad pH optima in the range pH 4.5--5.0 and shared a common susceptibility to inactivation by heat, extreme pH, and such divalent cations as Hg2+, Pb2+, and Mn2+, indicating close similarity in enzymatic properties.     

Modification of a minor glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho p-toluenesulfonate

In order to elucidate the structure-function relationship of glucoamylases [EC 3.2.1.3, alpha-D-(1-4)-glucan glucohydrolase] from Aspergillus saitoi, the reaction of a minor component, Gluc M2 with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho p-toluenesulfonate (CMC) was studied at pH 4.5. Inactivation of Gluc M2 with [14C]CMC proceeded with the incorporation of about 5 CMC moieties. From the results of analyses of amino acid and sulfhydryl contents of CMC-modified Gluc M2 and the hydroxylamine treatment of the CMC-modified Gluc M2 at pH 7.0, it was concluded that the sites of CMC-modification were carboxylic acids of Gluc M2. In the presence of maltose, when Gluc M2 was treated with [14C]CMC, ca. 4 CMC moieties were incorporated with a simultaneous decrease in activity (30%). The Gluc M2 modified in the presence of maltose was re-modified with CMC after elimination of maltose. The CMC-modified Gluc M2 (70% activity) was inactivated completely with the further incorporation of ca. 2 CMC moieties. The logarithm of the half-life of the inactivation of Gluc M2 by CMC was a linear function of log[CMC] indicating that one carboxyl group among the modified ones was crucial for the inactivation of Gluc M2. From the results of these modification reactions, it was concluded that one or two carboxylic acids in Gluc M2 were crucial for the catalysis of glucoamylase from A. saitoi. Based on the analysis of the pH-profile of CMC inactivation of Gluc M2, the participation of a carboxylic acid having pKa 5.7 in the active site is proposed.     

The effect of pH and hypertonic KC1 on the collection of the cyclic AMP-protein complex from rat skeletal muscle and erythrocyte extracts.

The effect of pH on the extent of binding of cyclic AMP was evaluated by membrane filtration, charcoal exclusion and Sephadex G-25 chromatography. The amount of binding activity found in hemolysates of rat erythrocytes and 105,000 × g supernatants of rat thigh muscle homogenates varies appreciably with pH and method of measurement. Measurements of binding activity in a muscle extract by exclusion from Sephadex G-25 indicated the presence of two pH optima, one at pH 4.5 and the other at pH 7.4 or higher. The filtration method gave higher values than the charcoal method at pH 4.5 while the reverse was true at pH 7.4. With the erythrocyte preparation no binding was evident above pH 5.0 by either procedure except in the presence of 0.8 M KCl. Hypertonic KCl raised the pH of optimum binding to 5–5.5 for both tissues as indicated by both the filtration and charcoal methods. It is apparent from these results that the determination of cyclic AMP binding proteins from various tissues requires that more attention be paid to the role of ionic strength, pH and the mode of collection of the bound complex.     

N-bromosuccinimide oxidation of a glucoamylase from Aspergillus saitoi

1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4) glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with NBS was studied. 2. The tryptophan residues in Glu M1 were oxidized at various NBS/Gluc M1 ratios. The enzymatic activity decreased to about 80% of that of the native Gluc M1 with the oxidation of the first 2 tryptophan residues. The oxidation of these 2 tryptophan residues occurred within 0.2-0.5 s. On further oxidation of ca. 4-5 more tryptophan residues of Glu M1, the enzymatic activity of Gluc M1 decreased to almost zero (NBS/Gluc M1 = 20). Thus, the most essential tryptophan residue(s) is amongst these 4-5 tryptophan residues. 3. 7.5 tryptophan residues were found to be eventually oxidized with increasing concentrations of NBS up to NBS/Gluc M1 = 50. This value is comparable to the number of tryptophan residues which are located on the surface of the enzyme as judged from the solvent perturbation difference spectrum with ethylene glycol as perturbant. 4. In the presence of 10% soluble starch, about 5 tryptophan residues in Gluc M1 were oxidized at an NBS/Gluc M1 ratio of 20. The remaining activity of Glu M1 at this stage of oxidation was about 76%. On further oxidation, after removal of soluble starch, the enzymatic activity decreased to zero with the concomitant oxidation of 2 tryptophan residues. The results indicated that the essential tryptophan residue(s) is amongst these 2 tryptophans. 5. The UV difference spectrum induced by addition of maltose and maltitol to Gluc M1 showed 4 troughs at 281, 289, 297, and 303 nm. The latter 3 troughs were probably due to tryptophan residues of Gluc M1 and decreased with NBS oxidation.     

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.     

Starch digestion and adsorption by beta-amylase of Emericella nidulans (Aspergillus nidulans).

A mutant strain of Emericella nidulans MNU 82 was isolated by multistep mutation. The beta-amylase produced by the mutant was able to digest raw starch. It was readily and strongly adsorbed onto raw starch at pH 5.0. The enzyme to starch ratio was 1950 U/g starch. The enzyme showed no correlation between the capacity of raw starch digestion and adsorption of the enzyme.     

Purification and characterization of an exo-polygalacturonase from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici

Abstract An exo-polygalacturonase (EC 3.2.1.15) was purified to apparent homogeneity from cultures of Fusarium oxysporum f.sp. lycopersici on synthetic medium supplemented with citrus pectin, using preparative isoelectric focusing. The enzyme, denominated PG2, had an apparent M _r of 74000 Da upon SDS-PAGE. The pI of the main PG2 isoform was 4.5, and pH and temperature optima were 5.0 and 55 °C, respectively. PG2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by anaysis of degradation products. The enzyme was N-glycosylated. The N-terminal amino acid sequence, L-A-F-N-V-P-S-K-P-P, has no identity to other known polygalacturonases.     

A novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization

This study reports the purification and characterization of a novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1. Maximum alpha-amylase activity (53 U mL(-1)) was obtained at 45 degrees C after 44 h of incubation. The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography, and showed a molecular weight of 56 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH 5.0, performed stability over a broad range of pH 4.5-11.0, and was optimally active at 40-50 degrees C. The enzyme preparation had a strong digesting ability towards various raw starches and efficiently hydrolyzed raw corn starch at a concentration of 20% and pH 5.0, which were normally used in the starch industries, in a period of 12h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel alpha-amylase.     

Starch digestion and adsorption by β-amylase of Emericella nidulans (Aspergillus nidulans)

B. CHATTERJEE, A. GHOSH AND A. DAS. 1992. A mutant strain of Emericella nidulans MNU 82 was isolated by multistep mutation. The β-amylase produced by the mutant was able to digest raw starch. It was readily and strongly adsorbed onto raw starch at pH 5.0. The enzyme to starch ratio was 1950 U/g starch. The enzyme showed no correlation between the capacity of raw starch digestion and adsorption of the enzyme.     

Behaviour of Endomycopsis fibuligera glucoamylase towards raw starch

An extracellular glucoamylase [exo-1,4-α-d-glucosidase, 1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] of Endomycopsis fibuligera has been purified and some of its properties studied. It had a very high debranching activity (0.63). The enzyme was completely adsorbed onto raw starch at all the pH values tested (pH 2.0–7.6). Amylase inhibitor from Streptomyces sp. did not prevent the adsorption of glucoamylase onto raw starch although the enzyme did not digest raw starch in the presence of amylase inhibitor. Sodium borate (0.1 m) eluted only 35% of the adsorbed enzyme from raw starch. The optimum pH for raw starch digestion was 4.5 whereas that of boiled soluble starch hydrolysis was 5.5. Waxy starches were more easily digested than non-waxy starches, and root starches were slowly digested by this enzyme.     

Modification of glucoamylases from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide

To investigate the role of carboxyl groups of glucoamylases [EC 3.2.1.3] from a Rhizopus sp. (Gluc1 and Gluc2), the modification of Gluc1 and Gluc2 with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), was studied. The inactivation of Gluc1 proceeded with the incorporation of about 3 CMC moieties. In the presence of maltose, the modification of about 2.2 carboxyl groups of Gluc1 proceeded with a slight loss of enzymatic activity. In the re-modification of Gluc1 modified in the presence of maltose, Gluc1 was inactivated by further modification of about 1.3 carboxyl groups. Therefore, one carboxyl group, which was protected by maltose, was thought to be a crucial one. The inactivation of Gluc2 proceeded similarly to that of Gluc1, but the number of CMC moieties incorporated was about one less than in the case of Gluc1. Thus, it was suggested that one of the reactive carboxyl groups of Gluc1 was located in the N-terminal part of Gluc1, which is deficient in Gluc2. From the results of kinetic studies on CMC-modified Gluc1, it was suggested that the hydrolysis mechanism of malto-oligomers differs somewhat from that of PNPG.     

Association of Influenza Virus Matrix Protein with Ribonucleoproteins

To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.     

Stepwise cleavage of rabbit immunoglobin G by papain and isolation of four types of biologically active Fc fragments

Four types of Fc fragments of different sizes were isolated by papain treatment of rabbit immunoglobulin G under various conditions and by subsequent chromatographic procedures. 1. Brief digestion at neutral pH without reduction produced a molecule in which the Fab and Fc fragments were still linked by a pair of labile disulphide bridges, and the Fc fragment released by cleaving these bonds, called 1Fc fragment, contained a portion of the ;hinge' region including an interchain disulphide bridge. Both complement-binding and guinea-pig skin-binding activities were retained by this fragment, which had mol. wt. 48000. 2. Prolonged digestion at neutral pH of immunoglobulin G whose labile inter-heavy-chain disulphide bridges had been reduced removed the ;hinge' region, giving mFc fragments (mol. wt. 46000), which lacked the capacity to bind guinea-pig skin but retained the antigenic as well as the complement-binding activities of 1Fc fragment completely. 3. Digestion at pH5.0 yielded a smaller fragment, sFc (mol. wt. 40000), which was no longer able to bind complement. Though the antigenic structure was intact, sFc fragment was curiously unable to precipitate with antibodies to the N-terminal determinants. 4. Fragment stFc (mol. wt. 25000), representing the C-terminal portion of Fc fragment, was formed from all the larger fragments by digestion at pH4.5. Only the C-terminal antigenic determinants were retained by stFc fragment.     

Effect of pH on MHC class II-peptide interactions.

The effect of pH on class II-peptide interactions has been analyzed using several mouse (IAd, IAk, IEd, IEk) and human (DR1, DR5, DR7) MHC specificities, and eight different class II-restricted determinants. In direct binding assays, acidic conditions led to increased binding capacity for many class II-peptide combinations. IE molecules seemed to bind optimally around pH 4.5, whereas IA molecules displayed binding optima in the 5.5 to 6.5 range. In contrast, the DR molecules studied were, in most cases, affected only marginally by pH changes in the 4.5 to 7.0 range. Despite these apparent isotype-specific trends, no general rule could be formulated, because even for the same class II molecules, the binding capacity could be increased for many peptides when the binding was performed under acidic conditions, was unaffected for some, and even decreased for others. The mechanisms responsible for this complex behavior were analyzed in more detail by kinetic and equilibrium analysis of three different class II-peptide combinations (IAd/OVA 323-339, IAk/HEL 46-61, and DR1/HA 307-319). It was found that acidic pH conditions could affect both on and off rates for class II-peptide complexes. Depending on the net balance of these effects, either increases, decreases, or no effect on overall affinities at equilibrium were detected. In the case of IAd/OVA 323-339, it was also found that acidic conditions influenced the binding capacity of class II molecules by increasing the fraction of sites available for peptide binding, presumably by favoring dissociation of endogenously bound, acid-sensitive peptides.     

A study of the binding of adenosine diphosphate to myosin subfragment-1.

The binding of ADP to subfragment-1 was investigated by the gel filtration method. The amount of bound ADP was determined as a function of free ADP concentration. Linear Scatchard plots were obtained. The maximum binding number, 0.55 mole of ADP per 10(5) g of protein, and the dissociation constant, 1.6 x 10(-6) M, were obtained, using subfragment-1 prepared by tryptic digestion, in the presence of 0.083 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 25 degrees. Similar maximum numbers, about 0.5 mole per 10(5) g of protein, were obtained with subfragment-1 prepared by chymotryptic digestion of myosin or papain digestion of myofibrils. The maximum number did not depend on the KCl concentration or the temperature, while the dissociation constant decreased on decreasing either the KCl concentration or the temperature. Adenylyl imidodiphosphate binding to subfragment-1 prepared by chymotryptic digestion was also measured by the gel filtration method. The maximum binding number, 0.41 mole per 10(5) g of subfragment-1, and the dissociation constant, less than 10(-7) M, were obtained in the presence of 0.7 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 8 degrees. The difference absorbance at 288 nm of the difference absorption spectrum induced by ADP of subfragment-1 prepared by tryptic digestion was proportional to the amount of bound ADP. The steady-state ATPase rate of subfragment-1 prepared by tryptic digestion was inhibited competitively by ADP in the presence of MgCl2. The extent of the initial burst of ATPase [EC 3.6.1.3] decreased from 0.46 +/- 0.06 to 0.30 +/- 0.09 mole of Pi per 10(5) g of subfragment-1 on adding ADP to a level of 0.6 mM. Subfragment-1 prepared by tryptic digestion bound F-actin with a mole ratio of 1/0.96 of actin monomer. The binding was depressed by the addition of ADP. On the basis of these results, subfragment-1 preparations were assumed to be a half-and-half mixture of two kinds of protein, and properties of each protein are discussed.     

Comparative characterization of two serine endopeptidases from Nocardiopsis sp. NCIM 5124

A protease-producing, crude oil degrading marine isolate was identified as Nocardiopsis sp. on the basis of the morphology, cell wall composition, mycolic acid analysis and DNA base composition. The Nocardiopsis produces two extracellular proteases, both of which are alkaline serine endopeptidases. Protease I was purified to homogeneity by chromatography on CM-Sephadex at pH 5.0 and pH 9.0. Protease II was purified using DEAE-cellulose, Sephadex G-50, phenyl-Sepharose and hydroxyapatite chromatography. Protease I and II had almost similar M(r) of 21 kDa (Protease I) and 23 kDa (Protease II), pI of 8.3 and 7.0 respectively with pH and temperature optima for activity between 10.0 and 11.0 and about 60 degrees C. Specific activities were 152 and 14 U/mg respectively on casein. However, Protease I was antigenically unrelated to Protease II. Both proteases were endopeptidases and required extended substrate binding for catalysis. Both proteases had collagenolytic and fibrinolytic activity but only Protease I had elastinolytic activity. The proteases were chymotrypsin-like with respect to their amino acid compositions and N-terminal sequences.     

Physical characteristics of hyaluronate binding to the surface of simian virus 40-transformed 3T3 cells

The binding of labeled hyaluronate to the surface of Simian virus 40-transformed 3T3 cells was studied as a function of 1) pH, 2) ionic strength, 3) temperature, and 4) molecular weight of the hyaluronate. Binding occurred over a wide range of pH values with optima at pH 7 and at less than pH 4. Binding at low pH was eliminated at high ionic strength whereas that at physiological pH was enhanced, with a maximum at 0.5 M NaCl. The enhancement of binding at pH 7 was reversible and independent of the particular salt used. Scatchard plot analysis showed that increasing the ionic strength resulted in both a decrease in the dissociation constant (Kd) and an increase in the amount bound at saturation (Bmax). Temperature also influenced the binding of hyaluronate to the cell surface. The amount bound at low temperatures (0 degrees C) was 3 to 5 times that bound at high temperatures (40 degrees C) with a sharp transition occurring at 18 degrees C, the temperature of phase transition of the plasma membrane. The temperature effect was primarily a change in the Bmax and was reversible. Finally the molecular weight of the ligand influenced the binding. High molecular weight preparations of hyaluronate had a higher binding affinity (lower Kd) and a lower Bmax than did smaller molecular weight preparations.     

Comparative properties of channel catfish (Ictalurus punctatus) and blue crab (Callinectes sapidus) acetylcholinesterases

1. Acetylcholinesterases (AChEs) from channel catfish and blue crabs were examined for substrate preference, KmS, effects of inhibitors, and pH and osmotic activity profiles. 2. Similarities were noted for substrate preference along with pH and osmotic optima. 3. Crab AChE had a lower Km (9 x 10(-5) vs 2 x 10(-4) M) and was more sensitive in terms of KI50S than fish AChE to eserine (2.6 x 10(-7) vs 3 x 10(-7) M), malathion (4.5 x 10(-5) vs 1.6 x 10(-4) M) and parathion (6.9 x 10(-5) vs 7 x 10(-4) M). 4. Fish AChE appeared easier to solubilize using Triton X-100.     

Characterization of two partially unfolded intermediates of the molecular chaperone DnaK at low pH

In this study, the effect of pH on the conformation and the reactivity of the Escherichia coli Hsp70 molecular chaperone DnaK was investigated using spectroscopic and chemical assays. DnaK exhibits negligible binding of the hydrophobic dye 1-anilino-naphthalene-8-sulfonate (ANS) between pH 7 to 5.0, whereas appreciable binding occurs between pH 4.5 to 4.0. The binding of ANS to a protein is diagnostic of the presence of accessible ordered hydrophobic surfaces. Such hydrophobic surfaces are often displayed by partially folded protein intermediates such as molten globules. Nucleotide inhibits 70% of the ANS binding at pH 4.5 but none of the ANS binding at pH 4.0. Proteolysis of nucleotide-free DnaK at pH 4.5 with cathepsin D yields detectable fragments (masses > 20 kDa) of the C-terminal peptide-binding domain but none of the N-terminal ATPase domain, thus the ATPase domain is preferentially targeted for proteolysis. In contrast, proteolysis of nucleotide-free DnaK at pH 4.0 with cathepsin D cuts near the linker region, yielding both functional domains. Our interpretation of these data is that incubation of DnaK at pH 4.5 produces a partially unfolded form of the ATPase domain, in which secondary structure is mainly intact, but tertiary structure is reduced. Incubation of the protein at pH 4.0 produces an intermediate in which both functional domains have collapsed and possibly separated. Nucleotide inhibits the conformational change that occurs at pH 4.5 but not at 4.0.     

Alcoholic fermentation of raw cassava starch by Rhizopus koji without cooking

Using only wheat bran koji from the Rhizopus strain, raw cassava starch and cassava pellets converted reasonably well to alcohol (ethanol) without cooking at 35 degrees C and pH 4.5-5.0. When the initial broth contained 30 g raw cassava starch, 10 g Rhizopus sp. koji, and 100 mL tap water, 12.1 g of alcohol was recovered by final distillation from fermented broth. In this case, 12.1 g alcohol corresponds to an 85.5% conversion rate based on the theoretical values of the starch content. When the initial broth contained 40 g cassava starch, 14.1 g of alcohol was recovered, where 14.1 g corresponds to a 74.5% conversion rate. The alcoholic fermentation process described in the present work is considered more effective and reasonable than the process using raw starch without cooking reported until now, since the new process makes it unnecessary to add yeast cells and glucoamylase preparation.