Fractionation,Purification and Some Properties of Proteolytic Enzymes from Stem Bromelain

The heterogeneity of the proteolytic enzymes in the stem bromelain was investigated by the isoelectric focusing with carrier ampholytes. The isoelectric focusing of the stem bromelain demonstrated the presence of two types of proteolytic enzymes which were distinguishable from each other by their isoelectric points. One of these was a basic protein having an isoelectric point of 9.45. This basic enzyme comprised almost all of basic protein which are found in stem bromelain. The other was an acidic protein having an isoelectric point near pH 4.7. This was a minor compooent. The purification of the two enzymes was carried out by use of chromatographies on CM-Sephadex, DEAE-Sephadex and Sephadex at pH 7.0.     

Changes in relative mobility of pancreatic amylase variants in isoelectric focusing

Using isoelectric focusing with one ampholytic solution, double- and single-banded amylase phenotypes were found in a sample of rhesus monkeys,Macaca mulatta. When applying different ampholytic solutions, these variants were shown to change their position relative to each other. Single-banded phenotypes showed either a position corresponding to one of the bands of the double-banded phenotype or to an intermediate one. Family studies, however, suggested that the differences between the observed patterns were not caused by genetic differences. This discloses a problem with respect to the interpretation of electrophoretic data, i.e. bands with different positions produced by isoelectric focusing may not necessarily represent genetic differences.     

Purification,partial characterization,and postembryonic levels of amylases from Sitophilus oryzae and Sitophilus granarius

Amylases from adults of Sitophilus oryzae (L.) and S. granarius (L.) were purified by using a sequential procedure of ammonium sulfate precipitation, glycogen-complex formation, and ion exchange chromatography. Amylase of S. oryaze was purified 47.4-fold to a specific activity of 478 units/mg protein. One amylase unit equals 1 mg maltose hydrate produced/min at 30°C. Amylase of S. granarius was purified 85.4-fold to a specific activity of 453 units/mg protein. Amylase of S. oryzae had a Km of 0.173% for soluble starch and consisted of two anionic isozyrnes with isoelectric points of pH 3.70 and pH 3.76. Amylase of S. granarius had a Km of 0.078% for starch and was a single protein with an isoelectric point of pH 3.76. Purified amylases of both species had molecular weights of 56,000 estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, were activated by chloride, and had double energies of activation calculated from Arrhenius plots. Based on fresh weights of adults feeding on whole wheat through 10 weeks of age, S. oryzae contained three-fold and eight-fold more amylase than S. granarius and S. zeamais Motschulsky, respectively. High amylase levels in S. oryzae may provide this species with an adaptive advantage when feeding on cereals containing naturally occurring amylase inhibitors.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: III. alpha-Amylase Isozymes

The formation of amylase isozymes in germinating rice (Oryza sativa) seeds was studied by isoelectric focusing on polyacrylamide gel disc electrophoresis. Time sequence comparisons of the amylase zymogram were made between extracts from gibberellic acid-treated embryoless and embryo-attached half-endosperm of rice seeds. In both cases, 4 major and 9 to 10 minor isozyme bands were detectable at the maximal stage of the enzyme induction. However, in the embryo-attached half-seeds, bands started to diminish after the 5th day of incubation, in agreement with the results of time sequence analyses of enzyme activities. Nearly identical patterns of amylase isozyme bands on a polyacrylamide gel disc electrophoresis in combination with isoelectric focusing indicate the intrinsic role of gibberellic acid in the starch breakdown in germinating rice seeds. We tentatively assign the newly synthesized enzymes to be α-amylases based on experimental results concerning the lability of the preparation on a prolonged treatment at pH 3.3 and the stability on heat treatment for 15 minutes at 70 C.     

Multiple forms of acid phosphatase in rat liver parenchymal,endothelial, and kupffer cells

Purified suspensions of highly viable parenchymal, endothelial, and Kupffer cells were prepared from rat liver. In the liver cell classes, total activities of acid phosphatase were determined with 4-methylumbelliferylphosphate, 1-naphthylphosphate, and p-nitrophenylphosphate. The specific enzyme activities were different for each type of cell and, even within one cell class, the enzymes showed different conversion rates for the three substrates. These results indicate the presence of multiple forms of acid phosphatase enzymes in each cell class. The inhibiting effects of tartrate, fluoride, and alloxan on the acid phosphatase activities were investigated. Depending on the substrate used, the inhibitors inactivated the enzymes at different rates, which also indicates the presence of multiple forms of acid phosphatase enzymes in the liver cell classes. By means of an isoelectric focusing technique, acid phosphatase enzymes could be separated on the basis of their differences in isoelectric points. One form with an isoelectric point around 4 is found in Kupffer cells, whereas another form with an isoelectric point of about 7 is found in parenchymal cells. Endothelial cells possess both forms. These findings suggest a specificity in the function of this lysosomal enzyme in each cell class.     

Demonstration of amylase from the zoophytophagous anthocorid Orius insidiosus

To better understand facultative phytophagy in the zoophytophagous anthocorid, Orius insidiosus, tests for amylase were conducted and the enzyme was partially purified. Three activity bands were detected with polyacrylamide-starch gel electrophoretic analysis of amylase in O. insidiosus. The major amylase was found to have a mean isoelectric point (pI) of 4.53. The presence of amylase indicates the ability of O. insidiosus to use starch, a nutrient, derived from plants, either by direct ingestion or by ingestion of plant material from the digestive system of their prey. The presence of amylase suggests that these predators are more committed to plant feeding than other species of predators that lack this enzyme.     

Multiple dextranases from the yeast <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The soil yeast Lipomyces starkeyi (NCYC 1436) secretes dextranase activity into the growth medium. Resolution of a dextranase-active protein fraction by SDS-PAGE produced three protein bands, of 66 kDa, 68 kDa and 78 kDa, and isoelectric focusing of the same fraction resulted in seven protein bands, of pIs 3.50, 3.85, 4.20, 4.80, 4.85, 5.00 and 5.30. Dextranase activity was demonstrated for all the isoelectric forms, and for the 78 kDa species in the presence of SDS. Amino acid compositions of the 66 kDa, 68 kDa and 78 kDa protein bands were determined, and the N-termini of the 66 kDa and 78 kDa protein bands were sequenced: the first two amino acids at the N-terminus of each protein were alanine and valine, respectively; an alanine-valine pair is seen early in the N-terminal coding sequences of the dextranases and the isopullulanase produced by the phylogenetically disparate organisms contributing to glycosyl hydrolase family 49.     

Variation in gene copy number and polymorphism of the human salivary amylase isoenzyme system in Caucasians

Summary The polymorphic patterns of human salivary amylase of a large number of individuals of Caucasian origin were determined by using isoelectric focusing and polyacrylamide gel electrophoresis. Nine different salivary amylase protein variants were found; three of them are recorded for the first time and their heredity is shown. Some of the variants are encoded by haplotypes expressing three allozymes. Most variants display low frequencies. Analysis of the relative intensities of variant-specific isozyme bands, combined with segregation analysis, show that extensive quantitative variation is present in the population. The numbers of salivary amylase genes in some families showing quantitative variation at the protein level have been estimated by the polymerase chain reaction. We present evidence that quantitative variations in amylase protein patterns do not always reflect variations in gene copy number but that other mechanisms are also involved.     

Purification and characterization of two alpha-amylases from Toxoplasma gondii.

Two distinct alpha-amylases have been identified in Toxoplasma gondii. They were purified close to homogeneity from cytoplasmic and membrane fractions. The apparent molecular weight of the cytoplasmic amylase was 22,300 Da and that of the membrane enzyme was 39,600 Da by gel filtration, and 25,000 and 41,000 Da by SDS gel electrophoresis, respectively. The physicochemical and catalytic properties of both enzymes showed them to be very different. Cytoplasmic alpha-amylase had an acid isoelectric point and its optimum pH was pH 5.0; its activity was unaffected by NaCl, Ca2+, or EDTA. The membrane alpha-amylase had an isoelectric point of 7.7 and an optimum pH of 8.0. It was affected by Ca2+, inhibited by EDTA, and activated eight-fold by NaCl. Both amylases were inactivated by temperatures above 65 degrees C, but cytoplasmic amylase was more resistant to thermal denaturation.     

Thermostable α‐Amylase and Glucoamylase from Thermomyces lanuginosus F1

An α‐amylase and a glucoamylase produced by Thermomyces lanuginosus F₁ were separated by ion‐exchange chromatography on Q‐Sepharose fast flow. The enzymes were further purified to electrophoretic homogeneity by chromatography on Sephadex G‐100 and Phenyl‐Sepharose CL‐4B.The molecular weights and isoelectric points of the enzymes were 55,000 Da and pHi 4.0 for α‐amylase and 70,000 Da and pH_i 4.0 for glucoamylase, respectively. The optimum pH and temperatures for the enzymes were found to be 5.0 and 60 °C for α‐amylase, and 6.0 and 70 °C for glucoamylase,respectively. Both enzymes were maximally stable at pH 4.0 and retained over 80% of their activity between pH 5.0 and 6.0 for 24 h. After incubation at 90 °C (1 h), the α‐amylase and glucoamylase retained only 6% and 16% of their activity, respectively. The enzymes readily hydrolyzed soluble starch, amylose, amylopectin and glycogen but hydrolyzed pullulan very slowly. Glucoamylase and α‐amylase had highest affinity for soluble starch with K_M values of 0.80 mg/ml and 0.67 mg/ml, respectively. The α‐amylase hydrolyzed raw starch granules with a predominant production of glucose and maltose. The activities of α‐amylase and glucoamylase increased in the presence of Mn²⁺, Co²⁺, Ca²⁺, Zn²⁺ and Fe²⁺, but were inhibited by guanidine‐HCl, urea and disodium EDTA. Both enzymes possess pH and thermal stability characteristics that may be of technological significance.     

Isolation and characterization of porcine parathyroid cathepsin B.

Cathepsin B was isolated from porcine parathyroid tissue and from liver by a procedure involving acetone precipitation, gel filtration, and carboxymethylcellulose chromatography. The final preparations of each migrated as single bands upon sodium dodecyl sulfate polyacrylamide gels but exhibited several minor active variants upon isoelectric focusing. The parathyroid and liver enzymes were similar to each other and also resembled cathepsin B from other sources. The molecular weights for the porcine enzymes were estimated as 25,000, and the isoelectric point was at pH 4.8. The parathyroid enzyme cleaved benzyloxycarbonyl-Val-Lys-Lys-Arg-(4-methoxy)-2-naphthylamide at pH 5.8 and 37 degrees C with a Km of 0.14 mM and a kcat of 68 s-1. The pH optimum for this reaction was pH 6 to 7. The enzyme was unstable above pH 7.5 and below pH 4.5. It was strongly inhibited by HgCl2, ZnSO4, iodoacetate, iodoacetamide, and N-ethylmaleimide which indicated that it is a thiol protease, and by leupeptin, a strong inhibitor of cathepsin B from other sources. Antibodies to the parathyroid enzyme were elicited in rabbits. The antisera formed single precipitin bands upon double diffusion in agar gels against both the parathyroid and liver enzymes. Precipitin bands were formed at both pH 6 and pH 8.5 which indicated that the antisera recognized both native and denatured forms of the enzymes.     

Starch Utilization by Bacteroides ovatus Isolated from the Human Large Intestine

Starch supported growth of continuous cultures of Bacteroides ovatus when this carbohydrate provided the sole source of carbon and energy. Inducible amylase and α-glucosidase activities were inversely related to dilution rate in starch-limited and starch-excess chemostats over the dilution rate (D) range D = 0.03/h to D = 0.20/h, and were partly repressed during growth under conditions of starch-excess. Preparative isoelectric focusing of B. ovatus cytoplasmic extracts indicated the existence of three distinct starch-hydrolyzing enzymes. Incubation of active fractions from the isoelectric focusing cell with maltose and a variety of low-molecular-weight oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose) identified a single amylase activity, an enzyme with combined β-amylase and glucoamylase/α-glucosidase properties, and also a possible pullulanase. The ability of B. ovatus to synthesize several starch-hydrolyzing enzymes with different specificities and activities may confer a significant competitive advantage to this organism in the colonic ecosystem. Received: 14 August 1996 / Accepted: 29 October 1996     

Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.     

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.     

Midgut amylase,lysozyme, aminopeptidase,and trehalase from larvae and adults of Musca domestica

Based on polyacrylamide gel electrophoresis, density-gradient ultracentrifugation and thermal inactivation, there is only one major molecular species of each of the following larval enzymes (soluble in water or solubilized in Triton X-100): membrane-bound aminopeptidase (pH optimum 8.5; K_m 0.21 mM L-leucine p-nitroanilide; M_r 322,000), amylase (pH optimum 6.5; K_m 0.14% starch; M_r 66,000), lysozyme (pH optimum 3.5; K_m 0.3 mg/ml; Mr 24,000); and membrane-bound trehalase (pH optimum 5.0; K_m 1.09 mM trehalose; M_r 94,000). Except for lysozyme, the properties of adult digestive enzymes are different from those described for larval enzymes. Larval aminopeptidase and trehalase were purified by electrophoresis and larval lysozyme (contaminated with amylase) by density-gradient ultracentrifugation, and were used to raise antibodies in a rabbit. Antibodies raised against larval aminopeptidase, trehalase, and amylase did not recognize the imaginal enzymes, whereas those against larval lysozyme recognize imaginal lysozyme. The data suggest that the genes coding for digestive enzymes (except for lysozyme) are different in larvae and imagoes.     

Mutants ofXanthomonas campestris defective in secretion of extracellular enzymes

Summary Mutants ofXanthomonas campestris B 1459 were isolated that are defective in secretion of both cellulase and amylase. Both enzymes accumulated in the periplasmic space. The defects in secretion of cellulase or amylase were partly overcome by introducing into the mutants specific multiple copies of DNA cloned fromX. campestris, and presumed to code for cellulase or amylase enzymes. The mutant strains also showed reduced amounts of extracellular pectinase and protease activities, as if the mutants were generally defective for secretion of extracellular enzymes. The mutants showed reduced pathogenesis for turnip seedlings. The secretion-defective mutants may allow production of xanthan gum with reduced cellulose, pectin, protein and starch-degrading enzyme activities, thereby allowing more widespread mixing of microbially produced xanthan gum with these commercially important water-soluble polymers.     

Monilidiols,Characteristic and Bioactive Metabolites of Benomyl-resistant Strains of Monilinia fructicola

A new polygalacturonase was found in a culture filtrate of Aureobasidium pullulans. The enzyme was purified and obtained in crystalline form with 10% recovery. The crystalline enzyme was a homogeneous protein by analyses by sedimentation and electrophoresis. The enzyme was most active around pH 4.5, and stable in the pH range of 4 to 6. Its molecular weight was 42,000 and its isoelectric point was pH 6.0. The enzyme was an endo-polygalacturonase, catalyzing the cleavage of glycosidic bonds of polygalacturonic acid at random.

The enzyme had less protopectinase activity than those of the endo-polygalacturonases that were isolated as protopectin-solubilizing enzymes from Trichosporon penicillatum, Kluyveromyces fragilis, and Galactomyces reessii. Some characteristics were compared with the endo-polygalacturonases, which have potent protopectinase activity.     

Purification of β-N-Acetylhexosaminidase from Egg White and the Microsomal and Lysosomal Fractions of Hen Oviduct

β-N-Acetylhexosaminidase (NAHase) was purified from egg white and the lysosomal and microsomal fractions of hen oviduct. The purification procedure included affinity chromatography using Sepharose 4B coupled with IgG specific for NAHase of hen oviduct. The isoelectric points of the three enzymes were different, but their antigen determinants were identical. In sodium dodecyl sulfate polyacrylamide gel electrophoresis, both the egg white and lysosomal enzyme gave only one protein band each, corresponding to a MW of 68000 and 53000, respectively, but the microsomal enzyme gave two protein bands, corresponding to those of the lysosomal and egg white enzymes.     

alpha-amylase from five strains of Bacillus amyloliquefaciens: evidence for identical primary structures.

The alpha-amylases from five strains of Bacillus amyloliquefaciens were compared to determine whether differences in primary structure are responsible for variations in catalytic properties previously reported among the enzymes. Amino acid analysis established virtually identical compositions for the proteins. Reaction with dimethylaminoaphthylene sulfonylchloride indicated the amino-terminal amino acid of each amylase to be valine. Carboxyl termini of the enzymes have been determined by digestion with carboxypeptidase A. The resulting kinetic data indicate tyrosine as the carboxyl terminus and leucine as the penultimate residue for all five proteins. Isoelectric focusing of the enzymes yielded isoelectric points in the pH range of 5.09 to 5.18. Tryptic digests of the enzymes chromatographed on a cation-exchange column showed identical elution patterns. It is concluded that the primary structure of the amylase from the five strains is identical or exhibits only conservative substitutions.     

Homology modeling and molecular dynamics study on Schwanniomyces occidentalis alpha-amylase

With consumers growing increasingly aware of environmental issues, industries find enzymes as a reasonable alternative over physical conditions and chemical catalysts. Amylases are important hydrolase enzymes, which have been widely used in variety of industrial process such as pharmaceutical, food, and fermentation industries. Among amylases α-Amylase is in maximum demand due to its wide range of applications. The homology modeling study on Schwanniomyces occidentalis amylase (AMY1, UniProt identifier number: P19269) was performed by Modeller using Aspergillus oryzae (6TAA) as the template. The resulting structure was analyzed for validity and subjected to 14 ns of molecular dynamics (MD) simulation trough GROMACS. The validity of obtained model may represent that utilized OPLS force field is suitable for calcium-containing enzymes. DSSP secondary structure and contact map analysis represent the conservation of domain A TIM barrel feature together with calcium ion coordination sphere. Investigating the covariance matrix followed by principle component analyses for the first five eigenvectors of both trajectories indicate a little more flexibility for AMY1 structure. The electrostatic calculation for the final structures shows similar isoelectric point and superimposed buffering zone in the 5–8 pH range.