Isolation of a detergent-solubilized maltase/glucoamylase from rat intestine and its comparison with a maltase/glucoamylase solubilized by papain

Maltase/glucoamylase from the rat intestinal brush-border membrane was solubilized by homogenization of the intestinal mucosa in buffer containing 0.5% Triton X-100. After removal of the detergent with butan-1-ol, the enzyme was purified by chromatography on Sepharose 4B and DEAE-cellulose. The final specific activity was 70.3 units/mg of protein in six preparations, comparing favourably with the specific activity of 65.0 units/mg of protein of a pure papain-solubilized maltase/glucoamylase previously isolated and characterized by us [Flanagan & Forstner (1978) Biochem. J. 173, 553–563]. The two enzymes were compared. Both migrated as single bands with the same mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were eluted at the same volume from Sepharose 4B, and had the same sedimentation pattern in mannitol gradients. The amino acid composition was similar; content of total apolar residues differed by 1.0mol%. Antibodies prepared against either enzyme gave identical precipitin lines with each. Neither enzyme bound tritiated Triton X-100. The only difference noted was the tendency of the detergent-solubilized enzyme to aggregate on storage, whereas the papain-solubilized enzyme remained unchanged. Both enzymes had two N-termini, glycine and arginine. When the two enzymes were dissociated by boiling in sodium dodecyl sulphate, each exhibited the same five species on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Single N-termini were found in the two smaller species, 1 (glycine) and 2 (arginine), whereas larger species (3–5) had both N-terminal amino acids. Both the Triton- and papain-solubilized enzymes appear to be oligomers of species 1 and 2, indicating that the native enzyme contains two subunit types. Aggregation in aqueous solutions does not depend on a proteolytically susceptible peptide fragment at the N-terminus of either subunit.     

Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH.

Maltase-glucoamylase, a microvillous membrane ectoenzyme, was solubilized from rat intestinal mucosa by digestion with papain and subsequently purified to homogeneity with an overall yield of 10--20%. An antibody to the purified enzyme formed a single precipitin line in immunodiffusion experiments with an intestinal homogenate. The enzyme was shown to be an acidic glycoprotein (20% sugar by weight) which contained low amounts of cysteine and no sialic acid. At pH3--6, maltase activity was slowly lost, but the enzyme was re-activated by re-adjustment of the pH to neutrality. However, in the presence of sodium dodecyl sulphate, acid pH values inactivated maltase irreversibly, and at the same time converted the enzyme (mol.wt. 500000 approx.) into five new species with apparent molecular weights ranging from 134000 to 480000 as judged by polyacrylamide-gel electrophoresis. The same five fragments were also formed by boiling the enzyme for brief periods in the presence of sodium dodecyl sulphate or urea either with or without reducing agents. The dissociated species were stable on re-electrophoresis, and amino acid analysis showed them to be very similar to each other and to the original enzyme. The bands migrated anomalously on polyacrylamide gels of different concentration, thereby preventing the assignment of precise molecular weights. It is possible that the five species may represent stable aggregates of a common monomer of the enzyme.     

Developmental pattern of rat intestinal brush-border enzymic proteins along the villus–crypt axis

At various postnatal stages, intestinal epithelial cells were isolated sequentially from villus tip to crypt base by successive EDTA treatments. According to the localization of marker enzymic activities, isolated cells were pooled into three cell compartments: villus (V), lower villus and upper crypt (VC) and crypt (C). Purified brush-border-membrane proteins were separated by 7.5%-polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Enzymic activities could be assigned to some protein bands: maltase/glucoamylase (protein band 3), sucrase-isomaltase (protein bands 3 and 6), lactase (protein band 5) and alkaline phosphatase (region of protein bands 8 and 9). The findings suggest the following. (1) Sucrase-isomaltase activities appeared in compartment C at 17 days with a simultaneous increase of the pre-existing protein band 3 and appearance of a well-defined protein band in position 6; the enzymic complex remained still present in the crypt cells until adulthood. From the day 21 onwards, sucrase-isomaltase was detected in compartments VC and V. (2) Lactase was only present in the three cell compartments until day 21; at this developmental stage its activity completely disappeared from compartment C, in spite of the persistence of a weak protein band. (3) Alkaline phosphatase activity could be detected as a single peak corresponding to protein band 9 in all three cell compartments until day 21; thereafter it was replaced by two peaks of activity showing a less precise correlation with the well-defined protein bands 8 and 9. In the crypt cells of the adult rat, however, the preweaning situation, which was regularly observed, is an unexpected phenomenon. (4) Maltase and glucoamylase did not display any marked qualitative or quantitative modifications either along the villus-crypt axis or during the period of postnatal development studied. Evidence is given from the present data that each brush-border enzyme investigated has a specific developmental pattern.     

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.     

Purification and characterization of rat adipose tissue lipoprotein lipase.

Lipoprotein lipase (EC 3.1.1.34) extracted from adipose tissue of glucose-fed rats with 5 mM-sodium barbital, pH 7.5, containing 20% (v/v) glycerol and 0.1% (v/v) Triton X-100, was partially purified by affinity chromatography on heparin linked to Sepharose 4B. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the partially purified enzyme preparation revealed the presence of two major Coomassie-staining bands (mol.wts. 62 000 and 56 000) as well as a number of minor bands. Treatment of partially purified enzyme with [1,3-3H]di-isopropyl fluorophosphate resulted in the incorporation of radiolabel into the band of mol.wt. 56 000, but not into the band of mol.wt. 62 000. Both the amount of the 56 000-mol.wt. polypeptide and the incorporation of [1,3-3H]di-isopropyl fluorophosphate into this band were greatly reduced in the enzyme preparations isolated from adipose tissue of 48 h-starved rats. whereas the amount of the 62 000-mol.wt. polypeptide was unaffected by starvation. Purification of lipoprotein lipase from adipose tissue of glucose-fed rats was also carried out using affinity chromatography on Sepharose 4B linked to heparin with low affinity for antithrombin-III. This procedure resulted in the presence of a single band of mol.wt. 56 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These results suggest that the polypeptide of mol.wt. 56 000 corresponds to the subunit of lipoprotein lipase, whereas the 62 000-mol.wt. polypeptide probably represents antithrombin-III.     

Albumin obscures sex differences in blood protein patterns of rats and humans.

Sex related differences in the blood protein patterns of male and female rats and humans have been studied by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In rats, a prominent band of mol.wt. 74000--78000 is seen in females in far greater quantity than in males, castrated males or ovariectomized females. A secondary band of 100000 is seen under non-reducing conditions in female rats that is absent in males. In humans, bands of 92000 and 88000 mol.wt. appear to be variable in concentration in men although relatively constant in women. The above differences are observable only if serum albumin is removed from the samples before electrophoresis. The results suggest that each sex has its own characteristic blood protein pattern.     

Affinity purification and some molecular properties of human liver alkaline phosphatase.

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.     

Pig heart mitochondrial ATPase : properties of purified and membrane-bound enzyme. Effects of flavonoids.

Soluble ATPase (F1) has been purified from pig heart mitochondria. The purified enzyme had a high specific activity and was homogeneous as checked by ultracentrifugation and electrofocusing. It could be dissociated into subunits by cold-treatment or sodium dodecyl sulfate denaturation. The molecular weights of the two major and three minor subunits could be estimated by sodium dodecyl sulfate gel electrophoresis. The native enzyme had an isoelectric point of 5.2 while the cold-denatured enzyme showed three main bands focusing at pH 5.0, 5.2, and 5.4. Kinetic properties (Vm and Km (atp) have been compared for the soluble and membrane bound ATPase in presence of various anions. Inhibitory effects of Quercetin and other flavonoids have been tested in order to get an insight on the interaction between ATPase and its natural inhibitor.     

Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta

1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate-glucuronic acid-N-acetyl[1-(3)H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-(3)H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.     

Purification and characterisation of alpha-L-fucosidase from human placenta. pH-dependent changes in molecular size.

alpha-L-Fucosidase has been purified 12 000 fold from human placenta. The enzyme is a glycoprotein containing, by weight: 0.9% galactose; 1.9% mannose, 1.9% N-acetylglucosamine and 1.9% N-acetylneuraminic acid. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate separated proteins with molecular weights ot 55 000, 51 400 and 25 000. Resolution of the two larger protein bands varied with the gel system and these proteins may differ only in carbohydrate content. Gel filtration of te purified enzyme failed to separate the three proteins. Treatments with the cross-linking reagent dimethyl suberimidate prior to electrophoresis, resulted in a diminution of the original protein bands and the formation of oligomers with molecular weights of 80 000, 100 000, 130 000, and 144 000. These results suggest that the heavy (55 000 and 51 400) and light (25 000) proteins are structurally associated. The molecular weight of the native enzyme, measured by gel filtration, was dependent on the pH of the eluting buffer. At pH 5.0 or 6.0 a catalytically active peak was observed, with a molecular weight of 305 000. At pH 7.5 this peak was completely absent and the enzyme eluted as an asymmetrical peak with an apparent molecular weight of about 60 000. The reduction in apparent molecular weight at pH 7.5 was reversible by dialysis of isolated fractions at pH 6.0. In agreement with these findings the sedimentation coefficient was 8.5 S at pH 5.0 but only 3.6 S at pH 7.5. The results can be accounted for by the existence of a pH-dependent equilibrium between aggregated and dissociated forms of the enzyme or by pH-depedent conformational changes.     

Some molecular properties of NAD(P)H dehydrogenase from rat liver

NAD(P)H dehydrogenase (EC 1.6.99.2) purified from rat liver cytosol revealed three discrete bands, of mol.wts. about 27000, 18000 and 9000, when subjected to polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate. Elution of the bands from the gel and individual re-electrophoresis on separate gels showed that the 27000-mol.wt. band yielded three bands similar to those obtained with the intact enzyme, whereas the 18000-mol.wt. band retained its characteristic mobility. Amino acid analysis of native enzyme and protein extracted from each of the three bands from sodium dodecyl sulphate/polyacrylamide gels suggests that the native enzyme is composed of two subunits and that each subunit consists of two dissimilar non-covalently bound polypeptides, so that altogether the enzyme is composed of four polypeptides, two of mol.wt. 18000 and two of mol.wt. 9000. NAD(P)H dehydrogenase was active over a wide pH range with no sharp optimum. The same K(m) value for NADH but different values for V(max.) were obtained for the enzyme purified from Sprague-Dawley and Wistar rats. In immunodiffusion, however, the enzymes from the two rat strains showed a reaction of complete identity. NAD(P)H dehydrogenase was effectively inhibited by thiol-blocking reagents, indicating that the activity is dependent on free thiol group(s). By amino acid analysis six cysteine residues were found per mol of enzyme. Guanidino-group- and amino-group-selective reagents had only moderate inactivating effects on the enzyme activity.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Purification and properties of the high-activity form of the enzyme.

1. The high-activity form of aminolaevulinate synthetase has been prepared from extracts of semi-anaerobically grown cells of Rhodopseudomonas spheroides, which were allowed to become activated in air. Specific activity was 130 000--170 000 nmol of aminolaevulinate/h per mg of protein at 37 degree C. 2. Enzyme fraction Ia prepared on DEAE-Sephadex was a mixture of four active enzymes, pI5.55, 5.45, 5.35 and 5.2, when prepared in either Tris or phosphate buffers and when extracts were activated by air or by cystine trisulphide. 3. The enzyme was further purified by preparative polyacrylamide-gel electrophoresis in imidazole/veronal buffer, pH 7.6, followed by gel filtration on Sephadex G-100 and concentration with DEAE-Sephadex. 4. The most active enzyme, pI 5.55, ran as a single protein band, mol.wt. 49 000, in sodium dodecyl sulphate and 2-mercaptoethanol. The apparent molecular weight under non-denaturing conditions was 62 000--68 000 on Sephadex G-100 or G-200, pH 7.5, and on polyacrylamide-gel electrophoresis, pH 8.5, at enzyme concentrations below 10 000 units/ml, i.e. less than 60 microgram of protein/ml, and the enzyme was mainly monomeric. 5. The enzyme was homogeneous by gel disc electrophoresis at pH 8.9 and 7.6, but a slightly more diffuse band of protein was obtained during electrophoresis in glycine buffer, pH 7.4. 6. Enzyme samples possessed an intrinsic yellow fluorescence when viewed under u.v. light and this fluorescence coincided exactly with enzymic activity on gel electrophoresis. Fluorescence maxima were 420 nm (excitation) and 495 nm (emission). 7. Radioactive 35S-labelled enzyme had 14 atoms of sulphur/mol of protein (or/40 leucine residues) of which 5--6 residues were cyst(e)ine and 8--9 residues were methionine. 8. Mo carbohydrate was detected apart from glucose, which prevented accurate determination of tryptophan with methanesulphonic acid and tryptamine.     

蚯蚓体内超氧化物歧化酶分析

蚯蚓体内SOD含量甚高,35℃饲养的蚯蚓其SOD比活最高,因此,纯化前将蚯蚓在35℃养殖4周以上.采用硫酸铵分级沉淀和柱层析的方法,从蚯蚓体内分离得到纯的铜锌超氧化物歧化酶.每100g组织得到SOD制品总活力为17,190 U,比活7995 U/mg,回收率为35%.该酶呈淡蓝绿色,最大紫外吸收波长为270nm.该酶分子量为33,000,亚基分子量为16,500.该酶亚基含156个氨基酸残基,不含酪氨酸.N-末端为丙氨酸,等电聚焦为三条谱带,等电点分别为5.30 、5.59和6.22.     

Purification and characterization of human transcortin.

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 X 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.     

Pig kidney phosphofructokinase. Purification to homogeneity and qualitative basic characterization.

Phosphofructokinase has been purified from pig kidney by extraction with phosphate buffer at pH 8, followed by alcohol treatment, affinity chromatography on matrix-bound Cibacron blue F3G-A, and gel chromatography on Sepharose 6B. Using sodium dodecyl sulphate electrophoresis the enzyme was found to be homogeneous and to have a specific activity of about 80 units/mg protein. Like other phosphofructokinases, at pH 7.0 the enzyme exhibits a sigmoidal dependence in its activity on the fructose 6-phosphate concentration and is strongly inhibited by ATP. The degree of citrate inhibition is influenced by the concentration of the two substrates. ATP strengthens and fructose 6-phosphate relieves the inhibition by citrate. AMP and cAMP are able to overcome the ATP inhibition. The ADP activation curve is biphasic. The molecular weight of the subunit of pig kidney phosphofructokinase was determined to be 88 000 by means of sodium dodecyl sulphate electrophoresis.     

PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

The inhibition of platelet cyclo-oxygenase by aspirin is associated with the acetylation of a 72kDa polypeptide in the intracellular membranes.

Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein.     

Purification and properties of a cellulase from Aspergillus niger.

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.     

Purification and characterization of a thermostable glucoamylase from Chaetomium thermophilum

Thermostable amylolytic enzymes are currently being investigated to improve industrial processes of starch degradation. A thermostable extracellular glucoamylase (exo-1, 4-alpha-D-glucanohydrolase, E.C.3.2.1.3) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by using ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography, and Phenyl-Sepharose Fast Flow chromatography. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 64 kDa. The glucoamylase exhibited optimum catalytic activity at pH 4.0 and 65 degrees C. It was thermostable at 50 degrees C and 60 degrees C, and retained 50% activity after 60 min at 65 degrees C. The half-life of the enzyme at 70 degrees C was 20 min. N-terminal amino acid sequencing (15 residues) was AVDSYIERETPIAWN. Different metal ions showed different effects on the glucoamylase activity. Ca2+, Mg2+, Na+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, and Hg2+ cause obvious inhibition. These properties make it applicable to other biotechnological purposes.