Affinity Chromatographic Purification of β-Glucosidase of Candida Guilliermondii

Abstract

A 8-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethylcneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacryl-amide disc electrophoresis of the purified enzyme gave a single band. A K_m of 1.25 × 10^?4M was obtained using p_-nitrophenyl-β-D_-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at a temperature of 37°C. Enzyme activity was completely inhibited by Hg⁺⁺, Pb⁺⁺, and Zn⁺⁺ ions. The molecular weight of the enzyme is 48, 000 as estimated by sucrose density gradient centri-fugation.     

Purification and Characterization of Invertase from Lactobacillus reuteri CRL 1100

The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45°C over a wide pH range (4.5–7.0) with maximum activity at pH 6.0 and 37°C. The invertase activity was significantly inhibited by bivalent metal ions (Ca⁺⁺, Cu⁺⁺, Cd⁺⁺, and Hg⁺⁺), β-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K _m and V_max values for sucrose were 6.66 mM and 0.028 μmol/min, respectively. An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a β-fructofuranosidase. Received: 13 August 1999 / Accepted: 15 September 1999     

Purification and Properties of Glucose-6-Phosphate Dehydrogenase from Bacillus subtilis Spores

Glucose-6-phosphate dehydrogenase [d-glucose-6-phosphate: NADP oxidoreductase, EC. 1. 1. 1. 49] obtained from spores of Bacillus subtilis PCI 219 strain was partially purified by filtration on Sephadex G-200, ammonium sulfate fractionation and chromatography on DEAE-Sephadex A-25 (about 54-fold). The optimum pH for stability of this enzyme was about 6.3 and the optimum pH for the reaction about 8.3. The apparent K_m values of the enzyme were 5.7 × 10^–4 M for glucose-6-phosphate and 2.4 × 10^–4 M for nicotinamide adenine dinucleotide phosphate (NADP). The isoelectric point was about pH 3.9. The enzyme activity was unaffected by the addition of Mg⁺⁺ or Ca⁺⁺. The inactive glucoses-6-phosphate dehydrogenase obtained from the spores heated at 85 C for 30 min was not reactivated by the addition of ethylenediaminetetraacetic acid, dipicolinic acid or some salts unlike inactive glucose dehydrogenase.     

Purification and enzymatic properties of α-galactosidase from Penicillium janthinellum

α-Galactosidase (E.C.3.2.1.22) from Penicillium janthinellum was purified by precipitation and fractionation with ammonium sulphate, cold acetone or ethanol, calcium phosphate gel, and column chromatographies on Sephadex G-100 and G-200. The enzyme was purified about 110.39-fold when Sephadex G-100 was used. α-Galactosidase exhibited the optimum pH and temperature at 4.5 and 60°C, respectively. The optimum enzyme stability was obtained at pH 3.5 for 24 h (at room temperature). The enzyme was found to be thermostable below 65°C up to 40 minutes and was gradually inactivated by increasing the temperature above this degree. The MICHAELIS constant was 0.55 mM for p-nitrophenyl-α-D-galactoside. The α-galactosidase activity was strongly inhibited by Hg⁺⁺ and slightly activated by Mn⁺⁺. The results show the possibility of producing a thermostable enzyme from a low-priced agricultural product, for instance, lupine.     

Purification and properties of an endopolygalacturonase produced byRhizopus stolonifer

Summary A polygalacturonase from culture filtrates of a strain ofRhizopus stolonifer was purified about 80 fold by ethanol precipitation, followed by ion exchange chromatography (CM-Sepharose 6B) and gel filtration (Sephadex G-100). The purified preparation was homogeneous when examined by PAGE. The enzyme is an endopolygalacturonase with an optimum catalytic activity at pH 5.0 and 45°C, and a molecular weight of 57,000±500 daltons. The activity was stimulated by Fe⁺⁺⁺, Mg⁺⁺, Co⁺⁺, and inhibited by Mn⁺⁺ and Zn⁺⁺. The enzyme was stable in the pH range of 3.0 to 5.0. The purified enzyme was specific for nonmethoxylate polygalacturonic acid, with K_m and V_max values respectively of 0.19 mg/ml and 1.3 mol/g/min. In addition, this enzymatic preparation degraded pectic substances in organge peel.     

Purification and properties of cis-aconitic decarboxylase

Summary Lyophilized and stored in a deep-freeze, the mycelial material was found to retain cis-aconitic decarboxylase activity unimpaired at the end of 2 months. Mycelia could be stored also in the frozen condition but after squeezing hard to remove as much of adherent water as possible. Extracts with maximum cis-aconitic decarboxylase activity were obtained when the frozen or better the lyophilized mycelia of Aspergillus terreus were ground in a mortar with phosphate buffer using pyrex glass powder as abrasive. Cis-aconitic decarboxylase was purified 25-fold by fractionation with ammonium sulfate, starting from extracts of the mycelia in phosphate buffer. The purified enzyme was considerably more stable than the crude extracts to storage and dialysis. The optimum pH was 5.8 using 0.2 m phosphate buffer; Km value was 5×10^-3 m at pH 5.8 and 37°C. EDTA and 8-hydroxyquinoline activated the enzyme; all metals tested inhibited the enzyme, Zn⁺⁺ and Cu⁺⁺ leading to complete inactivation. Fluoride, arsenite and azide also inhibited the enzyme activity.     

Isolation,partial purification,and some properties of protease I from a thermophilic mold Thermoascus aurantiacus var. levisporus

When the thermophilic mold Thermoascus aurantiacus var. levisporus was grown in a modified Czapek Dox medium containing casein the filtrate was found to contain proteolytic activity. The maximum production of activity occurred at 50 ° C in a medium containing 8% casein. The filtrate was subjected to ammonium sulfate fractionation and chromatography on DEAE-cellulose. Two proteases were separated. No further work was done on protease II. Protease I was further purified by gel filtration on Sephadex G 100–200. It showed a 40-fold purification with a final recovery of approximately 25%. It is a neutral protease with a pH optimum at 7.0. The optimal activity of the enzyme occurred in 0.02 M phosphate buffer but was completely inhibited at a concentration of 0.1 M. The optimum temperature for casein hydrolysis was found to be 55 ° C. The enzyme was inhibited by Hg⁺⁺ but was greatly stimulated by Cu⁺⁺ and mercaptoethanol. Metallo and sulfhydryl agents had no significant effect on enzyme activity.     

Characterization of a peptidase fromDiplococcus pneumoniae

Some properties of a purified peptidase fromDiplococcus pneumoniae have been studied. The enzyme has a broad pH optimum between 6 and 8 and a K_m (onl-leucylglycylglycine) of 2.8mm. It is activated by low levels of Hg⁺⁺ and is inhibited by Mn⁺⁺, Co⁺⁺, β-mercaptoethanol and EDTA. Substrate specificity studies show that the enzyme is an exopeptidase of the aminopeptidase type, most active on tripeptide substrates bearing bulky substituents at the NH₂ ⁻ terminal end.     

Studies on Metabolic Pathway of NAD in Yeast

The properties of yeast 5′-nucleotidase, one of NAD-metabolic system in yeast, were studied.

1) The enzyme has optimum pH at 5.8~6.1 for its activity and is most stable at pH 6. It is inactivated completely at 55°C for 6 min, pH 7, but never at 40°C for 6 min. 2) The enzyme hydrolyzes only 5′-nucleotides of guanine, adenine, hypoxanthine, uracil and cytosine, but never splits nicotinamide mononucleotide, thiamine monophosphate, ribose 5-monophosphate and flavin mononucleotide. 3) The enzyme seems to have specially high affinity for 5′-AMP. 4) The enzyme activity is accelerated by addition of Co⁺⁺ and Ni⁺⁺, but inhibited by Ag⁺, Cu⁺⁺, EDTA, I₂ and N-bromosuccimide. Mg⁺⁺, KCN, NaF and thiol reagents except p-chloromercuribenzoate have no effects. 5) Nucleosides have inhibitory effects, among which adenosine is most effective inhibitor. 6) The activity is reduced up to 30% by dialysis against 1 mm EDTA solution, and the reduced activity is completely reactivated by addition of Co⁺⁺ or Ni⁺⁺, but not by Mn⁺⁺ or Mg⁺⁺.     

Studies on the Nucleases Produced by Microorganisms

The properties of crude phosphodiesterase forming 5′-mononucleotide of Pellicularia H-II were investigated on its metal requirement, pH response for activity and so on. The dialyzate of crude PDase against distilled water became partly inactive, but was recovered with Zn⁺⁺, Mn⁺⁺ and Mg⁺⁺, whereas completely inactivated dialyzate against EDTA was restored specifically with only Zn⁺⁺

The optimum pH of PDase activity was 5.0 and that of ribonuclease 4.0. The crude PDase was partially purified by acetone fractionation and Amberlite IRC-50 (XE-64) or CM-cellulose column chromatography. Two PDase and a RNase activities were recognized.

Pellicularia PDase was found to be of new type according to its Zn⁺⁺ dependency and non-activity towards bis-p-nitrophenyl phosphate.     

A Branched-chain Amino Acid Aminotransferase from the Rumen Ciliate Genus Entodinium

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The K_m values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for α-ketoglutarate: and 0.1 μM for pyridoxal phosphate. Enzyme activity was inhibited by p-chloromercuribenzoate and HgCl₂. Gel filtration indicated the enzyme to have a molecular weight of 34,000.     

Purification and Characterization of an Acid Phosphatase from Mycoplasma fermentans

An acid phosphatase associated with the cell membranes of Mycoplasma fermentans was released from the membranes with Triton X-100, then purified by ion-exchange chromatography on DEAE-Sephacel and CM-Sepharose, followed by affinity chromatography on Con A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single band with a molecular mass of 31.2 kilodaltons. The enzyme activity toward p-nitrophenyl phosphate was enhanced remarkably by Cu²⁺, Co²⁺ and Mg²⁺, but the activity was not inhibited by EDTA. The enzyme dephosphorylated O-phospho-l -tyrosine as well as p-nitrophenyl phosphate, but not O-phospho-l -threonine, O-phospho-l -serine, glucose-1-phosphate, phosphoryl choline and adenosine triphosphate. The level of the O-phospho-l -tyrosine phosphatase activity was the highest in Mycoplasma faucium and the second highest in Mycoplasma fermentans of all tested human mycoplasmas.     

Purification and characterization of an acid phosphatase from Trichoderma harzianum

An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55°C. The enzyme retained 60% of its activity after incubation at 55°C for 60 min. The K _m and V _max values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min^?1, respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.     

Characterization of fructose-1,6-diphosphate-insensitive catabolic glycerol kinase ofPseudomonas aeruginosa

Glycerol kinase is induced in cells ofPseudomonas aeruginosa strain PAO when grown in the presence of glycerol or glycerol-3-phosphate. The enzyme was isolated from the soluble cytoplasmic fraction of cell extracts and purified 500-fold by ammonium sulfate precipitation and chromatography on columns of Sephadex G-25, DEAE-Sephadex, hydroxyapatite, and Sephadex G-200. A molecular weight of 120,000 was estimated by gel filtration of the catalytically active enzyme. In polyacrylamide gel electrophoresis the purified product contained one major band of Coomassie Blue staining material. The enzyme exhibited an apparent Km of 40 M for glycerol and 23 M for ATP. Of the nucleotide triphosphates tested, only ATP served as a phosphoryl group donor. Mg⁺⁺ or Mn⁺⁺ was required for activity, although a threefold greater concentration of Mn⁺⁺ was required when Mn⁺⁺ substituted for Mg⁺⁺. In contrast to most other catabolic glycerol kinases in bacteria, the enzyme was not inhibited by fructose-1,6-diphosphate nor by other tested metabolites.     

Studies on the Proteolytic Enzymes of Thermophilic Streptomyces

Proteolytic enzymes derived from thermophilic streptomyces sp. strain 1689 were purified and some properties were studied. A 8-fold purification was obtained from the culture supernatant by ammonium sulfate fractionation, acetone precipitation, and chromatography on CM-Sephadex. Two proteinases of almost identical properties were fractionated on CM-Sephadex chromatography. The purified preparations appeared to be homogeneous on ultracentrifugation. The optimum pH for proteolytic activity on casein was found to be pH 10.6~10.8. The stability was considerably increased by the addition of Ca⁺⁺, and the proteinases exhibited d relatively high thermal stability. Enzyme activity was inhibited by oxidizing agents, PCMB, potato inhibitor, DFP, and heavy metal ions. Na⁺, K⁺, Mg⁺⁺, and Fe⁺⁺ showed an activating effect.     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.     

Studies on Phosphoprotein Phosphatase in Chick Embryo

Several properties of partially purified phosphoprotein phosphatase from chick embryo are described. The enzyme was active toward casein, phosvitin and phosphopeptone, but not toward low molecular weight phosphate esters including aliphatic and aromatic phos-phomonoesters, a phosphodiester, pyrophosphates and phosphoamides. The enzyme was not activated by reducing compounds. Heavy metal ions and sulfhydryl inhibitors inhibited the enzyme activity, but the inhibited enzyme was partially reactivated with cysteine. Metal chelating agents also inhibited the activity. To the oxalate treated enzyme, Fe⁺⁺ and Co⁺⁺ had a stimulatory effect. Differences in property between phosphoprotein phosphatases of chick embryo and of mammalian tissues are discussed.     

Purification and Characterization of a Phytase from <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> MOK1

A phytase (EC 3.1.3.8) from Pseudomonas syringae MOK1 was purified to apparent homogeneity in two steps employing cation and an anion exchange chromatography. The molecular weight of the purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The optimal activity occurred at pH 5.5 and 40°C. The Michaelis constant (K _m ) and maximum reaction rate (V_max) for sodium phytate were 0.38 mM and 769 U/mg of protein, respectively. The enzyme was strongly inhibited by Cu²⁺, Cd²⁺, Mn²⁺, and ethylenediaminetetraacetic acid (EDTA). It showed a high substrate specificity for sodium phytate with little or no activity on other phosphate conjugates. The enzyme efficiently released orthophosphate from wheat bran and soybean meal.Received: 9 September 2002 / Accepted: 6 December 2002     

Purification and properties of an NADP+-specific isocitrate dehydrogenase from Rhizobium meliloti

An NADP⁺-specific isocitrate dehydrogenase has been purified and characterized from Rhizobium meliloti. The enzyme showed Mn⁺⁺ or Mg⁺⁺ requirement. The apparent K_m values were 2.00×10^-5 m and 1.51×10^-5 m for dl-isocitrate and NADP⁺, respectively. The enzyme was inhibited by ATP, to a lesser extent by ADP and AMP. -Ketoglutarate also inhibited the enzyme activity. Oxalacetate and glyoxylate together inhibited the enzyme activity. The inhibition was competitive. Studies with thiol inhibitors suggested that the enzyme contained a sulfhydryl group at or near the active site. The enzyme has an approximate molecular weight of 60 000. Fluorescence studies suggested that the enzyme contained tryptophan     

Studies on Bacterial Protease

The neutral protease of Bacillus amylosacchariticus was inactivated by low concentrations of several metal-chelating agents and the inactivated enzyme with EDTA restored its activity almost completely by the addition of Zn⁺⁺ or Co⁺⁺ and partially by Fe⁺⁺ or Mn⁺⁺, if these metal ions were added shortly after the EDTA-treatment. The native enzyme was found to contain 0.19% of zinc together with a significant amount of calcium. Parallel increase in specific activity and zinc content of enzyme preparation was observed throughout the purification procedure. The elution pattern of enzyme activity on a CM-cellulose column chromatography also completely coincided with that of protein-bound zinc. A zinc-free inactive enzyme was also reactivated by the addition of zinc or cobalt ions, clearly showing that the neutral protease of B. amylosacchariticus is a zinc mctalloenzyme.