Purification and properties of phloroglucinol reductase from Eubacterium oxidoreducens G-41

Phloroglucinol reductase was purified 90-fold to homogeneity from the anaerobic rumen organism Eubacterium oxidoreducens strain G-41. The enzyme is stable in the presence of air and is found in the soluble fraction after ultracentrifugation of cell extract. Ion-exchange, hydrophobic interaction, and affinity chromatography were used to purify the enzyme. The native Mr is 78,000, and the subunit Mr is 33,000 indicating an alpha 2 homodimer. The enzyme is specific for phloroglucinol and NADPH. The Km and Vmax are 600 microM and 640 mumol min-1 mg-1 (pH 7.2) for phloroglucinol, and 6.7 microM and 550 mumol min-1 mg-1 (pH 6.8) for NADPH; the Km and Vmax for the reverse direction are 290 microM and 140 mumol min-1 mg-1 (pH 7.2) for dihydrophloroglucinol, and 27 microM and 220 mumol min-1 mg-1 (pH 7.2) for NADP. Temperature and pH optima are 40 degrees C and 7.8 in the forward direction. The pure enzyme is colorless in solution and flavins are absent. Analysis for cobalt, manganese, molybdenum, vanadium, tungsten, selenium, copper, nickel, iron, and zinc indicated that these metals are not components of the phloroglucinol reductase. Cupric chloride, n-ethylmaleimide, and p-chloromercuribenzoate are potent inhibitors of enzyme activity. The properties of phloroglucinol reductase indicate that it functions in the pathway of anaerobic degradation of trihydroxybenzenes by catalyzing reduction of the aromatic nucleus prior to ring fission.     

Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities

The appA gene that was previously shown to code for an acid phosphatase instead codes for a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The purified enzyme with a molecular mass of 44,708 Da was further separated by chromatofocusing into two isoforms of identical size with isoelectric points of 6.5 and 6.3. The isoforms had identical pH optima of 4.5 and were stable at pH values from 2 to 10. The temperature optimum for both phytase isoforms was 60 degrees C. When heated at different pH values the enzyme showed the greatest thermal resistance at pH 3. The pH 6.5 isoform exhibited K(m) and Vmax values of 0.79 mM and 3165 U.mg-1 of protein for phytase activity and 5.5 mM and 712 U.mg-1 of protein for acid phosphatase, respectively. The pH 6.3 isoform exhibited slightly lower K(m) and Vmax values. The enzyme exhibited similar properties to the phytase purified by Greiner et al. (1993), except the specific activity of the enzyme was at least 3.5-fold less than that previously reported, and the N-terminal amino acid sequence was different. The Bradford assay, which was used by Greiner et al. (1993) for determination of enzyme concentration was, in our hands, underestimating protein concentration by a factor of 14. Phytase production using the T7 polymerase expression system was enhanced by selection of a mutant able to grow in a chemically defined medium with lactose as the carbon source and inducer. Using this strain in fed-batch fermentation, phytase production was increased to over 600 U.mL-1. The properties of the phytase including the low pH optimum, protease resistance, and high activity, demonstrates that the enzyme is a good candidate for industrial production as a feed enzyme.     

Purification and properties of NADH/NADPH-dependent p-hydroxybenzoate hydroxylase from Corynebacterium cyclohexanicum

Crude soluble extracts of Corynebacterium cyclohexanicum, grown on cyclohexanecarboxylic acid, were found to contain 4-hydroxybenzoate 3-hydroxylase which functions with NADH as well as NADPH. The purified enzyme preparation was electrophoretically homogeneous and contained FAD as prosthetic group. The relative molecular mass of the enzyme was estimated to be about 47000 by native and denaturated acrylamide gel electrophoresis, indicating that it is monomeric. The enzyme was stable at 60 degrees C for 10 min. The enzyme was highly specific for p-hydroxybenzoate. The activity was inhibited by several aromatic analogues of p-hydroxybenzoate such as p-aminobenzoate, p-fluorobenzoate, o-hydroxybenzoate, m-hydroxybenzoate, 2,4-dihydroxygenzoate, and 2,5-dihydroxybenzoate. The Km value for NADH was fairly constant, about 45 microM, in the pH range 7.0-8.4, whereas the Km value for NADPH increased from 63 microM to 170 microM as the pH rose from 7.0 to 8.4. V values in the same pH range, however, were approximately constant in both cases; about 30 mumol min-1 mg-1 for NADH, and 26 mumol min-1 mg-1 for NADPH. Mg2+ was required for full activity of the enzyme in low concentrations of phosphate buffer. The enzyme was inhibited by C1- which was non-competitive with respect to NADH, NADPH and p-hydroxybenzoate.     

Human insulin receptor beta-subunit transmembrane/cytoplasmic domain expressed in a baculovirus expression system: purification, characterization, and polylysine effects on the protein tyrosine kinase activity.

We have expressed, purified, and characterized the insulin receptor protein tyrosine kinase (PTK) retaining the transmembrane and downstream domains. The proteins expressed in insect cells using a baculovirus expression system were identified as membrane-bound by immunofluorescence staining and biochemical characterization. One-step purification by immunoaffinity chromatography from Triton X-100 cell extracts resulted in a approximately 360-fold increase in the specific kinase activity with a yield of approximately 50%. An appMr = approximately 60,000 protein was the major component identified by both silver staining of the purified enzyme and immunostaining of the crude extracts after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Using nondenaturing conditions, the molecular weight was estimated to be approximately 250,000 and approximately 500,000 by glycerol gradient centrifugation and gel permeation chromatography, respectively, suggesting that oligomers of the beta-subunit domains such as tetramers and octamers are formed. The basal PTK activity of this enzyme was much higher than those of previously reported soluble-form insulin receptor PTKs expressed in insect cells or the native receptor. Km and Vmax for two substrates, src-related peptide and poly(Glu, Tyr) (4:1), were 2.4 mM and 2.5 mumol min-1 mg-1 and 0.26 mM and 1.2 mumol min-1 mg-1, respectively. Specific activities measured under two previously reported conditions using histone H2B as a substrate were 100 or 135 nmol min-1 mg-1, in contrast to those of soluble PTKs which were reported to be 20 or 70 nmol min-1 mg-1, respectively. The purified enzyme was autophosphorylated at Tyr residues. Autophosphorylation activated the enzyme approximately 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of a novel raw starch degrading-cyclodextrin glycosyltransferase from Klebsiella pneumoniae AS- 22

A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The K_m and k_cat values for the pure enzyme were 1.35 mg ml⁻¹ and 249 μM mg⁻¹ min⁻¹, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l⁻¹ gelatinized soluble starch or 125 g l⁻¹ raw wheat starch at an enzyme concentration of 10 U g⁻¹ of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.     

Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans

A yeast strain, Aureobasidium pullulans, which could produce the high yield of protease was isolated from sediment of saltern in Qingdao, China. Maximum production of enzyme (623.1 U/mg protein; 7.2 U/ml) was obtained in a medium containing 2.5 g soluble starch and 2.0 g NaNO(3), 100ml seawater, initial pH 6.0, after fermentation at 24.5 degrees C for 30 h. The protease had the highest activity at pH 9.0 and 45 degrees C.     

黑曲霉菊糖酶的纯化及性质

黑曲霉(Aspergillus niger)319发酵液经硫酸铵分级沉淀、DEAE-纤维素52离子交换层析和Sephadex G-100分子筛层析,得到了电泳纯的菊糖酶组分。提纯倍数为67,收率为25.5％。菊糖酶的最适pH为5.0,最适温度为60℃。此酶为单亚基蛋白,凝胶过滤法测得分子量为28000,含糖13.9％,用等电聚焦法测得等电点为5.4,该酶对温度有较高的稳定性,对pH的稳定范围较窄。Hg²⁺、Pb²⁺和Cu²⁺对该酶有强烈的抑制作用。此酶对菊糖有较强的底物专一性,产物为果糖,但它也可作用于蔗糖,I/S值为0.348。当以菊糖为底物时,K_m为6.25mmol/L,V_m为67.11 μmol·mg~(-1)·min~(-1)。     

Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from maltose-grown Streptococcus bovis JB1 was purified to apparent homogeneity by ion-exchange chromatography (Mono Q). The enzyme had an isoelectric point of 4.50 and an apparent molecular mass of 77,000 Da, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was rich in acidic and hydrophobic amino acids. The 15-amino-acid NH2-terminal sequence was 40% homologous with the Bacillus subtilis saccharifying alpha-amylase and 27% homologous with the Clostridium acetobutylicum alpha-amylase. alpha-Amylase activity on soluble starch was optimal at pH 5.0 to 6.0. The enzyme was relatively stable between pH 5.5 and 8.5 and at temperatures below 50 degrees C. When soluble potato starch was used as the substrate, the enzyme had a Km of 0.88 mg.ml-1 and a kcat of 2,510 mumol of reducing sugar.min-1.mg of protein-1. The enzyme exhibited neither pullulanase nor dextranase activity and was 40 to 70% as active on amylopectin as on amylose. The major end products of amylose hydrolysis were maltose, maltotriose, and maltotetraose.     

Production and characterization of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean

Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications.     

Purification and characterization of acetyl esterase from Candida guilliermondii

An extracellular acetyl esterase (EC 3.1.1.6) from Candida guilliermondii NRRL Y-17257 was purified to homogeneity by acetone precipitation and QAE sepharose anion-exchange chromatography. The enzyme was a monomer with an apparent molecular weight of 67 kDa and a pI of 7.6. It had maximum activity at pH 7.5 and at 50-60 degrees C. It was relatively stable over a pH range of 5.8-8.0 and exhibited thermal stability up to 60 degrees C. The Km and Vmax values on alpha-naphthylacetate were 2.63 mM and 213.3 micromol alpha- naphthol min-1 mg-1 protein, respectively.     

Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.     

嗜热脂肪芽孢杆菌β-半乳糖苷酶的性质

利用硫酸铵分级沉淀、离子交换层析 (DEAE- 2 2 )、Sephadex G- 75凝胶过滤从嗜热脂肪芽孢杆菌胞内提纯得到 β-半乳糖苷酶。研究表明 ,该酶最适表观反应温度和最适 pH分别为 6 0℃和 6 .4。在 50℃该酶具有良好的热稳定性。碱金属和碱土金属盐对酶有激活作用 ,重金属 Zn²⁺、Fe³⁺、Cu²⁺抑制酶的活力。巯基保护剂能明显增强酶的活力 ,而巯基结合试剂强烈抑制酶的活性。该酶对 β-     

菠菜叶片中乙醇酸氧化酶3种同工酶的生化特性

By DEAE cellulose and Sepharose 6B chromatography, the proteins containing glycolate oxidase isozymes GOⅡ and GOⅢ were extracted from spinach green leaves. The protein containing GOⅡ showed two bands of 67±2 kD and 40±2 kD in SDS PAGE whose specific activity of glycolate oxidase was 33.4 U·mg -1 ·min -1 .It migrated towards cathode in Native PAGE in pH 8.3 buffer system. pI of GOⅡwas about 9.4 detected by IEF. The protein containing GOⅢ showed three bands of 67±2 kD, 40±2 kD and 38±2 kD in SDS PAGE whose specific activity of glycolate oxidase 14.4 U·mg -1 ·min -1 and could not migrate anywhere in the same Native PAGE. pI of GOⅢ was about 8.3 detected by IEF. The 40±2 kD might be the subunits of GOⅡ and GOⅢ. Antibodies of the protein containing GOⅡ and GOⅢ were prepared respectively. GOⅡ was very unstable and could change into GOⅢ artifact; GOⅢ was also unstable and could change into GOⅠartifact whose Mr ≈470 kD and pI ≈7.4 . This GOⅠ(specific activity: 9.8 U·mg -1 ·min -1 ), showing one 40±2 kD band in SDS PAGE, could be purified on another Sepharose 6B chromatography. The specific activity of GOⅡ decreased rapidly to about half of its original value and then was relatively stable when stored in 50% glycerol at -20℃. The results above explained why GOⅡ was extracted difficultly, and GOⅢ were easily confused with GOⅠ and GOⅡ.     

Purification and Characterization of the polygalacturo nase from Tomato Fruits

The polygalacturonase (PG) isolated fron the pericarp of fully ripe tomato fruits was purified by (NH4)2SO4 precipitation, carboxyme-thylsepharose ion-exchang column and sephadex G-75 gel filtration. Specific activity of purified PG was 1.5 μmol galacturonic acid mg-1 protein min-1, which was 30 times as high as that of the crude extract with 1.7mol NaC1. When the elution separated by second sephadex G-75 column was analyzed by SDS-PAGE, only a single protein band was detected. It was shown by heat and pH experiments of the purified enzyme that the enzyme activity retained 50%, after treatment with heat at 50℃ for 10 min, and that the optimal pH was 4.6.     

Direct identification and quantification of the cofactor in glutamate semialdehyde aminotransferase from pea leaves

Glutamate semialdehyde aminotransferase, a key enzyme in the synthetic pathway leading to chlorophyll was purified from pea (Pisum sativum) leaves. Although the preparation contained a single contaminant the enzyme could be unambiguously identified as a dimer of subunit molar mass 45 kDa having an absorption spectrum consistent with the presence of pyridoxamine phosphate as cofactor. The cofactor was released by treatment with strong phosphate at low pH and was identified and quantified fluorimetrically. The specific activity of the enzyme (1.4 mumol.min-1.mg-1; 23 nkatal.mg-1) is very much higher than previously reported.     

A specific short dextrin-hydrolyzing extracellular glucosidase from the thermophilic fungus Thermoascus aurantiacus 179-5

The thermophilic fungus Thermoascus aurantiacus 179-5 produced large quantities of a glucosidase which preferentially hydrolyzed maltose over starch. Enzyme production was high in submerged fermentation, with a maximal activity of 30 U/ml after 336 h of fermentation. In solid-state fermentation, the activity of the enzyme was 22 U/ml at 144 h in medium containing wheat bran and 5.8 U/ml at 48 h when cassava pulp was used as the culture medium. The enzyme was specific for maltose, very slowly hydrolyzed starch, dextrins (2-7G) and the synthetic substrate (alpha-PNPG), and did not hydrolyze sucrose. These properties suggest that the enzyme is a type II alpha-glucosidase. The optimum temperature of the enzyme was 70 degrees . In addition, the enzyme was highly thermostable (100% stability for 10 h at 60 degrees and a half-life of 15 min at 80 degrees), and stable within a wide pH range.     

Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase.

Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000-fold purification to apparent homogeneity gave a 14-kDa phosphatase with a specific activity of 3 micro mol.min-1.mg-1 at pH 7.5 with 7 micro m phosphopeptide I as substrate. Partial amino-acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNA-library followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA-insensitive phosphohistidine phosphatase activity of 9 micro mol.min-1.mg-1 towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine-, phosphothreonine-, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.     

Semisynthetic fluorohydrolases prepared by chemical modification of ribonuclease.

A process for conformational modification of protein, which we have previously reported, was investigated as a means of generating fluorohydrolase activity in bovine ribonuclease (RNase). The resulting modified RNase had catalytic activity that depended upon the chosen modifier. Bovine pancreatic ribonuclease, modified by addition of hexamethylphosphoramide (HMPA) at pH 3, was derivatized with diimidates of chain lengths from C1 to C8. The derivative with the highest activity was obtained when RNase was crosslinked with dimethyl pimelimidate (C5). This derivative, which was active over a pH range of 6.5 to 8.0 with an optimum pH of 7.4, hydrolyzed phenylmethylsulfonylfluoride (PMSF) and the potent acetylcholinesterase inhibitor, diisopropyl phosphorofluoridate (DFP). The mean fluorohydrolase activity for four preparations using dimethyl pimelimidate was 0.8 +/- 0.2 U mg-1. Gel filtration on G-75 Sephadex and SDS-polyacrylamide gel electrophoresis showed components having a molecular weight of 13,000 and 27,000, with activity restricted to the 27,000 molecular weight fraction. After gel filtration, the specific activity was 9.1 +/- 2.4 U mg-1, resulting in a molecular activity of 125 min-1. The mechanism of this unique transformation of RNase into a fluorohydrolase is not known, nor has the location of the active site been determined.     

Biochemical properties of a thermostable phytase from Neurospora crassa

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.