Purification and characterization of an acetyl xylan esterase from Bacillus pumilus

Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55 degrees C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis contant (Km) and Vmax for alpha-naphthyl acetate were 1.54 mM and 360 micromol min-1 mg of protein-1, respectively.     

Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus

AIMS: An investigation was carried out on the purification and characterization of an alkaline protease from Bacillus pumilus MK6-5. METHODS AND RESULTS: An alkalophilic Bacillus pumilus MK6-5 was grown in a laboratory fermenter containing 1% reverse osmosis concentrated cheese whey powder, 0.25% corn steep liquor, 1% glucose, 0.5% tryptone, 1% sodium citrate, 0.02% MgSO4.7H2O and 0.65% Na2CO3 at 35 degrees C and pH 9.6, agitation at 250 rev min(-1) and aeration of 1 vvm for 60 h. When the enzyme was purified using ammonium sulphate precipitation, ion exchange and gel filtration chromatographies, a 26.2% recovery of enzyme with 36.6-fold purification was recorded. The purified protease was found to be homogenous by SDS-PAGE with molecular mass estimate of 28 kDa. The enzyme was optimally active at pH 11.5 and temperature of 55-60 degrees C. The Km and kcat values observed with synthetic substrates at 37 degrees C and pH 8.0 were 1.1 mmol l(-1) and 624 s(-1) for Glu-Gly-Ala-Phe-pNA and 3.7 mmol l(-1) and 826 s(-1) for Glu-Ala-Ala-Ala-pNA, respectively. The kinetic data revealed that small aliphatic and aromatic residues were the preferred residues at the P1 position. Inhibition profile exhibited by PMSF suggested the B. pumilus protease to be an alkaline serine protease. CONCLUSIONS: Bacillus pumilus MK6-5 produced a calcium-dependent, thermostable alkaline serine protease. SIGNIFICANCE AND IMPACT OF THE STUDY: The thermostable alkaline protease from Bacillus pumilus MK6-5 will be extremely useful in ultrafiltration membrane cleaning due to its ability to work in broad pH and temperature ranges, and tolerance to detergents, unlike the mesophilic proteases which face these limitations.     

Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67

Bacillus pumilus TYO-67 was isolated from tofu (soybean curd) as the best producer of a soybean-milk-coagulating enzyme, induced by the addition of soybean protein to the growth medium. The enzyme was purified approximately 30-fold with an 11% yield. The homogeneous preparation of the enzyme showed that it is a monomer with a molecular mass of about 30 kDa and has an isoelectric point at pH 9.75. The results of amino acid composition analyses showed that the enzyme is rich in alanine, aspartic acid, glycine, serine and valine. Although the amino-terminal amino acid (alanine) was identical with that of subtilisins, the amino-terminal sequence was different from those of subtilisins. The α-helix content of the enzyme was calculated to be 28.2%. The optimum pH and temperature were observed at 6.0–6.1 and 65 °C respectively. The enzyme was significantly activated by the addition of 1 mM Mn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺ ions in the reaction mixture, and its thermal stability was significantly increased by Ca²⁺ ion. Received: 31 August 1998 / Received last revision: 1 December 1998 / Accepted: 20 December 1998     

Purification and characterization of manganese-dependent alkaline serine protease from Bacillus pumilus TMS55

The purification and characterization of a Mn2+-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be 60 degreesC. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. Mn2+ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants (H2O2, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.     

Some properties of p-coumarate decarboxylase from Cladosporium phlei.

The optimal pH and temperature of p-coumarate decarboxylase were 6.0 and 23 degrees C respectively. The enzyme activity was reduced to three quarters by heat treatment at 35 degrees C for 5 min and by half at 25 degrees C in 24 h, but kept almost unchanged at -20 degrees C at least for 10 days. The activity was not inhibited by potassium cyanide, sodium diethyldithiocarbamate, ethylenediaminetetraacetic acid disodium salt, or sodium citrate at 10 mM concentration, but was inhibited by p-chloromercuribenzoate or iodoacetate at 0.1 mM, the inhibition by the former being prevented to a great extent by the presence of reduced glutathione or dithiothreitol. The activity was inhibited by maleic acid cinnamic acid, or p-methoxycinnamic acid, but not by fumaric acid, acrylic acid, p-hydroxystyrene, furcatin p-hydroxyphenylacetic acid, or phloretic acid. An unsubstituted p-hydroxy group on the benzene ring and an acrylic acid side chain were required for the enzyme activity. Km value for trans-p-coumaric acid was about 6.5 X 10(-4) M.     

Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2

An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3+/-4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC-MS of PL revealed 15% homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 degrees C in Tris/HCl buffer. It showed a half-life at 30 degrees C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 degrees C. The enzyme did not require Ca2+ ions for activity, and was strongly inhibited by EDTA and Co2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50% of esterification. The best substrate was 29.5% methylated pectin. PL cleaved polygalacturonic acid via a beta-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a Km of 0.24 gl(-1) and a vmax of 0.72 gl(-1)min(-1). The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80% of pectin was proven by means of ruthenium red dyeing and HPAEC (65%). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.     

Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1

AIMS: The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. METHODS AND RESULTS: Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11.0. In BPP-A, zein was better substrate than casein at pH 13.0, whereas casein was better one than zein at pH 11.0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. CONCLUSION: A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.     

Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus

An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography, ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal activity at pH 10 and with a temperature of 55 degrees C; the enzyme activity can be completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP). The first 20 amino acid residues of the purified DHAP have been determined with a sequence of AQTVPYGIPQIKAPAVHAQG. Alignment of this sequence with other alkaline protease demonstrates its high homology with protease from another B. pumilus strain.     

Purification and Some Properties of Dipeptidyl Carboxypeptidase from Bacillus pumilus

An intracellular protease from a bacterium, Bacillus pumilus HL721, was purified about 5000-fold by Chromatography with a Q-Sepharose Fast Flow column, TSK-gel HA-1000 glass column, and TSK-gel G3000SWXL column using Bz-Gly-Ala-Pro as a substrate. The enzyme was the most active at pH around 7.5 and stable from 4.5 and 8.0. The enzyme activity was inhibited by Cu²⁺, EDTA, N-ethylmaleimide, o-phenanthroline, and p-chloromercuribenzoic acid. The molecular weight of the enzyme was 155,000 by gel filtration. The enzyme removed dipeptide from the carboxyl end of some peptides used as substrates. From these results the enzyme seems to be a dipeptidyl carboxypeptidase.     

短小芽孢杆菌2080碱性蛋白酶的纯化与性质

短小芽孢杆菌（Bacillus pumilus）2080碱性蛋白酶的发酵液经超滤、硫酸铵沉淀、CM Sepharose Fast Flow和DEAE Sepharose Fast Flow离子交换层析得到了纯化的组分。SDS-PAGE电泳分析显示其分子量约为61kDa。酶学性质研究表明,该纯化酶的最适pH为10．5,最适温度为50℃。     

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (K_m) and V_max for α-naphthyl acetate were 1.54 mM and 360 μmol min⁻¹ mg of protein⁻¹, respectively.     

Purification and characterization of histidine decarboxylase from mouse kidney.

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.     

Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi.

Pyruvate decarboxylase from the obligate anaerobe Sarcina ventriculi was purified eightfold. The subunit Mr was 57,000 +/- 3000 as estimated from SDS-PAGE, and the native Mr estimated by gel filtration on a Superose 6 column was 240,000, indicating that the enzyme is a tetramer. The Mr values are comparable to those for pyruvate decarboxylase from Zymomonas mobilis and Saccharomyces cerevisiae, which are also tetrameric enzymes. The enzyme was oxygen stable, and had a pH optimum within the range 6.3-6.7. It displayed sigmoidal kinetics for pyruvate, with a S0.5 of 13 mM, kinetic properties also found for pyruvate decarboxylase from yeast and differing from the Michaelis-Menten kinetics of the enzyme from Z. mobilis. No activators were found. p-Chloromercuribenzoate inhibited activity and the inhibition was reversed by the addition of dithiothreitol, indicating that cysteine is important in the active site. The N-terminal amino acid sequence of pyruvate decarboxylase was more similar to the sequence of S. cerevisiae than Z. mobilis pyruvate decarboxylase.     

Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1.

A cyanide-degrading enzyme from Bacillus pumilus C1 has been purified and characterized. This enzyme consisted of three polypeptides of 45.6, 44.6, and 41.2 kDa; the molecular mass by gel filtration was 417 kDa. Electron microscopy revealed a multimeric, rod-shaped protein approximately 9 by 50 nm. Cyanide was rapidly degraded to formate and ammonia. Enzyme activity was optimal at 37 degrees C and pH 7.8 to 8.0. Activity was enhanced by Sc3+, Cr3+, Fe3+, and Tb3+; enhancement was independent of metal ion concentration at concentrations above 5 microM. Reversible enhancement of enzymatic activity by azide was maximal at 4.5 mM azide and increased with time. No activity was recorded with the cyanide substrate analogs CNO-, SCN-, CH3CN, and N3- and the possible degradation intermediate HCONH2. Kinetic studies indicated a Km of 2.56 +/- 0.48 mM for cyanide and a Vmax of 88.03 +/- 4.67 mmol of cyanide per min/mg/liter. The Km increased approximately twofold in the presence of 10 microM Cr3+ to 5.28 +/- 0.38 mM for cyanide, and the Vmax increased to 197.11 +/- 8.51 mmol of cyanide per min/mg/liter. We propose naming this enzyme cyanide dihydratase.     

Purification and characterization of a new sodium-transport decarboxylase. Methylmalonyl-CoA decarboxylase from Veillonella alcalescens

Upon resolution of the particulate cell fraction of Veillonella alcalescens by gel chromatography, membranes and ribosomes were clearly resolved. Methylmalonyl-CoA decarboxylase was bound to the membranes and not to ribosomes as reported earlier. Membrane vesicles containing methylmalonyl-CoA decarboxylase were prepared by disrupting V. alcalescens cells with a French pressure chamber. About 64% of the decarboxylase was oriented in these vesicles with the substrate binding site facing to the outside. The vesicles performed a rapid accumulation of Na+ ions in response to the decarboxylation of methylmalonyl-CoA. Decarboxylation and transport were highly uncoupled. The efficiency of the transport was considerably increased if methylmalonyl-CoA decarboxylation was retarded by using a low temperature or by slowly generating the substrate enzymically from propionyl-CoA. Under optimized conditions Na+ was concentrated inside the inverted vesicles eight-times higher than in the incubation medium. Methylmalonyl-CoA decarboxylase was solubilized from the membranes with Triton X-100 and purified about 20-fold by affinity chromatography on monomeric avidin-Sepharose columns. The decarboxylase was specifically activated by Na+ ions (apparent Km approximately equal to 0.6 mM). Whereas (S)-methylmalonyl-CoA was the superior substrate (apparent Km approximately equal to 7 microM), malonyl-CoA was also decarboxylated (apparent Km approximately equal to 35 microM). The decarboxylation of methylmalonyl-CoA yielded CO2 and not HCO-3 as the primary reaction product. Analysis of the purified enzyme by dodecylsulfate gel electrophoresis indicated the presence of four different polypeptides alpha, beta, gamma, delta with Mr 60 000, 33 000, 18 5000 and 14 000. The latter of these polypeptides was clearly visible only after silver staining but not after staining with Coomassie brilliant blue. A low molecular weight polypeptide with similar staining properties was also found in oxaloacetate decarboxylase. Methylmalonyl-CoA decarboxylase contained about 1 mol covalently bound biotin per 125 500 g protein which was localized exclusively in the gamma-subunit. This subunit therefore represents the biotin carboxyl carrier protein of methylmalonyl-CoA decarboxylase. A new very sensitive method for the detection of biotin-containing proteins is described.     

M-26碱性木聚糖酶的分离纯化及酶学性质研究

从Bacillus pumilus M-26发酵液中分离纯化碱性木聚糖酶,进行酶学性质研究,同时制备工业用碱性木聚糖酶制剂。首先将M-26发酵液进行硫酸铵盐析,制备工业用碱性木聚糖酶干品;然后进行sephadexG-25层析脱盐和cellulose DE-52层析得以纯化。硫酸铵的饱和度50%,酶制剂的酶活可达9 000 IU/g,收率为85%;分离纯化使酶的比活为126.32 IU/mg蛋白,纯化倍数为19.89,酶的回收率12.83%;分子量约为20 ku;M-26碱性木聚糖酶的最适温度和pH分别是55℃和pH 8.0,具有一定的耐碱性;该酶无纤维素酶活性,Fe2＋对其有激活作用;Mn2＋、Zn2＋、Fe3＋、Cu2＋对其具有抑制作用。短小芽胞杆菌M-26碱性木聚糖酶具有纸浆生物漂白应用前景。     

短小芽孢杆菌A-30耐碱性木聚糖酶的纯化及性质研究

木聚糖广泛存在于自然界 ,通常占高等植物干重的 1 5%～ 30 % ,由木糖经β- 1 ,4-糖苷键连接起来形成主链 ,并由阿拉伯糖、乙酰基甘露糖、葡萄糖醛酸等复杂侧链共同组成 .在众多可降解木聚糖的酶中 ,β-内切木聚糖酶 ( E.C3.2 .1 .8,β- 1 ,4- xylanxylanohydrolase)起主要作用 .在纺织、制浆造纸、饲料及食品等工业中具有潜在的应用价值 .近年来 ,欧美等国已将其应用在造纸制浆工业 ,降低了漂白时氯的用量 ,改善了纸张性能 ,并且减少了环境污染 .对木聚糖酶的研究成为生物技术领域研究的热点之一 .国内外对来源于不同菌种的木聚糖酶的分离…     

Purification and characterization of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes.

Oxalyl-coenzyme A (oxalyl-CoA) decarboxylase was purified from Oxalobacter formigenes by high-pressure liquid chromatography with hydrophobic interaction chromatography, DEAE anion-exchange chromatography, and gel permeation chromatography. The enzyme is made up of four identical subunits (Mr, 65,000) to give the active enzyme (Mr, 260,000). The enzyme catalyzed the thiamine PPi-dependent decarboxylation of oxalyl-CoA to formate and carbon dioxide. Apparent Km and Vmax values, respectively, were 0.24 mM and 0.25 mumol/min for oxalyl-CoA and 1.1 pM and 0.14 mumol/min for thiamine pyrophosphate. The maximum specific activity was 13.5 microM oxalyl-CoA decarboxylated per min per mg of protein.     

Purification and characterization of glutamate decarboxylase from Neurospora crassa conidia.

L-Glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of Neurospora crassa, was purified to homogeneity from conidia. The purification procedure included ammonium sulfate fractionation and DEAE-Sephadex and cellulose phosphate column chromatography. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. A single band coincident with enzyme activity was found on native 7.5% polyacrylamide gels. The molecular weight of glutamate decarboxylase was 30,500 as determined by gel permeation column chromatography at pH 6.0. The enzyme had an acidic pH optimum and showed hyperbolic kinetics at pH 5.5 with a Km for glutamic acid of 2.2 mM and a Km for pyridoxal-5'-phosphate of 0.04 microM.