Purification and properties of invertase from the flowers of Woodfordia fruticosa

Invertase (beta-fructofuranosidase, EC 3.2.1.26) was purified from the flowers of Woodfordia fruticosa, which is used to prepare certain fermented Ayurvedic drugs. The enzyme was purified to near homogeneity as judged by native PAGE with a yield of 10.7%, using (NH4)2SO4 fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulose chromatography at pH 6.8 and 4.42. The molecular mass of the purified enzyme as determined by elution through Sepharose 4B gel column was found to be approximately 280 kDa. SDS-PAGE of the purified enzyme showed that the enzyme is composed of three subunits with molecular mass of 66, 43 and 40 kDa. The enzyme showed a broad pH optimum between 4.0-7.0. Optimum assay temperature was 37 degrees C and above 45 degrees C, the enzyme activity slowly declined and inactivated around 80 degrees C. The apparent Km value of the enzyme for sucrose was 160 mM.     

The purification and characterization of a dextranase from Lipomyces starkeyi

Dextranase produced by Lipomyces starkeyi was purified 43-fold, by carboxymethyl-Sepharose chromatography followed by agarose gel-filtration chromatography. The purified enzyme showed four bands by SDS/polyacrylamide gel electrophoresis with estimated mass 74 kDa, 71 kDa, 68 kDa and 65 kDa. This preparation exhibited multiple isoelectric points between 5.6 and 6.1. All the isoelectric forms were active and catalytically similar. The dextranase contained a carbohydrate moiety (8%). The physical properties of the enzyme were pH and temperature optima of 5.0 and 55 degrees C, respectively. This dextranase was stable between pH 2.5 and 7.0 at temperatures below 40 degrees C. Lipomyces dextranase was a typical endodextranase with the final product of dextran hydrolysis being isomalto-oligosaccharides from glucose to isomaltotetrose.     

Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus

A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus. This enzyme was purified 100-fold from a soluble extract obtained at 100,000g. The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps. The native enzyme had a molecular mass of 46 kDa assessed through gel filtration. This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4-8, its optimal temperature was 45 degrees C and the enzyme became unstable at temperatures above 55 degrees C. The isoelectric point of the purified enzyme was 4.4. Michaelis constant and Vmax for L-lysine-p-nitroanilide were 0.33 mM and 2.2 mM min(-1) per milligram of protein, respectively. The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease. Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.     

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum

Heparinase I has been purified from F. heparinum by a novel scheme with 10mM CaCl(2) added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4-7.0 and 41 degrees C. CaCl(2) is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 degrees C or freezing-thawing.     

Purification and properties of a thermostable fumarate hydratase from the archaeobacterium Sulfolobus solfataricus

Fumarate hydratase (EC 4.2.1.2) from the extremely thermophilic archaeobacterium Solfolobus solfataricus has been purified to homogeneity by a rapid purification procedure using affinity chromatography and high-performance size-exclusion chromatography, and the enzyme's physical and biochemical properties have been determined. The native enzyme has a molecular mass of 170 kDa and is composed of identical subunits with a molecular mass of 45 kDa, thus indicating a tetrameric structure similar to fumarases isolated from other organisms. The enzyme was active at temperatures ranging from 40 degrees C to 90 degrees C, with a maximum activity at 85 degrees C. The pH optimum for generation of fumarate was found to be pH 8.0. The enzyme showed high stability to denaturation by heat and organic solvents.     

Overproduction and characterization of xylanase B from Aspergillus niger

The xynB gene, which encodes endo-beta-1,4-xylanase XynB, in Aspergillus niger BRFM281 was amplified by RT-PCR using mRNA isolated from a culture containing sugar beet pulp as an inducer. The cDNA was cloned into an expression cassette under the control of the strong and constitutive glyceraldhehyde-3-phosphate dehydrogenase gene promoter. The expression system was designed to produce the recombinant enzyme XynB with a six-histidine peptide fused to the carboxy end of the protein. Homologous overproduction of XynB was successfully achieved in shake flask cultures, and the secretion yield was estimated to be 900 mg x L(-1). The recombinant XynB was purified 1.5-fold by immobilized metal affinity chromatography to homogeneity using a one-step purification protocol with 71% recovery. The purified recombinant enzyme was fully characterized and has a molecular mass of 23 kDa and an optimal activity at pH 5.5 and 50 degrees C with stability in the pH range 4.0-7.0 and temperature up to 50 degrees C. Using soluble oat spelts xylan, the determined Km and Vmax values were 7.1 mg x mL(-1) and 3881 U x mg(-1), respectively.     

Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp.

A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S0-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.     

Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein

Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones. Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E. coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography. The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150+/-40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4%. The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration. Our results indicate that the active recombinant enzyme expressed in E. coli is a homodimer.Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate. The optimum pH ranged from pH 7 to 8, and the optimum temperature 40-45 degrees C. Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45 degrees C for 15 min. The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM. The K(m) value for DHEA was 1.9+/-0.3 microM and V(max)=190+/-18 nmol/min per mg of protein at 37 degrees C, pH 7.5.     

Thermostable D-carbamoylase from Sinorhizobium morelens S-5: purification, characterization and gene expression in Escherichia coli

A d-carbamoylase from Sinorhizobium morelens S-5 was purified and characterized. The enzyme was purified 189-fold to homogeneity with a yield of 19.1% by aqueous two-phase extraction and two steps of column chromatography. The enzyme is a homotetramer with a native molecular mass of 150 kDa and a subunit relative molecular mass of 38 kDa. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. The enzyme showed high thermal and oxidative stability. It was found to have a K(m) of 3.76 mM and a V(max) of 383 U/mg for N-carbamoyl-d-p-hydroxyphenylglycine. The hyuC gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of d-carbamoylase from other sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity from the recombinant. Our results show that the enzyme has great potential for industrial application.     

Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli.

The gene encoding an extremely heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei was cloned and expressed in Escherichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chromatography on a maltotriose-coupled Sepharose 6B column, and anion-exchange chromatography on Mono Q. The pullulanase, which was purified 90-fold with a final yield of 15%, is composed of a single polypeptide chain with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzyme activation up to 370% is achieved in the presence of calcium ions and reducing agents such as beta-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The high rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sodium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme seems to switch from the compact to the unfolded form, which is accompanied by an apparent shift in the molecular mass from 45 to 90 kDa.     

Glycosulphatase from Pseudomonas carrageenovora. Purification and some properties

A glycosulphatase present in the soluble fraction of disrupted Pseudomonas carrageenovora has been purified 500-fold by gel filtration on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose CL-6B. By dodecylsulphate/polyacrylamide gel electrophoresis the enzyme is practically homogeneous and has a molecular weight of 55 000. Conditions of optimal sodium chloride concentration and pH at 25 degrees C were 0.25--0.50 mol dm-3 and pH 7.0 respectively. The purified enzyme was inhibited by inorganic phosphate. Preparation is described of neocarrabiose 4-O-[35S]sulphate and neocarratetraose 4-O-[35S]sulphate from labelled Chondrus crispus. The purified glycosulphatase is active against both these substrates although only one of the two sulphate esters in the tetrasaccharide is hydrolysed. Analysis of the reaction products was by gel filtration, electrophoresis and 13C nuclear magnetic resonance spectroscopy. The results are consistent with the products of desulphation being respectively neocarrabiose and neocarratetraose 4-O-monosulphate with the sulphate ester proximal to the reducing end [3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-beta-D-galactopyranosyl-(1 leads to 4)-3,6-anhydro-alpha-D-galactopyranosyl-(1 leads to 3)-D-galactose 4-O-sulphate].     

Functional expression of amylosucrase, a glucan-synthesizing enzyme, from Arthrobacter chlorophenolicus A6

A gene (acas) designated as alpha-amylase was cloned from Arthrobacter chlorophenolicus A6. The multiple amino acid sequence analysis and functional expression of acas revealed that this gene really encoded an amylosucrase (ASase) instead of alpha-amylase. In fact, the recombinant enzyme exhibited typical ASase activity by showing both sucrose hydrolysis and glucosyltransferase activities. The purified enzyme has a molecular mass of 72 kDa and exhibits optimal hydrolysis activity at 45 degrees C and a pH of 8.0. The analysis of the oligomeric state of ACAS with gel permeation chromatography revealed that the ACAS existed as a monomer.     

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.     

Purification and characterization of an extracellular alpha-amylase from Clostridium perfringens type A.

An alpha-amylase (EC 3.2.1.1) secreted by Clostridium perfringens NCTC 8679 type A was purified to homogeneity and characterized. It was isolated from concentrated cell-free culture medium by ion-exchange and gel permeation chromatography. The enzyme exhibited maximal activity at pH 6.5 and 30 degrees C without the presence of calcium. The pI of the enzyme was 4.75. The estimated molecular weight of the purified enzyme was 76 kDa. The purified enzyme was inactivated between 35 and 40 degrees C, which increased to between 45 and 50 degrees C in the presence of calcium (5 mM). The purified enzyme produced a mixture of oligosaccharides as major end products of starch hydrolysis, indicating alpha-amylase activity.     

Purification and characterization of rat liver glycosylasparaginase.

1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.     

Purification and characterization of carboxymethyl cellulase from Bacillus sp. isolated from a paddy field

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.     

Purification and partial characterization of polygalacturonase from Streptomyces lydicus

Polygalacturonase produced by Streptomyces lydicus was purified to homogeneity by ultrafiltration and a combination of ion exchange and gel filtration chromatographic procedures. The purified enzyme was an exo-polygalacturonase with a molecular weight of 43 kDa. It was optimally active at 50 degrees C and pH 6.0. The enzyme was stable from pH 4.0 to 7.0 and at or below 45 degrees C for 90 min. K(m) value for polygalacturonic acid was 1.63 mg/mL and the corresponding V(max) was 677.8 microM min(-1) mg(-1). The inhibition constant (K(i)) for gluconic acid d-lactone was 20.75 mM. Purified enzyme had been inhibited by N-bromosuccinimide, while l-tryptophan could induce enzyme activity, indicating the involvement of tryptophan at the active site.     

Purification and characterization of a major secretory cellobiase, Cba2, from Cellulomonas biazotea

A novel cellobiase (Cba2) was purified from the culture supernatant of Cellulomonas biazotea and characterized. Cba2 appeared to be a major secretory cellobiase in C. biazotea as its enzymatic activity was estimated to represent over 40% of the total extracellular beta-glucosidase activity. The enzyme was purified over 260-fold subsequent to ammonium sulfate precipitation, gel-filtration chromatography, anion-exchange chromatography, and reversed-phase high-performance liquid chromatography. Cba2 was shown by SDS-PAGE to have a large molecular mass of 109 kDa, which makes it one of the largest secretory cellobiases characterized. Its homogeneity was confirmed by N-terminal amino acid sequencing. The K(m) and V(max) values were 0.025 mM and 0.0048 mM min(-1), respectively, for the Cba2 hydrolysis of p-nitrophenyl-beta-d-glucopyranoside, and 0.73 mM and 0.00033 mM min(-1), respectively, for the hydrolysis of cellobiose (at 37 degrees C and pH 7.0). The purified enzyme has a pH optimum of 4.8 and the optimum temperature for activity is 70 degrees C. In view of the secretory nature of Cba2 and the fact that it is a major component of secretory cellobiases of C. biazotea, it is potentially important in the enzymatic degradation of cellulose, and its availability as a recombinant protein may facilitate the studies of its biotechnological applications.     

Agmatine deiminase from cucumber seedlings is a mono-specific enzyme: purification and characteristics

Agmatine deiminase was purified to homogeneity from cucumber seedlings. The purification procedures included treatment with DE52, ammonium sulfate precipitation, DE52 column chromatography, Superdex 200 column chromatography, and agmatine-(CNBr)-diaminohexane-CNBr-activated-Sepharose 4B column chromatography. The purified agmatine deiminase exhibited a specific activity of 242nkat/mg protein at 30 degrees C, pH 7.0, with a yield of 33%. The molecular mass of the native enzyme was 67kDa, as estimated by Superdex 200 column chromatography. On the other hand, SDS-PAGE showed that the molecular masses of the subunits with 1% SDS and 5% of 2-mercaptoethanol treatment and with additional N-glycosidase F treatment were 47 and 36kDa, respectively. These results suggest that agmatine deiminase from cucumber is a glycoprotein. The Km of the enzyme for agmatine was 16microM and arcaine was a potent competitive inhibitor of the enzyme, with a Ki of 7.1microM. The enzyme was stable for 2 months at 4 degrees C. The enzyme does not have putrescine synthase activity or the activities of its components ornithine and putrescine transcarbamylase. The characteristics of the enzyme purified from cucumber were like those of the enzyme from maize. These results indicate that agmatine deiminase is distinctly different from putrescine synthase in higher plants.     

Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants （Camellia sinensis （L.） O. Kuntze）

β-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity,with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50℃ and was stable at temperatures lower than 40℃. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.