Purification of extracellular lipases from Trichosporon fermentans WU-C12

Two types of extracellular lipases (I and II) from Trichosporon fermentans WU-C12 were purified by acetone precipitation and successive chromatographies on Butyl-Toyopearl 650 M, Toyopearl HW-55F and Q-Sepharose FF. The molecular weight of lipase I was 53 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and 160 kDa by gel filtration, while that of lipase II was 55 kDa by SDS-PAGE and 60 kDa by gel filtration. For the hydrolysis of olive oil, the optimum pH and temperature of both the lipases were 5.5 and 35°C, respectively. The lipases showed stable activities after incubation at 30°C for 24 h in a pH range from 4.0 to 8.0. The thermostability of lipase I for 30 min at a reaction pH of 5.5 was up to 40°C, while that of lipase II under the same conditions was up to 50°C. Both lipases could hydrolyze the 1-, 2-, and 3-positions of triolein, and cleave all three ester bonds, regardless of the position in the triglyceride.     

Studies on a thermostable alpha-amylase from the thermophilic fungus Scytalidium thermophilum

An alpha-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration. The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE. The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B). Optima of pH and temperature were 6.0 and 60 degrees C, respectively. In the absence of substrate the purified alpha-amylase was stable for 1 h at 50 degrees C and had a half-life of 12 min at 60 degrees C, but was fully stable in the presence of starch. The enzyme was not activated by several metal ions tested, including Ca(2+) (up to 10 mM), but HgCl(2 )and CuCl(2) inhibited its activity. The alpha-amylase produced by S. thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order. The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses. Maltose and traces of glucose were formed only after 3 h of reaction. These results confirm the character of the enzyme studied to be an alpha-amylase (1,4-alpha-glucan glucanohydrolase).     

Aberrant mobility phenomena of the DNA repair protein XPA

The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time-resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post-translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS-PAGE (multiple bands from approximately 40-45 kDa) and gel filtration chromatography ( approximately 92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a approximately 32-kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA +/- cisplatin +/- mismatches. xXPA showed at least 10-fold higher affinity for cisplatin DNA +/- mismatches compared to undamaged DNA +/- mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu-rich region (EEEEAEE) and/or disordered N- and C-terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from approximately 5 to 25 kDa separated by reverse-phase HPLC and precisely determined by ESI-FTICR MS were correlated with their migration on SDS-PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%-50% larger molecular weights than expected. Thus, both the disordered domains and the Glu-rich region in xXPA are primarily responsible for the aberrant mobility phenomena.     

Identification and characterization of Chromobacterium viscosum lipase

Separation of a commercial preparation of Chromobacterium viscosum by hydrophobic interaction chromatography yields two active fractions, one corresponding to a lipase of 33.0 ± 1.0 kDa by SDS-PAGE and the other to a high molecular weight aggregate (> 250 kDa) of the lipase with some impurities absorbing at 436 nm. Partial disaggregation of this complex occurs on gel filtration chromatography in the presence of 1% (w/v) CHAPS. On gel filtration under non reducing conditions the lipase behaves like a 17 kDa protein; in the presence of a strong denaturant and of a reducing agent a molecular size of 36 kDa is obtained, in accordance with SDS-PAGE results.     

Purification and characterization of xylanase from a thermophilic Streptomyces sp. K37

Extracellular xylanase (EC 3.2.1.8) from Streptomyces sp. K37 was purified 33.53 by ultrafiltration and cation exchange chromatography followed by gel filtration chromatography. The optimum pH and temperature for purified xylanase were found to be pH 6.0 and 60 degrees C. The Km and V(max) values of the purified xylanase were 15.4 mg ml(-1) and 0.67 micromole reducing sugar min(-1) ml(-1). High performance liquid chromatography (HPLC) gel filtration of the purified xylanase eluted xylanase activity as a peak corresponding to the molecular weight of about 24.3 kDa while the molecular weight determined by SDS-PAGE was found to be 26.4 kDa. The purified xylanase of Streptomyces sp. K37 was found to be endoxylanase and non arabinose liberating enzyme and was highly glycosylated (73.97%).     

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.     

A Novel PHB Depolymerase from a Thermophilic Streptomyces Sp.

A novel PHB depolymerase from a thermophilic Streptomyces sp. MG was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The molecular mass of the purified enzyme was 43 kDa as determined by size exclusion chromatography and 41 kDa by SDS-PAGE. The optimum pH and temperature were 8.5 and 60 °C respectively. The enzyme was stable at 50 °C and from pH 6.5–8.5. The enzyme hydrolyzed not only bacterial polyesters, i.e. poly(3-hydroxybutyric acid and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), but also synthetic, aliphatic polyesters such as polypropiolactone, poly(ethylene adipate) and poly(ethylene succinate). Revisions requested 9 November 2005; Revisions received 12 December 2005     

Purification of an active gelatinase from collagen fiber fraction of granulation tissue in rats

Gelatinase was extracted at 60 degrees C from the collagen fiber-rich fraction of granulation tissue induced by carrageenin in rats. A large part of the extracted gelatinase was unbound to Zn-chelating Sepharose. The unbound gelatinase gave a single band corresponding to a molecular mass of 57 kDa on SDS-substrate PAGE, but showed a much higher molecular mass (greater than 200 kDa) on Sephadex G-150 gel filtration. In addition, that unbound fraction contained gelatin fragments was revealed by SDS-PAGE. When the unbound fraction of Zn-chelating Sepharose was incubated at 37 degrees C, the gelatin fragments disappeared and the apparent molecular mass of gelatinase in gel filtration decreased. This gelatin degradation of the unbound fraction was enhanced by treatment with a 4-aminophenylmercuric acetate (APMA). The results suggest that the gelatinase is bound to gelatin fragments in the unbound fraction. After the treatment with APMA, the gelatinase was purified to to homogeneity; the purified gelatinase gave a single band corresponding to a molecular mass of 57 or 67 kDa on SDS-PAGE under nonreducing or reducing conditions, respectively. The purified gelatinase is a metalloproteinase, and extensively degraded gelatin, but showed no proteolytic activity toward alpha-casein or types I and IV collagens. The results suggest that the 67-kDa active gelatinase is bound to collagen fibers and plays an important role in a rapid degradation of collagen fibers in granulation tissue.     

Purification and subunit composition of a GTP-binding protein from maize root plasma membranes

When frozen plasma membranes isolated from maize seedling roots are thawed, a significant portion of GTP-binding activity goes into solution. The GTP-binding protein was purified by ion exchange chromatography on Mono-Q and gel filtration on Superose 6. Its molecular weight was estimated at 61 kDa by gel filtration. The same molecular weight was obtained upon solubilization of the GTP-binding protein with cholic acid followed by gel filtration in the presence of this detergent. SDS-PAGE demonstrated that the isolated GTP-binding protein consists of two types of subunit of molecular weights 27 kDa and 34 kDa.     

Isolation of a Raw Starch-Binding Fragment from Barley α-Amylase

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α₇-and α₈-helices of the (α/β)₈ domain.     

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H₂ plus hydrogenase purified from strain MC. Received: 5 February 1997 / Accepted: 17 April 1997     

The enzyme defect in bovine protoporphyria. Studies with purified ferrochelatase

Ferrochelatase was purified from the livers of normal and protoporphyria cattle by chromatography on Blue Sepharose CL-6B in order to investigate the enzyme defect in this disorder. The increase in specific activity (up to 2900-fold) indicated that the normal and protoporphyria enzymes were purified to a similar degree. The mutant enzyme had catalytic activity which was 10 to 15% of normal ferrochelatase, although the Michaelis constants for protoporphyrin and iron were similar. The molecular mass of the normal and protoporphyria enzyme protein was 40 kDa as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the presence of 15 mM sodium cholate, gel filtration demonstrated a similar size. However, at a lower concentration of sodium cholate (4 mM) the molecular mass was about 240 kDa, suggesting that the purified enzymes aggregate under this condition. Polyvalent antibodies were raised in rabbits using as antigens purified normal native enzyme and normal 40-kDa protein which had been further purified by preparative SDS-PAGE. In Western blots these antibodies complexed with both the normal and mutant 40-kDa proteins. The amount of 40-kDa protein in normal and protoporphyria mitochondrial fractions was also similar as evaluated by Western blots. These studies indicate that the ferrochelatase defect in bovine protoporphyria probably results from a point gene mutation that causes a minor change in enzyme structure.     

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.     

Purification of rosmarinic acid synthase from cell cultures of Coleus blumei Benth

Rosmarinic acid synthase from cell cultures of Coleus blumei Benth. was purified to apparent homogeneity by fractionated ammonium sulfate precipitation (60–80% saturation), hydrophobic interaction chromatography, affinity chromatography and gel filtration. This purification procedure resulted in a 225-fold-enriched specific enzyme activity with a yield of 9%. The protein preparation was apparently pure according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis. The apparent molecular mass determined by gel filtration and SDS-PAGE was 77 kDa, indicating that rosmarinic acid synthase is a monomeric enzyme.Abbreviations DTT dithiothreitol - HIC hydrophobic interaction chromatography - RA rosmarinic acid - RAS rosmarinic acid synthase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged. Two-dimensional gel electrophoresis was done with the help of Dr. Guy Bauw, University of Gent, Belgium.     

Identification of a novel gentisate 1,2-dioxygenase from <Emphasis Type="Italic">Silicibacter pomeroyi</Emphasis>

A 1,125-bp long ORF encoding a novel gentisate 1,2-dioxygenase with two-domain bicupins was cloned from Silicibacter pomeroyi DSS-3 and expressed in Escherichia coli. The resulting product was purified to homogeneity and partially characterized. Non-reductive SDS-PAGE and gel filtration showed that the active recombinant gentisate 1,2-dioxygenase had an estimated molecular mass of 132 kDa, and reductive SDS-PAGE indicated an approximate size of 45 kDa. The enzyme thus appears to be a homotrimeric protein. This is in contrast to the homotetrameric or dimeric protein of the gentisate 1,2-dioxygenases that have been characterized thus far. The K (m) and K (cat)/K (m) for gentisate were 12 muM and 653 x 10(4) M(-1 )s(-1); the pI was 4.6-4.8. It was optimally active at 40 degrees C and pH 8.0.     

Affinity purification and partial characterization of IgM-like immunoglobulins of African catfish, Clarias gariepinus (Burchell, 1822)

IgM like macroglobulin from bovine serum albumin (BSA)-immunized African catfish C. gariepinus was purified by affinity chromatography and partially characterized. The molecular weight of this macroglobulin was 840 kDa, as estimated by gel filtration chromatography. Purified macroglobulin was analyzed using SDS-PAGE under reducing and non-reducing conditions. The molecular weight (MW) of heavy and light chain was 74.8 kDa and 27.2 kDa respectively, in presence of a reducing agent. In non-reducing SDS-PAGE, a single high MW band was observed representing tetrameric form.     

The formylpeptide chemoattractant receptor copurifies with a GTP-binding protein containing a distinct 40-kDa pertussis toxin substrate

Detergent extraction of plasma membranes from differentiated HL60 cells, specifically labeled with the chemoattractant, formyl-Nle-Leu-Phe-Nle-[125I-Tyr] Lys, resulted in the solubilization of a receptor-radioligand complex. GTP-binding activity coeluted with the radioligand when the sodium cholate extract was purified by chromatography on wheat germ agglutinin-Sepharose 6MB. A molecular size of approximately 59 A was estimated for the lectin-Sepharose-purified receptor complex by gel filtration chromatography on Ultrogel AcA 34. The isolated complex eluted from the gel filtration column exhibited an enhanced rate of ligand dissociation in response to GTP gamma S. Approximately 0.65 mol of pertussis toxin substrate/mol of receptor was estimated following partial purification of the receptor-ligand complex by sequential chromatography on wheat germ agglutinin-Sepharose, DEAE-Fractogel, and Ultrogel AcA 34. The pertussis toxin substrate which copurified with the receptor was compared with two distinct G proteins, containing alpha-subunits of 40 and 41 kDa, previously purified from HL60 cell plasma membranes. Approximately 86% of the pertussis toxin substrate identified in the receptor preparation consisted of the 40-kDa polypeptide. Differences in the peptide maps indicate that the predominant G protein which coelutes with the receptor is distinct from the purified G protein with an alpha-subunit of 41 kDa but homologous to the purified G protein with an alpha-subunit of 40 kDa.     

Purification and characterisation of adenosine nucleosidase from Coffea arabica young leaves

An adenosine nucleosidase (ANase) (EC 3.2.2.7) was purified from young leaves of Coffea arabica L. cv. Catimor. A sequence of fractionating steps was used starting with ammonium sulphate salting-out, followed by anion exchange, hydrophobic interaction and gel filtration chromatography. The enzyme was purified 5804-fold and a specific activity of 8333 nkat mg-1 protein was measured. The native enzyme is a homodimer with an apparent molecular weight of 72 kDa estimated by gel filtration and each monomer has a molecular weight of 34.6 kDa, estimated by SDS-PAGE. The enzyme showed maximum activity at pH 6.0 in citrate-phosphate buffer (50 mM). The calculated Km is 6.3 microM and Vmax 9.8 nKat.     

A normal mucin-binding lectin from the sponge Craniella australiensis

A lectin, Craniella australiensis (CAL), was isolated from sponge C. australiensis by ion-exchange on DEAE-Sephacel and purified by gel filtration on Sephadex G-150 and HPLC on DEAE-5PW. The purified lectin was a trimeric protein as revealed by SDS-PAGE and MALDI-TOF analysis. SDS-PAGE showed that the CAL protein had a molecular mass of 54 kDa, and consisted of three 18 kDa subunits. Gel filtration of purified lectin on Sephadex G-200 indicates that it exists as a 54 kDa protein in its native state. The amino acid composition was rich in Thr and Glx. CAL was found to agglutinate native and trypsinized human A, B erythrocytes, and agglutinate native erythrocytes of mouse, sheep, rabbit and chicken, and trypsinized erythrocytes of sheep and rabbit. The hemagglutination activity was inhibited by glycoproteins such as PSM and asialo-PSM, but not by any of the monosaccharides tested. The activity was stable between 20 and 70 degrees C. Significant CAL activity was observed between pH 5 and 8. The lectin reaction is independent of the presence of divalent cations Ca2+ and Mg2+. The sequence of N-terminal residues of CAL was determined as TSSCQSIVVE. The lectin showed a potent mitogenic response towards BALB/c splenocytes.     

Identification of precursor peptide of aculeacin A acylase as a protein with proteolytic activity

Abstract A protein with the proteolytic activity was isolated from culture filtrate of the aculeacin A acylase producing strain, Actinoplanes utahensis NRRL12052. The purified protein showed a single band of molecular mass of 87 kDa in SDS-PAGE and gel filtration using HPLC, and reacted with anti-aculeacin A acylase antiserum. The 87-kDa protein was degraded to two peptides of molecular mass of 60 kDa and 19 kDa by incubation at 37°C in the presence of 0.1% SDS and the former band also responded to the antiserum. These results indicate that the 87-kDa protein possessing the proteolytic activity is a precursor of aculeacin A acylase.