Purification and biochemical characterization of a laccase from the aquatic fungus <Emphasis Type="Italic">Myrioconium</Emphasis> sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene

Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO₄ and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k _cat/K _m) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3, 46.9, and 28.2 s⁻¹ mM⁻¹, respectively, with K _m values of 4.2, 67.8, and 104.9 μM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15°C. The laccase-encoding gene was identified and sequenced at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.     

A surfactant tolerant laccase of <Emphasis Type="Italic">Meripilus giganteus</Emphasis>

A laccase (Lcc1) from the white-rot fungus Meripilus giganteus was purified with superior yields of 34% and 90% by conventional chromatography or by foam separation, respectively. Size exclusion chromatography (SEC) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) yielded a molecular mass of 55 kDa. The enzyme possessed an isoelectric point of 3.1 and was able to oxidize the common laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at a pH of 2.0, whereas the enzyme was still able to oxidize ABTS and 2,6-dimethoxyphenol (DMP) at pH 6.0. Lcc1 exhibited low K _m values of 8 μM (ABTS) and 80 μM (DMP) and remarkable catalytic efficiency towards the non-phenolic substrate ABTS of 37,437 k _cat/k _m (s⁻¹ mM⁻¹). The laccase showed a high stability towards high concentrations of various metal ions, EDTA and surfactants indicating a considerable biotechnological potential. Furthermore, Lcc1 exhibited an increased activity as well as a striking boost of stability in the presence of surfactants. Degenerated primers were deduced from peptide fragments. The complete coding sequence of lcc1 was determined to 1,551 bp and confirmed via amplification of the 2,214 bp genomic sequence which included 12 introns. The deduced 516 amino acid (aa) sequence of the lcc1 gene shared 82% identity and 90% similarity with a laccase from Rigidoporus microporus. The sequence data may aid theoretical studies and enzyme engineering efforts to create laccases with an improved stability towards metal ions and bipolar compounds.     

Efficient production of laccases by Trametes sp. AH28-2 in cocultivation with a Trichoderma strain

A biocontrol fungus isolated from rotting wood was identified as a Trichoderma strain (named as Trichoderma sp. ZH1) by internal transcribed spacer (ITS) sequences of rRNA genes. The laccase yield of Trametes sp. AH28-2 in cocultivation with Trichoderma sp. ZH1 reached 6,210 U l⁻¹, approximately identical to those induced by toxic aromatic inducers. Cocultures maintained 60–70 % of their highest laccase activity obtained at 5 days after inoculation of the biocontrol fungus, at least for 20 days. Furthermore, a novel laccase isozyme (LacC) was obtained through the fungal interactions. The molecular weight of LacC is about 64 kDa, and its isoelectric point is 6.6. The temperature and pH optimum for LacC to oxidize guaiacol are 55 °C and 5.0, respectively. LacC is stable both at 60 °C and pH 4.0–8.0. Furthermore, the K _m values of LacC for various substrates were also determined. Our work demonstrates a safe strategy for the production of industrial laccases, instead of the traditional method of chemical induction.     

Cloning,purification and biochemical characterization of beta agarase from the marine bacterium <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> sp. AG4

A gene (agrP) encoding a β-agarase from Pseudoalteromonas sp. AG4 was cloned and expressed in Escherichia coli. The agrP primary structure consists of an 870-bp open reading frame (ORF) encoding 290 amino acids (aa). The predicted molecular mass and isoelectric point were determined at 33 kDa and 5.9, respectively. The signal peptide was predicted to be 21 aa. The deduced aa sequence showed 98.6% identity to β-agarase from Pseudoalteromonas atlantica. The recombinant protein was purified as a fusion protein and biochemically characterized. The purified β-agarase (AgaP) had specific activity of 204.4 and 207.5 units/mg towards agar and agarose, respectively. The enzyme showed maximum activity at 55°C and pH 5.5. It was stable at pH 4.5 to 8.0 and below 55°C for 1 h. The enzyme produced neoagarohexaose and neoagarotetraose from agar and in addition to that neoagarobiose from the agarose. The neoagarooligosaccharides were biologically active. Hence, AgaP is a useful enzyme source for use by cosmetic and pharmaceutical industries.     

Purification,molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete <Emphasis Type="Italic">Trametes</Emphasis> sp. strain AH28-2

A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.     

Purification and Characterization of Thermostable Pectate Lyase with Protopectinase Activity from Thermophilic Bacillus sp. TS 47

A strain of thermophilic bacterium, Bacillus sp., with pectolytic activity has been isolated. It produced an extracellular endo-polygalacturonate trans-eliminase (PL, EC 4.2.2.1) when grown at 60°C on a medium containing polygalacturonate (PGA). The PL was purified by hydrophobic, cation exchange, and size exclusion column chromatographies. The molecular mass of the enzyme was 50 kDa by SDS-PAGE. The isoelectric point of the enzyme was pH 5.3. The enzyme had a half-life of 13 and 1 h at 65 and 70°C, respectively, and showed optimal activity around at 70°C and pH 8.0. It had protopectinase activity, besides PL activity, on lemon protopectin and cotton fibers. The first 20 amino acids sequence of the enzyme had significant similarity with that of PL from methophilic Bacillus subtilis, with 50% identity.     

Purification and characterization of a new type of serine carboxypeptidase from<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K_m, V_max, K_cat, and K_cat/K_m values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min^–1, 291 s^–1, and 339 mM^–1 s^–1, respectively.     

Selective induction, purification and characterization of a laccase isozyme from the basidiomycete Trametes sp. AH28-2

The white-rot fungus Trametes sp. AH28-2 can synthesize extracellular laccase by induction in cellobiose-based liquid culture medium. Both yields and composition of laccase isozymes, produced by Trametes sp. AH28-2, would be quite different with induction by different small-molecule aromatic compounds, o-toluidine, guaiacol and 3,5-dihydroxytoluene, which affected microbial growth and the synthesis of laccase isozymes differentially. Higher concentrations of the three inducers could considerably increase laccase isozymes yields but not change the laccase composition. Coculturing of Trametes sp. AH28-2 with either Aspergillus oryzae or Gloeophyllum trabeum showed a few effects on laccase production. Laccase isozyme, laccase B, was selectively induced by 3,5-dihydroxytoluene and purified to homogeneity by two-step chromatography. Purified laccase B appeared as blue, with a broad peak at about 600 nm and a shoulder peak at about 330 nm. The ratio of absorbance at 280 nm to that at 600 nm was 21. Every molecule of laccase B had approximately four copper atoms. Molecular mass of laccase B was estimated to be 74 kDa on SDS-PAGE, 72 kDa by FPLC and was determined to be 71?454 Da by mass spectrum. After being treated with N-glycosidase F, laccase B lost 25% of its molecular mass. The isoelectric point of laccase B was 4.0. Its optimal pH and temperature for oxidizing guaiacol were respectively 4.7 and 45 C. The half-life of the enzyme at 60 C was 14.0 min. The enzyme showed a good stability in a range of pH value of 3.5-7.5. The K(m) values of the enzyme toward substrates syringaldazine, guaiacol, ABTS, and DMOP were respectively 28.0, 1249.0, 177.0 and 109.8 μM. The corresponding V(max) are 504.0, 1910.0, 117.4 and 159.0 μM min(-1) mg(-1). In addition, activity of laccase B was inhibited strongly by sodium azide and cyanide, mildly by SDS and trifluoroacetic acid, but only weakly by dimethyl sulfoxide.     

Purification and characterization of an extracellular laccase from a Pseudomonas sp. LBC1 and its application for the removal of bisphenol A

The Pseudomonas sp. LBC1 produced extracellular laccase when grown in the nutrient broth. The enzyme was purified using acetone precipitation and an anion-exchange chromatography. The molecular weight of the purified laccase was estimated as 70 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. An enzyme showed maximum substrate specificity towards o-tolidine than other substrates of laccase including 2,2′-azinobis, 3-ethylbenzothiazoline-6-sulfonic acid, hydroquinone, N,N′-dimethyl phenylene diamine, syringic acid and veratryl alcohol. The optimum pH and temperature for the laccase activity were 4.0 and 40 °C, respectively. Cyclic voltammogram revealed the redox potential of purified enzyme as 0.30 V. The laccase was stable up to 40 °C and within pH range 6.0–8.0. Sodium azide and EDTA strongly inhibited laccase activity. The purified laccase completely degraded the higher concentration of bisphenol A within 5 h. Biodegradation metabolites of bisphenol A were characterized by using FTIR, HPLC and GC–MS.     

Production,purification and characterization of a thermostable laccase from a tropical white-rot fungus

A thermostable laccase was isolated from a tropical white-rot fungus Polyporus sp. which produced as high as 69,738 units of laccase l⁻¹ in an optimized medium containing 20 g of malt extract l⁻¹, 2 g of yeast extract l⁻¹, 1.5 mM CuSO₄. The laccase was purified to electrophoretic purity with a final purification of 44.70-fold and a recovery yield of 21.04%. The purified laccase was shown to be a monomeric enzyme with a molecular mass of 60 kDa. The optimum temperature and pH value of the laccase were 75°C and pH 4.0, respectively, for 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS). The Michaelis–Menten constant (K _m ) of the laccase was 18 μM for ABTS substrate. The laccase was stable at pH values between 5.5 and 7.5. About 80% of the initial enzyme activity was retained after incubation of the laccase at 70°C for 2 h, indicating that the laccase was intrinsically highly thermostable and with valuable potential applications. The laccase activity was promoted by 4.0 mM of Mg²⁺, Mn²⁺, Zn²⁺ and Ca²⁺, while inhibited by 4.0 mM of Co²⁺, Al³⁺, Cu²⁺, and Fe²⁺, showing different profiles of metal ion effects.     

Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum

Benzyl alcohol and starch-free commercial wheat bran were effective inducers of the laccase activity in cultures of Fusarium proliferatum (MUCL 31970). Initial pH value in the cultures was also an overriding factor for increasing its production. By gel permeation high-performance liquid chromatography, the enzyme eluted as an apparently homogeneous peak with a molecular mass of 54 kDa, but by isoelectrofocusing, two proteins with pI values of 5.17 and 5.07 were revealed. Two different phenoloxidase activities were also detected after nondenaturing polyacrylamide gel electrophoresis. By matrix-assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOF-MS), both proteins showed unique fingerprints, so they were classifiable as isozymes, and were named laccase 1 (Lac1, pI 5.17) and laccase 2 (Lac2, pI 5.07). No clear matches were found when compared with other proteins. The tandem mass spectrometry of some peptides from both isozymes reanalyzed by nanoelectron ionization–ion trap–mass spectrometry (nESI-IT-MS) confirmed their unique character. The following interesting properties, particularly its stability at alkaline pH, make this laccase a promising industrial enzyme for biotechnological applications: maximum activity at 60°C, thermal stability for 2 h at 40°C, optimum pH 3.5 (km=62 μM) measured on 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonate), and pH stability 4–8 (75% stability at pH levels 2.2 and 9) for 2 h at 25°C.     

Isolation and characterization of a novel intracellular glucosyltransferase from the acarbose producer <Emphasis Type="Italic">Actinoplanes</Emphasis> sp. CKD485-16

A novel intracellular glucosyltransferase (GTase) was isolated from cells of Actinoplanes sp. CKD485-16—acarbose-producing cells. The enzyme was purified by DEAE-cellulose and G75-40 Sephadex chromatography. The molecular mass of the enzyme was estimated to be 62 kDa by SDS-polyacrylamide gel electrophoresis, and its isoelectric point (pI) was pH 4.3. The N-terminal sequence of the GTase consisted of NH₂-Ser-Val-Pro-Leu-Ser-Leu-Pro-Ala-Glu-Trp. The optimum pH and temperature were 7.5 and 30°C. The enzyme was stable in a pH range of 5.5–9.0 and below 40°C. Enzymatic reactions were performed by incubating the GTase with various substrates. The GTase converted acarbose into component C, maltose into trehalose, and maltooligosaccharides into maltooligosyl trehaloses. The reactions were reversible. Various acarbose analogs were tested as inhibitors against the GTase as a means to suppress component C formation. Valienamine was the most potent, with an IC₅₀ value of 2.4×10^–3 mM and showed a competitive inhibition mode.     

The headspace-GC/MS: Alternative methodology employed in the bioreduction of (4S)-(+)-carvone mediated by human skin fungus

Abstract

The development of more efficient and environmentally friendly analytical methods represents a current focus for the fine chemical industry. In particular, microscale methodologies that are free of solvents/reagents. The headspace-GC/MS (HS-GC) methodology was employed in this study as a tool for monitoring a biocatalysed reaction of (4S)-(+)-carvone using Phoma sp., a filamentous fungus from human skin. Biocatalysis provides some advantages, such as high efficiency, high degrees of regioselectivity, chemoselectivity, and enantioselectivity. In order to optimize the small scale biocatalytic reaction of the (4S)-(+)-carvone by the filamentous fungus Phoma sp. was used headspace GC/MS methodology, factorial design of experiments and the response surface methodology (RSM) was performed using the biomass of the fungus, substrate mass and pH as parameters. It was observed that for all reactions conditions tested, forming the products (1?R,4S)-dihydrocarvone and (1S,4S)-dihydrocarvone. The most influential factor was pH, with the highest conversion rate (>95%) and diastereomeric excess (d.e.) (>80%) obtained at pH 5.0. Thus, it was demonstrated that human skin Phoma sp. fungus showed significant bioreduction activity and that headspace GC/MS is an efficient approach for real-time monitoring the biocatalysed reactions.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Purification and characterization of a new L-methioninase from solid cultures of <Emphasis Type="Italic">Aspergillus flavipes</Emphasis>

L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anion-exchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8–8.0 at 35°C. The enzyme appeared to be catalytically stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol, Hg⁺, Cu²⁺, and Fe²⁺, with slight inhibition by Triton X-₁₀₀. A flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S⁻¹) followed by a relative demethiolating activity to L-homo-cysteine (Km, 12 mM and Kcat, 9.3 S⁻¹). The enzyme has two absorption maxima at 280 and 420 nm, typical of other PLP-enzymes. Apo-L-methioninase has the ability to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme.     

Purification and characterization of laccase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> and decolorization of an anthraquinone dye by the enzyme

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0. Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization.     

Effect of Nutritional Parameters on Laccase Production by the Culinary and Medicinal Mushroom, <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M _r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Overexpression,one-step purification,and characterization of a type II cholesterol oxidase from a local isolate <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. PTCC 1633

A 1.6 kb gene encoding a cholesterol oxidase (choR) from a local isolate, Rhodococcus sp. PTCC 1633 was cloned into pET23a and the highly expressed recombinant enzyme was purified from the cell lysate of IPTG-induced Escherichia coli BL21(DE3)pLysS with one-step absorption on cholesterol. The purified protein had a molecular mass of 55 kDa, isoelectric point at about pH 9.0 and absorption peaks at 280, 380 and 460 nm, indicating that the enzyme is a flavoprotein. The optimum pH and temperature for the recombinant enzyme were 7.0 and 50°C, respectively. Steady-state kinetic revealed that the cholesterol oxidase had a K _m of 32 μM. This study is the first report concerning expression and one-step purification of a gene encoding cholesterol oxidase from Rhodococcus spp. This study revealed that this enzyme is a type II cholesterol oxidase.     

Characterization of cold-active uracil-DNA glycosylase from Bacillus sp. HJ171 and its use for contamination control in PCR

In this study, the gene encoding Bacillus sp. HJ171 uracil-DNA glycosylase (Bsp HJ171 UDG) was cloned and sequenced. The Bsp HJ171 UDG gene consists of a 738-bp DNA sequence, which encodes for a protein that is 245-amino-acid residues in length. The deduced amino acid sequence of the Bsp HJ171 UDG had a high sequence similarity with other bacterial UDGs. The molecular mass of the protein derived from this amino acid sequence was 27.218 kDa. The Bsp HJ171 UDG gene was expressed under the control of a T7lac promoter in the pTYB1 plasmid in Escherichia coli BL21 (DE3). The expressed enzyme was purified in one step using the Intein Mediated Purification with an Affinity Chitin-binding Tag purification system. The optimal temperature range, pH, NaCl concentration, and KCl concentration of the purified enzyme was 20–25°C, 8.0, 25 and 25 mM, respectively. The half-life of the enzyme at 40°C and 50°C were approximately 131 and 45 s, respectively. These heat-labile characteristics enabled Bsp HJ171 UDG to control carry-over contamination in the polymerase chain reaction product (PCR) without losing the PCR product. G.A. Kim and M.S. Lee contributed equally to this work.     

Detection of a unique fibrinolytic enzyme in <Emphasis Type="Italic">Aeromonas</Emphasis> sp. JH1

A fibrinolytic enzyme was found in a Gram-negative bacterium, Aeromonas sp. JH1. SDS-PAGE and fibrinzymography showed that it was a 36 kDa, monomeric protein. Of note, the enzyme was highly specific for fibrinogen molecules and the hydrolysis rate of fibrinogen subunits was highest for α, β, and γ chains in that order. The first 15 amino acids of N-terminal sequence were X-D-A-T-G-P-G-G-N-V-X-T-G-K-Y, which was distinguishable from other fibrinolytic enzymes. The optimum pH and temperature of the enzyme were approximately 8.0 and 40°C, respectively. Therefore, these results provide a fibrinolytic enzyme with potent thrombolytic activity from the Aeromonas genus.