Carbon,nitrogen and pH regulate the production and activity of a polygalacturonase isozyme produced by Penicillium expansum

The influence of carbon, nitrogen and pH on polygalacturonase (PG) activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized lyophilised fruit tissue and the highest PG activity occurred in apple pectin medium. Nitrogen source influenced PG activity and was highest with ammonia while the greatest mycelial mass was supported by glutamate or glutamine. PG activity and mycelial mass peaked 5 five days after inoculation as polyuronide content decreased and the pH and ammonium levels increased in apple pectin medium. A single active PG isozyme with an isoelectric point of ～7.6 was produced in apple pectin medium and a partial cDNA clone was obtained that was most homologous to the pggII gene from Penicillium. griseoroseum. The results from this study indicate that P. expansum can modulate the activity of PG in response to nutrient sources and ambient pH through signalling pathways that modulate nutrient acquisition, uptake and metabolism.     

Tomato Fruit Polygalacturonase Isozyme 1 (Characterization of the [beta] Subunit and Its State of Assembly in Vivo)

Polygalacturonase isozyme 1 (PG1) is a heterodimer comprising a catalytic and noncatalytic or [beta] subunit, whereas polygalacturonase isozyme 2 (PG2) comprises only the catalytic subunit. To assess the state of assembly of PG1 in vivo, both subunits were purified to homogeneity and used to study assembly of the heterodimer. PG1 could be reconstituted in vitro from purified [beta] subunit and purified PG2 under a wide range of salt and pH conditions, and PG1 reconstituted in vitro was indistinguishable from PG1 isolated from tomato (Lycopersicon esculentum) fruit. Specific antibodies indicated that the [beta] subunit was present in fruit of all developmental stages, but absent in vegetative tissue. The state of assembly of PG1 in vivo was tested based on the differential thermal stability of PG1 and PG2 by heating segments of ripe fruit pericarp tissue. Temperatures well below those required to inactivate PG1 in vitro caused the loss of activity of both PG1 and PG2, suggesting that only heat-labile PG2 is present in vivo. In addition, when extracts of ripe fruit were rigorously maintained and analyzed at 4[deg]C, PG1 was absent or barely detectable. These results are consistent with the hypothesis that PG1 can assemble spontaneously and is essentially absent in intact tomato fruit but forms artifactually from PG2 and the [beta] subunit during the extraction of tomato fruit tissue when low temperatures are not rigorously maintained.     

Presence of rhodanese in the cytosolic fraction of the fruit bat (Eidolon helvum) liver

Rhodanese was isolated and purified from the cytosolic fraction of liver tissue homogenate of the fruit bat, Eidolon helvum, by using ammonium sulphate precipitation and CM-Sephadex C-50 ion exchange chromatography. The specific activity was increased 130-fold with a 53% recovery. The K(m) values for KCN and Na(2)S(2)O(3) as substrates were 13.5 +/- 2.2mM and 19.5 +/- 0.7 mM, respectively. The apparent molecular weight was estimated by gel filtration on a Sephadex G-100 column to be 36,000 Da. The optimal activity was found at a high pH (pH 9.0) and the temperature optimum was 35 degrees C. An Arrhenius plot of the heat stability data consisted of two linear segments with a break occurring at 35 degrees C. The apparent activation energy values from these slopes were 11.5 kcal/mol and 76.6 kcal/mol. Inhibition studies on the enzyme with a number of cations showed that Mg(2+), Mn(2+), Ca(2+), and Co(2+) did not affect the activity of the enzyme, but Hg(2+) and Ba(2+) inhibited the enzyme.     

Purification and characterization of pectate lyase from banana (Musa acuminata) fruits

Pectate lyase (PEL) has been purified by hydrophobic, cation exchange and size exclusion column chromatographies from ripe banana fruit. The purified enzyme has specific activity of 680 +/- 50 pkat mg protein(-1). The molecular mass of the enzyme is 43 kDa by SDS-PAGE. The pI of the enzyme is 8 with optimum activity at pH 8.5. Analysis of the reaction products by paper and anion exchange chromatographies reveal that the enzyme releases several oligomers of unsaturated galacturonane from polygalacturonate. The K(m) values of the enzyme for polygalacturonate and citrus pectin (7.2% methylation) are 0.40 +/- 0.04 and 0.77 +/- 0.08 g l(-1), respectively. PEL is sensitive to inhibition by different phenolic compounds, thiols, reducing agents, iodoacetate and N-bromosuccinimide. The enzyme has a requirement for Ca(2+) ions. However, Mg(2+) and Mn(2+) can substitute equally well. Additive effect on the enzyme activity was observed when any two metal ions (out of Mg(2+), Ca(2+) and Mn(2+)) are present together. The banana PEL is a enzyme requiring Mg(2+), in addition to Ca(2+), for exhibiting maximum activity.     

酸性α-淀粉酶的分离纯化与酶学性质研究

纯化了枯草芽胞杆菌xm-1菌株酸性α-淀粉酶,并对其酶学性质进行了研究。通过硫酸铵沉淀和Sephadex G-75凝胶层析将酸性α-淀粉酶粗酶液纯化了32.5倍,活力回收率为10.0%。酶性质测定结果表明,该酸性α-淀粉酶分子量约为60kD,最适反应温度为45℃、最适作用pH5.0,该酶在pH3.4-6.0下稳定,高温耐受性差。Cu2+、Zn2+、EDTA对酶有不同程度的抑制作用,Ca2+和Mn2+对酶具有较强的激活作用。     

Purification and characterization of the laccase secreted by the white rot fungus Perenniporia tephropora and its role in the decolourization of synthetic dyes

AIMS: To characterize the white rot fungus Perenniporia tephropora with respect to its laccase and to test its ability to decolourize synthetic dyes. METHODS AND RESULTS: Under the culture conditions utilized, P. tephropora produced one laccase isozyme, which was purified to electrophoretic homogeneity by ammonium sulfate precipitation, size-exclusion chromatography and anion-exchange chromatography. The protein was monomeric with a molecular mass of 63 kDa (SDS-PAGE) and had an isoelectric point of 3.3. The N-terminal amino acid sequence was SIGPVADLTVTNANI and the highest similarity value was found to the laccase from Lentinus tigrinus (86.6%). The optimum pH of the enzyme varied and was substrate dependent. It was 4.0 and 5.0 for 2,6-dimethoxyphenol (DMP) and 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS), respectively. Under standard assay conditions, K(m) values of the enzyme were 7.3 and 0.4 mmol l(-1) towards DMP and ABTS, respectively. The laccase was inhibited by NaN(3), EDTA and p-coumarate but not by SDS and NaBr. Laccase was stable in the presence of some metal ions such as Cu(2+), Co(2+), Ca(2+), Cd(2+), Mg(2+), Mn(2+), Mo(2+), Ni(2+), Li(+) and Al(3+). The crude enzyme as well as the purified laccase was able to decolourize dyes from the textile industries, including remazol brilliant blue R, neolane blue and neolane pink. However, several other dyes were partially or not decolourized. In the presence of 1-hydroxybenzotriazole as mediator, only the decolourization of neolane yellow was achieved, while the decolourization of most of the dyes was just slightly improved. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the purification and the characterization of the laccase from the white rot fungus P. tephropora. The high levels of laccase secreted by this fungal strain as well as its stability suggest that it could be a useful tool for environmental applications.     

Heterologous expression of polygalacturonase genes isolated from Galactomyces citri-aurantii IJ-1 in Pichia pastoris

ABSTACT: The objective of this work was to isolate the polygalacturonase genes of Galactomyces citri-aurantii IJ-1 harvested from rotten citrus peels and to heterologously express these genes in Pichia pastoris. Two polygalacturonase (PG) genes from G. citri-aurantii IJ-1 were obtained and tentatively named PG1 and PG2. The genes were cloned into pPICZαC, and expressed in Pichia pastoris strain GS115 with a native signal peptide or the α-factor secretion signal peptide of Saccharomyces cerevisiae. All of the recombinant proteins were successfully secreted into the culture media and confirmed as a single band with a molecular weight of 35 to 38 kDa by SDS-PAGE. The specific enzyme activities of recombinant PG1 and PG2 purified by His-tag affinity resin were 4,749 and 6,719 U/mg, respectively, with an optimal pH and temperature of pH 4.0 and 50°C. The Michaelis-Menten kinetic constants for PG1 and PG2, K (m), were confirmed to be 0.94 and 0.84 mM, respectively. In the presence of Mn(2+), the activity of PG1 and PG2 were increased to 160.8 and 146.4% of normal levels, respectively. In contrast, Cu(2+) and Fe(3+) acted as strong inhibitors to the PGs.     

Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli

The SAP6 gene (without signal sequence) encoding Metschnikowia reukaufii acid protease was amplified by PCR and fused to the expression vector pET-24a(+). The carboxy-terminal 6x His-tagged recombinant acid protease (rSAP6) was expressed from pET-24a(+)SAP6-6His in Escherichia coli BL21 (DE3) and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified rSAP6 was 54kDa. The optimal temperature and pH of the purified rSAP6 were 40 degrees C and 3.4, respectively. The enzyme was stable below 45 degrees C and between pH 2.6 and 5.0. The results show that Mn(2+) had an activating effect on the enzyme, while Cu(2+), Mg(2+), Zn(2+) and Ag(+) acted as inhibitors of the enzyme. However, Ca(2+) had no effect on the enzyme activity. The purified rSAP6 was characterized as an aspartic protease as it was inhibited by aspartic protease-specific inhibitors, such as pepstatin. It was also found that the purified rSAP6 had milk-clotting activity.     

1株高效降解纤维素的槲寄生内生真菌研究

利用刚果红-CMC平板染色法、DNS法从槲寄生内生真菌中筛选获得1株高效纤维素酶产生菌H-3-3,通过分析其菌落形态特征和18S rDNA序列的同源性,将菌株H-3-3鉴定为拟茎点霉属Phom opsissp.;对其酶学性质研究得到：槲寄生内生真菌H-3-3中CMC酶、β-葡糖苷酶、滤纸酶这3种组分酶最适温度均为50℃,β-葡糖苷酶活和滤纸酶活最适pH值均为4.8,CMC酶最适pH值为4.4;3种酶在30～45℃,pH4.0～8.0稳定性较强。金属离子Mn^2＋、Co^2＋、Mg^2＋和Ca^2＋对3组分酶都有不同程度的促进作用,其中以Co^2＋效果最佳;而Ba^2＋、Zn^2＋、Cu^2＋对3组分酶分均表现出明显的抑制作用。     

Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.     

Purification and properties of α-d-mannosidase from rat epididymis

1. alpha-d-Mannosidase from rat epididymis was purified 300-fold. beta-N-Acetyl-glucosaminidase and beta-galactosidase were removed from the preparation by treatment with pyridine. Zn(2+) was added during the purification to stabilize the alpha-mannosidase. 2. Mammalian alpha-mannosidase is most stable at pH6. At lower pH values it undergoes reversible spontaneous inactivation. The enzyme is also subject to irreversible inactivation, which is delayed by the addition of albumin. 3. Reversible inactivation of alpha-mannosidase is accelerated by EDTA and reversed or prevented by Zn(2+). Other cations, such as Co(2+), Cd(2+) and Cu(2+), accelerate inactivation and the action of a toxic cation can be prevented by Zn(2+) or by EDTA in suitable concentration. 4. The enzyme is stabilized by substrate and neither Zn(2+), EDTA nor a toxic cation has more than a small effect in the assay of an untreated preparation. The addition of Zn(2+) is necessary, however, for a constant rate of hydrolysis during prolonged incubation of the enzyme with substrate. In an EDTA-treated preparation, Zn(2+) reactivates the enzyme during the assay. 5. Evidence is presented that alpha-mannosidase is a dissociable Zn(2+)-protein complex, in which Zn(2+) is essential for enzyme activity.     

Screening, purification, and characterization of an extracellular prolyl oligopeptidase from Coprinopsis clastophylla

Culture filtrates of 22 mushrooms were screened for extracellular prolyl oligopeptidase activity. Four strains with relatively high enzyme activity were all from inky cap mushrooms. The production of Coprinopsis clastophylla prolyl oligopeptidase was associated with the growth of the fungus and the enzyme was not released by cell lysis. The enzyme was purified 285-fold to a specific activity of 52.05 U/mg. It was purified to a single band on a native polyacrylamide gel. However, the enzyme separated into three bands on a sodium dodecyl sulfate-polyacrylamide gel with mobility corresponding to molecular weights of approximately 84, 60, and 26 kDa. The results of tandem mass spectrometric analysis revealed that the 60 kDa protein was likely a degradation product of the 84 kDa protein. The isoelectric point of the purified enzyme was 5.2. The purified enzyme had an optimal pH and temperature of 8.0 and 37°C, respectively. Diisopropyl fluorophosphate (DFP), p-chloromercuribenzoaic acid (PCMB), Hg(2+), and Cu(2+) strongly inhibited C. clastophylla prolyl oligopeptidase. This enzyme is a serine peptidase and one or more cysteine residues of the enzyme are close to the active site.     

Purification and Characterization of a Maltotetraose-Forming Alkaline (alpha)-Amylase from an Alkalophilic Bacillus Strain, GM8901

An alkalophilic bacterium, Bacillus sp. strain GM8901, grown at pH 10.5 and 50(deg)C, produced five alkaline amylases in culture broth. At an early stage of the bacterial growth, amylase I (Amyl I) was produced initially and then, as cultivation progressed, four alkaline amylases, Amyl II, Amyl III, Amyl IV, and Amyl V, were produced from proteolytic degradation of Amyl I. A serine protease present in the culture medium was believed to be involved in Amyl I degradation. We purified Amyl I from the culture supernatant by ammonium sulfate precipitation, heparin-Sepharose CL-6B column chromatography, phenyl-Toyopearl column chromatography, and Mono Q HR5/5 high-performance liquid chromatography. The molecular weight of Amyl I was estimated to be about 97,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amyl I had an extremely high optimal pH of 11.0 to 12.0 and was stable in a broad pH range of 6.0 to 13.0. Amyl I had an optimal temperature of 60(deg)C and was stable up to 50(deg)C. Thermostability was increased in the presence of Ca(sup2+) and soluble starch. The enzyme required metal ions such as Ca(sup2+), Mg(sup2+), Cu(sup2+), Co(sup2+), Ag(sup+), Zn(sup2+), and Fe(sup2+) for its enzyme activity and was inhibited by 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. According to the mode of action of Amyl I on starch, Amyl I was classified as an (alpha)- and exo-amylase. Amyl I produced maltotetraose predominantly from starch via intermediates such as maltohexaose and maltopentaose.     

Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis

The production of polygalacturonase (PGase) by Sporotrichum thermophile Apinis in stirred submerged fermentation (SmF) was high in comparison with that in static conditions. Yeast extract (0.25%) and citrus pectin (2%) at pH 7.0 and 45 degrees C supported a high enzyme production in flasks agitated at 200 rpm. An overall 1.75-fold enhancement in PGase production was achieved as a result of optimization. The enzyme was optimally active at pH 7.0 and 55 degrees C, and exhibited t(1/2) of 4 h at 65 degrees C. The Km and Vmax values of the enzyme (for pectin) were 0.416 mg ml(-1) and 0.52 micromol mg(-1)min(-1), respectively. The PGase activity was stimulated by Mn(2+) and Fe(2+), but inhibited strongly by Mg(2+), and slightly by Tween 80 and Triton X-100. Among the additives tested, beta-mercaptoethanol exerted a strong inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. An increase in the yield of banana, grape and apple juices was recorded due to the treatment of fruit pulps with the mixture of enzymes (pectinase, xylanases and cellulase) of S. thermophile as compared to that with only pectinase. The yield of fruit juices did not increase with enhanced titre of cellulase in the enzyme mixture.     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k (cat)/K (m) values at pH 7.0 and 40°C. Maximum proteolytic activity (59?U mL(-1)) was achieved after 48?hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca(2+) and Mg(2+), and inhibited by Cu(2+), Zn(2+), Cd(2+), and Fe(2+.).     

Purification and properties of a glucuronan lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472).

A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50 degrees C. Zn(2+), Cu(2+), and Hg(2+) (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified from S. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.     

蜗牛酶中一种β-葡萄糖苷酶的纯化及酶学性质的研究

通过DEAE-Sepharose离子交换层析和Sephadex G-100凝胶过滤层析的联用从中华白玉蜗牛消化酶中分离出1种具有人参皂苷Rb_1水解活性的β-葡萄糖苷酶.纯化后该酶在SDS-PAGE上呈单一蛋白质条带.反应最适pH为5.6,最适温度是80 ℃.pH稳定范围很广,在pH为4.0～11.0的溶液中和温度60 ℃以下保持长时间稳定状态,是一个耐碱和中等耐热的糖苷酶.Na~+、K~+、Li~+、Ca~(2+)、Mg~(2+)、EDTA、DTT和SDS不影响该酶活性,而Cu~(2+)、Ag~+和Fe~(3+)对该酶则具有明显的抑制作用.pNPG为底物的动力学参数Km和Vmax分别为0.182 mmol/L和0.189 μmol/(min·mg).     

Enhancement of endothelial cell migration by constitutively active LPA(1)-expressing tumor cells

2,3-Dihydroxybiphenyl-1,2-dioxygenase plays an important role in the degradation of polychlorinated biphenyls. The gene (BsbphCI) encoding a 2,3-DHBP dioxygenase from Bacillus sp. JF8 is 960 bp. We synthesized a 960 bp BsbphCI gene encoding a 2,3-DHBP dioxygenase derived from Bacillus sp. JF8 and expressed it in Escherichiacoli. The recombinant protein was about 36 kDa, confirmed by SDS-PAGE. The concentration of the purified protein was about 1.8 mg/mL. With 2,3-DHBP as a substrate, the optimal temperature for enzyme activity at pH 8.5 was 50 °C. The optimal pH for the 2,3-DHBP dioxygenase was 8.5. The enzyme retained 33% activity after heating at 60 °C for 60 min. We found that Cu(2+), K(+), Zn(2+), Mg(2+), Ni(2+), Co(2+), and Cd(2+) activated the enzyme. However, Ca(2+), Fe(2+), Li(+), and Cr(3+) inhibited it. Enzyme activity was reduced by exposure to H(2)O(2), SDS, and KI. The results of HPLC indicated that the transgenic E. coli strain with the BsbphCI gene degraded 2,3-DHBP more quickly than the wild type strain.     

Expression and purification of organic solvent stable lipase from soil metagenomic library

Gene for organic solvent stable lipase was overexpressed from soil metagenomic library. The clone with maximum activity was selected, and enzyme was purified by gel-permeation chromatography with a molecular mass of approx. 40 kDa. The deduced aminoacid sequence indicated that the protein belongs to the lipase family I.3 and containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca(2+) binding sites. The enzyme expressed maximum activity at 30 °C and pH 7.0 and found to be stable in pH and temperature ranging from 6.0-9.0 and 20-60 °C, respectively. Furthermore, the enzyme was found highly resistant to many organic solvents, especially isopropanol, DMSO, methanol, xylene and hexane. The enzyme showed enhanced activity in the presence of divalent cations (Mg(2+), Mn(2+), Ca(2+), Hg(2+), Cu(2+)), whereas the presence of trivalent cation (Fe(3+)) inhibited the activity.     

Characterisation of a novel white laccase from the deuteromycete fungus Myrothecium verrucaria NF-05 and its decolourisation of dyes

A novel 'white' laccase was purified from the deuteromycete fungus, Myrothecium verrucaria NF-05, which was a high laccase-producing strain (40.2 U·ml(-1) on the thirteenth day during fermentation). SDS-PAGE and native-PAGE revealed a single band with laccase activity corresponding to a molecular weight of approximately 66 kDa. The enzyme had three copper and one iron atoms per protein molecule determined by ICP-AES. Furthermore, both UV/visible and EPR spectroscopy remained silence, indicating the enzyme a novel laccase with new metal compositions of active centre and spectral properties. The N-terminal amino acid sequence of the purified protein was APQISPQYPM. Together with MALDI-TOF analysis, the protein revealed a high homology of the protein with that from reported M. verrucaria. The highest activity was detected at pH 4.0 and at 30°C. The enzyme activity was significantly enhanced by Na(+), Mn(2+), Cu(2+) and Zn(2+) while inhibited by DTT, NaN(3) and halogen anions. The kinetic constant (Km) showed the enzyme was more affinitive to ABTS than other tested aromatic substrates. Twelve structurally different dyes could be effectively decolourised by the laccase within 10 min. The high production of the strain and novel properties of the laccase suggested its potential for biotechnological applications.