Purification and partial characterization of serine protease from thermostable alkalophilic <Emphasis Type="Italic">Bacillus laterosporus</Emphasis>-AK1

An extracellular thermostable alkaline protease isolated from Bacillus laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced by the metal ions Ca²⁺ and Mg²⁺ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain completely when used with Wheel detergent.     

Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068

Aims: To prepare, purify and characterize an extracellular enzyme from Myxococcus fulvus ANSM068, designated as myxobacteria aflatoxin degradation enzyme (MADE), which possesses degradation activity against aflatoxin B₁ (AFB₁), G₁ (AFG₁) and M₁ (AFM₁) in solution. Methods and Results: The culture supernatant of strain M. fulvus demonstrated high degradation ability against AFB₁ (71·89%), AFG₁ (68·13%) and AFM₁ (63·82%) after 48 h of incubation. An enzyme was purified from the supernatant of M. fulvus using ethanol precipitation and chromatography on DEAE‐Sepharose and Superdex 75. An overall 166‐fold purification of the enzyme with a recovery of 57% and a final specific activity of 569·44 × 10³ U mg^?1 was obtained using the present purification protocol. The apparent molecular mass of MADE was estimated to be 32 kDa by SDS‐PAGE. AFG₁ and AFM₁ were significantly degraded, by 96·96 and 95·80%, respectively, when treated with pure MADE (100 U ml^?1) produced by strain ANSM068. MADE exhibited the largest amount of activity at 35°C and pH 6·0, with Mg²⁺ ions greatly promoting and Zn²⁺ strongly inhibiting MADE activity. Conclusions: An aflatoxin degradation enzyme from bacterial isolates can effectively remove aflatoxin B₁, G₁ and M₁ in solution. Significance and Impact of the Study: The high activity and wide temperature and pH range of MADE for the degradation of aflatoxin have promising applications in control of mycotoxins during food and feed processing.     

Involvement of two cytosolic enzymes and a novel intermediate, 5'-oxoaverantin, in the pathway from 5'-hydroxyaverantin to averufin in aflatoxin biosynthesis

During aflatoxin biosynthesis, 5'-hydroxyaverantin (HAVN) is converted to averufin (AVR). Although we had previously suggested that this occurs in one enzymatic step, we demonstrate here that this conversion is composed of two enzymatic steps by showing that the two enzyme activities in the cytosol fraction of Aspergillus parasiticus were clearly separated by Mono Q column chromatography. An enzyme, HAVN dehydrogenase, catalyzes the first reaction from HAVN to a novel intermediate, another new enzyme catalyzes the next reaction from the intermediate to AVR, and the intermediate is a novel substance, 5'-oxoaverantin (OAVN), which was determined by physicochemical methods. We also purified both of the enzymes, HAVN dehydrogenase and OAVN cyclase, from the cytosol fraction of A. parasiticus by using ammonium sulfate fractionation and successive chromatographic steps. The HAVN dehydrogenase is a homodimer composed of 28-kDa subunits, and it requires NAD, but not NADP, as a cofactor for its activity. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme coincides with a protein deduced from the adhA gene in the aflatoxin gene cluster of A. parasiticus. Also, the OAVN cyclase enzyme is a homodimer composed of 79-kDa subunits which does not require any cofactor for its activity. Further characterizations of both enzymes were performed.     

Utilization of diets amended with yeast and amino acids for the control of aflatoxicosis

Although the effects of aflatoxin on animal performance have been well established in previous studies, there are few studies reporting on the relationship between aflatoxin and Saccharomyces cerevisiae. The ability of Saccharomyces cerevisiae to minimize aflatoxicosis was evaluated. An aflatoxin-free diet and six contaminated diets (400 μg kg⁻¹ aflatoxin) were formulated with five diets containing the viable yeast (Y1026 or Y904). A 28-day bioassay using 21-day-old and 60-g body weight Wistar rats was conducted. The results showed that there were no significant (P > 0.05) differences for: food consumption; daily weight gain; food conversion, and enzyme activity. Hepatic tissues from the aflatoxin control group suffered from hepatotoxicity, cellular disorganization, and hepatocyte necrosis. The inclusion of yeast or yeast and amino acids (methionine and cysteine) reduced the toxicity. A. S. Baptista received fellowship from FAPESP.     

The appearance of an enzyme activity catalysing the conversion of norsolorinic acid to averantin in Aspergillus parasiticus cultures

The activity of the enzyme responsible for the conversion of norsolorinic acid to averantin was studied in two strains of Aspergillus parasiticus. Cell-free extracts of the enzyme were purified from different aged mycelia and little activity was found prior to 24 hours after inoculation but this quickly reached a maximum at 48 hours and declined thereafter. Both strains of A. parasiticus, one in aflatoxin producing strain, the other a versicolorin A accumulating mutant, showed this trend. It was concluded that the enzyme responsible for this conversion was a secondary metabolic enzyme and was distinct from alcohol and mannitol dehydrogenases.     

Degradation of cellulose by the major endoglucanase produced from the brown-rot fungus Fomitopsis pinicola

An endoglucanase that is able to degrade both crystalline and amorphous cellulose was purified from the culture filtrates of the brown-rot fungus Fomitopsis pinicola grown on cellulose. An apparent molecular weight of the purified enzyme was approximately 32 kDa by SDS-PAGE analysis. The enzyme was purified 11-fold with a specific activity of 944 U/mg protein against CMC. The partial amino acid sequences of the purified endoglucanase had high homology with endo-beta-1,4-glucanase of glycosyl hydrolase family 5 from other fungi. The K(m) and K(cat)values for CMC were 12 mg CMC/ml and 670/s, respectively. The purified EG hydrolyzed both cellotetraose (G4) and cellopentaose (G5), but did not degrade either cellobiose (G2) or cellotriose (G3).     

Reduction of aflatoxin B1 in chicken feed by using Saccharomyces cerevisiae, Rhizopus oligosporus and their combination

Aflatoxin B1 is a toxigenic and carcinogenic compound produced by Aspergillus flavus and Aspergillus parasiticus. An approach to prevent aflatoxin contamination in feed was carried out by using Saccharomyces cerevisiae (Sc) and Rhizopus oligosporus (Ro). Aspergillus flavus was cultured together with Sc, Ro and their combination (ScRo) in chicken feed. The aflatoxin B1 content was observed at day 0, 5, 10 and 15. The result showed that aflatoxin B1 contaminations in feed were reduced by Sc, Ro and ScRo addition. The highest reduction of aflatoxin B1 content was shown at day 5 for all treatments with Sc, Ro and ScRo. The best activity of reducing aflatoxin B1 was shown by Ro. Although the ability of reducing aflatoxin B1 of Sc, Ro or ScRo was not significantly different, Sc or Ro gave the better result than ScRo and they are better used individually.     

Purification of rat-liver microsomal UDP-glucuronyltransferase. Separation of two enzyme forms inducible by 3-methylcholanthrene or phenobarbital.

Glucuronidation reactions catalysed by rat liver microsomal UDP-glucuronyltransferase are differentially inducible by 3-methylcholanthrene and phenobarbital. To elucidate the molecular basis of this functional heterogeneity the enzyme was purified from livers of rats pretreated with the inducing agents. Using cholate solubilization, chromatography on Bio-Gel A-1.5m and on DEAE-cellulose in the presence of the nonionic detergent Brij 58, two enzyme forms could be separated. Both forms were subsequently purified to apparent homogeneity by affinity chromatography on UDP-hexanolamine Sepharose 4B, 3-Methylcholanthrene-inducible enzyme activity towards 1-naphthol, 4-nitrophenol, 3-hydroxybenzo(a)pyrene and N-hydroxy-2-naphthylamine copurified with one enzyme form (enzyme 1). In contrast phenobarbital-inducible enzyme activity towards morphine, chloramphenicol and 4-hydroxybiphenyl was associated with the other enzyme fraction (enzyme 2). Sodium dodecylsulfate/polyacrylamide gels showed similar molecular weights of 54000 for enzyme 1 and 56000 for enzyme 2. The results suggest the presence of at least two forms of UDP-glucuronyltransferase in rat liver. Factors affecting enzyme activity in purified and membrane-bound states are discussed.     

Involvement of Two Cytosolic Enzymes and a Novel Intermediate, 5′-Oxoaverantin, in the Pathway from 5′-Hydroxyaverantin to Averufin in Aflatoxin Biosynthesis

During aflatoxin biosynthesis, 5′-hydroxyaverantin (HAVN) is converted to averufin (AVR). Although we had previously suggested that this occurs in one enzymatic step, we demonstrate here that this conversion is composed of two enzymatic steps by showing that the two enzyme activities in the cytosol fraction of Aspergillus parasiticus were clearly separated by Mono Q column chromatography. An enzyme, HAVN dehydrogenase, catalyzes the first reaction from HAVN to a novel intermediate, another new enzyme catalyzes the next reaction from the intermediate to AVR, and the intermediate is a novel substance, 5′-oxoaverantin (OAVN), which was determined by physicochemical methods. We also purified both of the enzymes, HAVN dehydrogenase and OAVN cyclase, from the cytosol fraction of A. parasiticus by using ammonium sulfate fractionation and successive chromatographic steps. The HAVN dehydrogenase is a homodimer composed of 28-kDa subunits, and it requires NAD, but not NADP, as a cofactor for its activity. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of tryptic peptides of the purified HAVN dehydrogenase revealed that this enzyme coincides with a protein deduced from the adhA gene in the aflatoxin gene cluster of A. parasiticus. Also, the OAVN cyclase enzyme is a homodimer composed of 79-kDa subunits which does not require any cofactor for its activity. Further characterizations of both enzymes were performed.     

Effects of lipidic carbon sources on the extracellular lipolytic activity of a newly isolated strain of Bacillus subtilis

An isolate exhibiting high extracellular lipolytic activity was identified as Bacillus subtilis by 16S rRNA gene sequence analysis. The enzyme activity of the isolate was improved by using different concentrations of lipidic carbon sources such as vegetable oils, fatty acids and triglycerides. Lipolytic activity was assayed spectrophotometrically using p-nitrophenyl palmitate. One percent (v/v) of sesame oil provided the highest activity with 80 and 98% enhancements with respect to 1% (v/v) concentrations of linoleic acid and triolein as the favored fatty acid and triglyceride, respectively. Glucose presented a repressive effect on lipase production. Lipase secreted by B. subtilis was partially purified by ultrafiltration and anion exchange chromatography; and the purified enzyme was tested for its residual activity in the presence of EDTA, SDS, Triton X-100, Tween 20, Tween 80 and protease. The present work reports, for the first time, that the lipolytic activity of a B. subtilis strain can be improved by using inexpensive vegetable oils; and also that B. subtilis lipase is suitable for use in detergents.     

Aflatoxin B1 metabolism by 3-methylcholanthrene-induced hamster hepatic cytochrome P-450s

We have studied the activation of aflatoxin B1 by hamster liver microsomes and purified hamster cytochrome P-450 isozymes using a umu mutagen test. The hamster liver microsomes or S-9 fractions were much more active than rat liver microsomes or S-9 fractions in the activation of umu gene expression by aflatoxin B1 metabolites. 3-Methyl-cholanthrene treatment increased aflatoxin B1 activation by hamster liver microsomes. Two major 3-methylcholanthrene-inducible cytochrome P-450 isozymes, P-450 MC1 (IIA) and P-450 MC4 (IA2), were purified from 3-methylcholanthrene-treated hamster liver microsomes, and the metabolism of aflatoxin B1 by these two cytochromes was studied. In the reconstituted enzyme system, both P-450 MC1 and P-450 MC4 were highly active in the activation of aflatoxin B1, and antibodies against these P-450s specifically inhibited these activities. Antibody against P-450 MC1 inhibited the activation of aflatoxin B1 by 20% in the presence of 3-methyl-cholanthrene-treated hamster liver microsomes. In contrast, antibody against P-450 MC4 stimulated the activity by 175%. These results indicated that hamster P-450 MC1 might convert aflatoxin B1 to more toxic metabolite(s), whereas P-450 MC4 might convert aflatoxin B1 to less toxic metabolite(s), than aflatoxin B1 in liver microsomes. The metabolite(s) produced by both hamster cytochrome P-450 MC1 and MC4 were genotoxic in the umu mutagen test.     

Enzymes in atlatoxin B1 biosynthesis: Strategies for identifying pertinent genes

Recent work on the aflatoxin biosynthetic pathway is reviewed, with special emphasis on the enzymes of the late stages of the pathway involving conversion of sterigmatocystin (ST) to aflatoxin B₁ (AFB₁) through an O-methylsterigmatocystin intermediate. Two enzyme activities were discovered in subcellular fractions of cell-free extracts of a mutant strain ofAspergillus parasiticus (SRRC 163): 1)A post-microsomal methyltransferase (MT) catalyzed conversion of ST to OMST, and 2) a microsomal-associated activity (oxido-reductase) converted OMST to AFB₁. The 168 KDa, anionic MT was purified to homogeneity and characterized (two subunits, 110 KDa and 58 KDa). Preliminary evidence indicated the presence of a cationic isozyme of the MT in mycelial extracts. The oxido-reductase has been partially purified and characterized. Polyclonal antibodies were prepared to the anionic MT and the enzyme's amino acid composition determined. A cDNA library has been constructed from mRNA isolated fromAspergillus parasiticus mycelia during the onset of AFB₁ biosynthesis for the purpose of identifying the genes responsible for aflatoxin biosynthesis.     

Partial Purification and Characterization of a 37 kDa Extracellular Proteinase from Trichophyton vanbreuseghemii

An exocellular proteinase synthesized by the geophilic dermatophyte Trichophyton vanbreuseghemii has been purified and characterized. The fungus obtained from soil in Iran was cultivated in modified Czapek–Dox liquid medium containing 0.1% bacteriological peptone and 1% glucose as the nitrogen and carbon sources. Partial purification of the proteinase was accomplished by (NH₄)₂SO₄ precipitation, followed by ion exchange chromatography. Analysis of the enzyme by SDS-PAGE revealed a single polypeptide chain with an apparent molecular mass of 37 kDa. Proteinase activity was optimum at pH 8, but remained high in the range of pH 7–11. Moreover, the partially purified enzyme presented a keratinolytic activity as evidenced by the keratin azure test. The inhibition profile and the good activity of the enzyme towards the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide suggested that it belonged to the chymotrypsin/subtilisin group of serine proteinases. The keratinolytic properties of T. vanbreuseghemii suggest that this fungus may be an alternative for the recycling of industrial keratinic wastes.     

Purification and characterization of an extracellular alpha-amylase from Bacillus subtilis AX20

A Bacillus subtilis AX20 from soil with ability to produce extracellular alpha-amylases was isolated. The characterization of microorganism was performed by biochemical tests as well as 16S rDNA sequencing. Maximum amylase activity (38 U/ml) was obtained at stationery phase when the culture was grown at 37 degrees C. The enzyme was purified to homogeneity with an overall recovery of 24.2% and specific activity of 4133 U/mg. The native protein showed a molecular mass of 149 kDa composed of a homodimer of 78 kDa polypeptide by SDS-PAGE. The optimum pH and temperature of the amylase were 6 and 55 degrees C, respectively. The enzyme was inhibited by Hg(2+), Ag(2+), and Cu(2+) and it did not show an obligate requirement of metal ions. The enzyme was not inhibited by EDTA or EGTA, suggesting that this enzyme is not a metalloenzyme. The end products of corn starch and soluble starch were glucose (70-75%) and maltose (20-25%). Rapid reduction of blue value and the end products suggest an endo mode of action for the amylase. The purified amylase shows interesting properties useful for industrial applications.     

An anti-peptide antibody targeted to a specific region of rat cytochrome P-450IA2 inhibits enzyme activity.

An anti-peptide antibody has been produced which binds to and specifically inhibits the activity of cytochrome P-450IA2 in rat hepatic microsomes. This was achieved by raising an antibody against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn), the sequence of which occurs in cytochrome P-450IA2 at positions 290-296. The selection of this region of cytochrome P-450IA2 was based on several criteria, including prediction of surface and loop areas, identification of variable regions between cytochromes P-450IA2 and P-450IA1, and consideration of a site on cytochrome P-450IA1 where chemical modification has been shown to cause substantial enzyme inactivation. The specificity of antibody binding was determined by enzyme-linked immunosorbent assay and by immunoblotting using hepatic microsomal preparations and purified cytochrome P-450 isoenzymes. This showed that the antibody binds specifically to rat and mouse cytochrome P-450IA2 and to no other cytochrome P-450, as was predicted from the amino acid sequences of the peptide and the cytochromes P-450. The effect of the antibody upon enzyme activity was studied in hepatic microsomes from rats treated with 3-methylcholanthrene. The antibody was shown to inhibit specifically the activity of reactions catalysed by cytochrome P-450IA2 (phenacetin O-de-ethylase and 2-acetylaminofluorene activation), but had no effect on aryl hydrocarbon hydroxylase activity, which is catalysed by cytochrome P-450IA1, or on aflatoxin B1 activation.     

Peroxidase activity in mycelia of Aspergillus parasiticus that degrade aflatoxin

Summary Extracts of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 were assayed for peroxidase activity and for their ability to degrade aflatoxin. A positive relationship existed between rates of aflatoxin degradation and amount of peroxidase activity in these extracts. The supernatant fluid of homogenates from mycelia grown under similar conditions varied in amount of peroxidase present (170 to 2215 U/g). The fraction obtained, by precipitation with (NH₄)₂SO₄ at 45% of saturation, from six different homogenates prepared from three mycelial mats contained peroxidase and degraded aflatoxin. Rates of aflatoxin degradation by and amounts of peroxidase activity in each sample obtained from mycelial homogenates with (NH₄)₂SO₄ at 60% of saturation varied; however, when increased amounts of peroxidase activity were present, more aflatoxin was degraded and vice versa. Relatively little peroxidase activity was present in the fraction obtained with (NH₄)₂SO₄ at 30% of saturation and little or no aflatoxin was degraded by this precipitate. Trends for degradation of aflatoxin when more or less peroxidase activity was present in mycelial preparations suggest that the enzyme may be involved in degradation of aflatoxin by the Aspergillus.     

Protein kinase CK1 from Trypanosoma cruzi

A protein kinase activity, which uses casein as a substrate, has been purified to homogeneity from the epimastigote stage of Trypanosoma cruzi, by sequential chromatography on Q sepharose, heparin sepharose, phenyl sepharose, and alpha-casein agarose. An apparent molecular weight of 36,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography and sedimentation analyses demonstrated that the purified native enzyme is a monomer with a sedimentation coefficient of 2.9 S. The hydrodynamic parameters indicated that the shape of the protein is globular with a frictional ratio f/f(o) = 1.36 and a Stokes radius of 27.7 A. When two selective peptide substrates for protein kinases CK1 and CK2 were used (RRKDLHDDEEDEAM. SITA and RRRADDSDDDDD, respectively), the purified kinase was shown to predominantly phosphorylate the CK1-specific peptide. Additionally, the enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inactivator of CK1s from mammals. Based on these results, we concluded that the purified kinase corresponds to a parasite CK1.     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Positive correlation exists between glutathione S-transferase activity and aflatoxin formation in Aspergillus flavus.

The presence of glutathione (GSH) S-transferase activity, using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate, has been established in the cytosolic fraction of the toxigenic (aflatoxin producing) and nontoxigenic strains of Aspergillus flavus. Significant differences in the GSH S-transferase activity were observed between the toxigenic and non-toxigenic strains. A positive correlation has been demonstrated for the first time between aflatoxin formation and a biochemical parameter, namely GSH S-transferase activity. The evidence in support of A. flavus GSH S-transferase induction by endogenous aflatoxins is as follows: (i) the age-related production of aflatoxin follows the same pattern as the cytosolic GSH S-transferase activity profile; (ii) significantly higher enzyme activity was associated with mycelia of a toxigenic strain grown in medium supporting high aflatoxin production (sucrose-low-salts medium) while the enzyme activity was low in medium producing less aflatoxin (glucose-ammonium nitrate medium). The GSH S-transferase activity of the non-toxigenic strain was hardly affected by a change in the medium as it produces no aflatoxins; and (iii) the toxigenic strain demonstrated significantly higher apparent Vmax. with no change in Km as compared with the non-toxigenic strain. This indicates that the enzyme induction by endogenous aflatoxins is similar to the action of phenobarbitol and other inducing drugs (Kaplowitz et al., 1975).     

A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1

An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was purified by Ni-chelation affinity chromatography. It showed a single band with a molecular mass of 18kDa in SDS-PAGE. The purified enzyme exhibited both phospholipase A(2) and esterase activities with the optimal catalytic temperature at 90 degrees C. The enzyme activity was Ca(2+)-independent. Kinetic analysis revealed its Km, k cat, and Vm for the p-nitrophenyl propionate substrate were 103microM, 39s(-1), and 249micromol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100 degrees C was about 1h.