Purification and characterization of an N-acetylglucosaminidase produced by a Trichoderma harzianum strain which controls Crinipellis perniciosa.

Isolate 1051 of Trichoderma harzianum, a mycoparasitic fungus, was found to impair development of the phytopathogen, Crinipellis perniciosa, in the field. This Trichoderma strain growing in liquid medium containing chitin produced substantial amounts of chitinases. The N-acetylglucosaminidase present in the culture-supernatant was purified to homogeneity by gel filtration and hydrophobic interaction chromatography, as demonstrated by SDS-PAGE analysis. The enzyme had a molecular mass of 36 kDa and hydrolyzed the synthetic substrate rho-nitrophenyl-N-acetylglucosaminide (rhoNGlcNAc) with Michaelis-Menten kinetics. Maximal activities were determined at pH 4.0 and a temperature range of 50-60 degrees C. Km and Vmax values for rhoNGlcNAc hydrolysis were 8.06 micromoles ml(-1) and 3.36 micromoles ml(-1) min(-1), respectively, at pH 6.0 and 37 degrees C. The enzyme was very sensitive to Fe3+, Mn2+ and Co2+ ions, but less sensitive to Zn2+, Al3+, Cu2+ and Ca2+. Glucose at a final concentration of 1 mM inhibited 65% of the original activity of the purified enzyme. Determination of the product (reducing sugar) of hydrolysis of C. perniciosa mycelium and scanning electron microscopic analysis revealed that the N-acetylglucosaminidase hydrolyses the C. perniciosa cell wall.

Characterization of alpha-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides.

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. Enzymic hydrolysis of the RO is accomplished by alpha-galactosidase. While the content of RO decreases during seed germination, the activity of alpha-galactosidase increases substantially. Two alpha-galactosidases were isolated from germinating seeds by partition in an aqueous two-phase system followed by ion-exchange and affinity chromatography. One of the enzyme preparations (P1) showed a single protein with M(r) of 33 kDa, and the second (P2) had two proteins with M(r) of 31 and 33 kDa. Maximal activities against the synthetic substrate rho-nitrophenyl-alpha-D-galactopyranoside (rhoNPGal) were detected at pH 5.0-5.5 and 45-50 degrees C. Both enzymes were fairly stable at 40 degrees C, but lost most of their activities after 30 min at 50 degrees C. The K(m) values for hydrolysis of rhoNPGal by the P1 and P2 enzymes were 1.55 and 0.76 mM, respectively. The K(m) values determined for hydrolysis of raffinose and melibiose by the P2 enzyme were 5.53 and 5.34 mM, respectively and galactose was a competitive inhibitor (K(i)=0.65 mM). To different extents, both enzymes were sensitive to inhibition by galactose, melibiose, CuSO(4), and SDS. Sucrose and beta-mercaptoethanol showed discrete inhibitory effects on both enzymes.

Characterization of an amylase produced by a Trichoderma harzianum isolate with antagonistic activity against Crinipellis perniciosa, the causal agent of witches' broom of cocoa.

An isolate of Trichoderma harzianum showing antagonistic activity against Crinipellis perniciosa, the causal agent of the witches' broom disease of cocoa, produces substantial amounts of hydrolytic enzymes. An amylase purified from isolate 1051 had a molecular mass of about 68.7 kDa. Maximal activity against soluble starch was determined at pH 4.0 and 60 degrees C. The K(m) and V(max) values were 3.5 mg ml(-1) and 1.67 mg min(-1) of reducing sugar. The end products were mostly malto-oligosaccharides. The enzyme also hydrolyzed glycogen, amylopectin, maltotriose, and maltotetraose, but not pullulan or cellobiose. Maltose was only barely hydrolyzed. The purified amylase exerted a discrete hydrolytic effect on the C. perniciosa cell wall in vitro as observed by scanning electron microscopic analysis. While Fe(3+), Al(3+), Zn(2+), and Cu(2+) were effective in inhibiting the purified amylase, Mn(2+) considerably enhanced the activity. Ca(2+), Mg(2+), and Co(2+) showed no substantial effect on enzyme activity.

Synthesis of a Trichoderma chitinase which affects the Sclerotium rolfsii and Rhizoctonia solani cell walls.

A Trichoderma sp. isolate, hereafter called T6, produces a 46-kDa endochitinase (CHIT 46) which had been shown to drastically affect in vitro the cell walls of the phytopathogens Sclerotium rolfsii and Rhizoctonia solani. We attempted to gain insight into its properties. The CHIT 46 N-terminal amino acid sequence shares a very high homology with other fungal chitinases. Western blot analysis using polyclonal antibodies anti-CHIT 46 revealed that this enzyme is immunologically distinct from other proteins produced by the same Trichoderma isolate T6, but is immunologically identical with proteins having equivalent molar mass, probably chitinases, produced by other Trichoderma spp. isolates. In addition, the antibodies revealed also that a substantial amount of this enzyme is secreted into the culture medium 2 d after the Trichoderma isolate T6 comes into contact with chitin.

Keratinolytic activity of Aspergillus fumigatus fresenius.

Aspergillus fumigatus can utilize chicken feather keratin as its sole carbon and nitrogen source. Because enzymatic conversion of native keratin into readily usable products is of economic interest, this fungus was studied for its capacity to produce and secrete keratin-hydrolyzing proteinases. Substantial keratin-azure hydrolyzing activity was present in the culture fluid of keratin-containing media. Considerably lower activity was present in cultures containing glucose and nitrate as the carbon and nitrogen sources, or keratin plus glucose and nitrate. Secretion of keratin-hydrolyzing activity in A. fumigatus was induced by keratin but repressed by low-molecular-weight carbon and nitrogen sources. The amount of keratinolytic enzyme present in the culture fluid was dependent on the initial pH of the culture medium. The crude enzyme also hydrolyzed native keratin and casein in vitro. Hydrolysis was optimal at pH 9 and 45 degrees C. The crude enzyme was remarkably thermostable. At 70 degrees C, it retained about 90% of its original activity for 1.5 h. The obtained results indicated that the A. fumigatus keratinolytic enzyme may be suitable for enzymatic improvement of feather meal.

Purification and characterization of a truncated Bacillus subtilis alpha-amylase produced by Escherichia coli.

A Bacillus subtilis amylase gene was inserted into a plasmid which was transferred to Escherichia coli. During cloning, a 3' region encoding 171 carboxy-terminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The transformed cells produced substantial amylolytic activity. The active protein was purified to apparent homogeneity. Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of the B. subtilis complete alpha-amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with a Km of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50 degrees C, and the purified enzyme was stable at temperatures up to 50 degrees C. While Hg2+, Fe3+ and Al+3 were effective in inhibiting the truncated enzyme, Mn2+ and Co2+ considerably enhanced the activity.

Purification and Characterization of a Glucoamylase from Humicola grisea.

A thermostable extracellular glucoamylase from the thermophilic fungus Humicola grisea was purified to homogeneity. Its molecular mass and isoelectric point were 74 kDa and 8.4, respectively. The enzyme contained 5% carbohydrate, showed maximal activities at pH 6.0 and 60(deg)C, and was stable at 55(deg)C and pH 6.0 for 2 h. The K(infm) of soluble starch hydrolysis at 50(deg)C and pH 6.0 was 0.14 mg/ml. The purified enzyme was remarkably insensitive to glucose.

The cellulosome: the exocellular organelle of Clostridium.

The cellulolytic enzyme complex of the anaerobic thermophile Clostridium thermocellum is reviewed. This complex, called the cellulosome, is cell associated and has a mass of from 2 x 10(6) to 6.5 x 10(6) Daltons. It consists of from 14 to 26 different polypeptides. Cellulosomes form larger complexes, polycellulosomes, with masses from 50 x 10(6) to 80 x 10(6) Daltons. The cellulosome efficiently hydrolyzes crystalline cellulose whereas individual polypeptides alone or in mixtures do not. Many of the polypeptides are catalytically active and can be characterized as endoglucanases, xylanases, and cellodextrinases. Several of the polypeptides have been sequenced including the largest subunit, CipA, that is a glycoprotein with a mass of 210 kDa. CipA has a cellulose-binding domain and nine internal repeated sequences postulated to bind eight catalytic subunits and a special peptide (ORF3p). The ORF3p anchors the CipA to the cell surface. CipA can be characterized as a scaffold holding the catalytic subunits that line up with the cellulose fiber. This arrangement allows a multiple cutting of the cellulose glucan chain. A similar system has been observed for other cellulosome-like complexes, notably Clostridium cellulovorans.

Amylolytic activity of Humicola sp.

The thermophilic and cellulolytic fungus Humicola sp. secretes amylase in the liquid culture medium. This activity is induced by starch, maltose and cellobiose. Glucose impairs accumulation of amylolytic activity in the culture medium. The enzyme hydrolyzes starch, maltose and pullulan to glucose as the end-product.

Incorporation of ammonium in amino acids by Trypanosoma cruzi.

Ammonium ions were incorporated into L-glutamate and alpha-ketoglutarate in epimastigote forms of Trypanosoma cruzi through the following enzymatic systems: NADPH and NADH-dependent glutamate dehydrogenase, NADPH-dependent glutamate synthase, L-glutamine synthetase and NADH-dependent glutamate synthase in order of decreasing specific activity (mumoles of product formed/min/mg protein). The pH optima and Km's for the glutamate dehydrogenase system were determined. Disc electrophoresis showed the presence of cathodic bands of GDH activity, which were highly dependent on NADP+.

Purine metabolism in trypanosomatids.

Purine nucleotide biosynthesis was studied in culture forms of Trypanosoma cruzi strain Y, Crithidia deanei (a reduviid trypanosomatid with an endosymbiote) and an aposymbiotic strain of C. deanei (obtained by curing C. deanei with chloramphenicol). Trypanosoma cruzi was found to synthesize purine nucleotides only fring incorporated into both adenine and guanine nucleotides. Similar results were obtained with guanine, indicating that this flagellate has a system for the interconversion of purine nucleotides. Crithidia deanei was able to synthesize purine and pyrimidine nucleotides from glycine ("de novo" pathway) and purine nucleotides from adenine and guanine ("salvage" pathway). Adenine was incorporated into both adenine and guanine nucleotides, while guanine was incorporated into guanine nucleotides only, indicating the presence of a metabolic block at the level of GMP reductase. The aposymbiotic C. deanei strain was unable to utilize glycine for the synthesis of purine nucleotides, although glycine was utilized for synthesizing pyrimidine nucleotides. These results suggest that the endosymbiote is implicated in the de novo purine nucleotide pathway of the C. deanei-endosymbiote complex. The incorporation of adenine and guanine by aposymbiotic C. deanei strain followed a pattern similar to that observed for C. deanei.