Purification and biochemical characterization of Î²-xylosidase from<i>Humicola grisea</i>var. thermoidea

Almeida, Elzira Maria de; Polizeli, Maria de Lourdes Teixeira de Moraes; Terenzi, Héctor Francisco; Jorge, João Atı́lio

doi:10.1111/j.1574-6968.1995.tb07715.x

Cited by 44 publications

(29 citation statements)

References 15 publications

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“…Furthermore, the presence of cycloheximide inhibited the synthesis of b-D-xylosidase, suggesting that the enzyme produced in the presence of xylan required de novo protein synthesis. b-D-Xylosidase synthesis is also affected by catabolic repression in the thermophilic fungi H. grisea [1], Aspergillus phoenicis [29] and Aspergillus sydowii [10].…”

Section: Resultsmentioning

confidence: 99%

“…Most of the purified b-D-xylosidases exhibit molecular masses above 100 kDa [33], although some exhibit lower molecular masses [1,7,15,25,33]. In addition, dimeric and monomeric forms of b-D-xylosidases are widespread among the enzymes purified from filamentous fungi [1,15,29,34]. The carbohydrate content of the purified b-D-xylosidase was estimated to be 12%, similar to that reported for other b-D-xylosidases purified from fungi, which are also glycoproteins [4,13].…”

Section: Purification and Molecular Properties Of Mycelial B-d-xylosimentioning

confidence: 99%

“…Molecular sieving of the purified enzyme in Bio-Sil Sec-400 produced an apparent molecular mass of 38 kDa, which was in good agreement with the value found using SDS-PAGE, suggesting that the enzyme was a monomer. Most of the purified b-D-xylosidases exhibit molecular masses above 100 kDa [33], although some exhibit lower molecular masses [1,7,15,25,33]. In addition, dimeric and monomeric forms of b-D-xylosidases are widespread among the enzymes purified from filamentous fungi [1,15,29,34].…”

Section: Purification and Molecular Properties Of Mycelial B-d-xylosimentioning

confidence: 99%

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Purification and biochemical properties of a thermostable xylose-tolerant β-D-xylosidase from Scytalidium thermophilum

Zanoelo

Polizeli

Terenzi

et al. 2004

J IND MICROBIOL BIOTECHNOL

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The thermophilic fungus Scytalidium thermophilum produced large amounts of periplasmic beta- D-xylosidase activity when grown on xylan as carbon source. The presence of glucose in the fresh culture medium drastically reduced the level of beta- D-xylosidase activity, while cycloheximide prevented induction of the enzyme by xylan. The mycelial beta-xylosidase induced by xylan was purified using a procedure that included heating at 50 degrees C, ammonium sulfate fractioning (30-75%), and chromatography on Sephadex G-100 and DEAE-Sephadex A-50. The purified beta- D-xylosidase is a monomer with an estimated molecular mass of 45 kDa (SDS-PAGE) or 38 kDa (gel filtration). The enzyme is a neutral protein (pI 7.1), with a carbohydrate content of 12% and optima of temperature and pH of 60 degrees C and 5.0, respectively. beta- D-Xylosidase activity is strongly stimulated and protected against heat inactivation by calcium ions. In the absence of substrate, the enzyme is stable for 1 h at 60 degrees C and has half-lives of 11 and 30 min at 65 degrees C in the absence or presence of calcium, respectively. The purified beta- D-xylosidase hydrolyzed p-nitrophenol-beta- D-xylopyranoside and p-nitrophenol-beta- D-glucopyranoside, exhibiting apparent K(m) and V(max) values of 1.3 mM, 88 micromol min(-1) protein(-1) and 0.5 mM, 20 micromol min(-1) protein(-1), respectively. The purified enzyme hydrolyzed xylobiose, xylotriose, and xylotetraose, and is therefore a true beta- D-xylosidase. Enzyme activity was completely insensitive to xylose, which inhibits most beta-xylosidases, at concentrations up to 200 mM. Its thermal stability and high xylose tolerance qualify this enzyme for industrial applications. The high tolerance of S. thermophilum beta-xylosidase to xylose inhibition is a positive characteristic that distinguishes this enzyme from all others described in the literature.

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Section: Resultsmentioning

confidence: 99%

Section: Purification and Molecular Properties Of Mycelial B-d-xylosimentioning

confidence: 99%

Section: Purification and Molecular Properties Of Mycelial B-d-xylosimentioning

confidence: 99%

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Purification and biochemical properties of a thermostable xylose-tolerant β-D-xylosidase from Scytalidium thermophilum

Zanoelo

Polizeli

Terenzi

et al. 2004

J IND MICROBIOL BIOTECHNOL

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“…sulfurea also produced an extracellular xylosidase (162)(163)(164), but in H. grisea var. thermoidea (12,179) and Talaromyces emersonii (253) the xylosidase was periplasmic. Interestingly, the best xylanase-producing strains of T. lanuginosus secreted small amounts of xylan-debranching enzymes and did not produce ␤-mannan-and arabinan-degrading enzymes, whereas the low-xylanase-producing strains exhibited a higher degree of xylan utilization and also the ability to produce a mannan-degrading enzyme system (204).…”

Section: Xylanasementioning

confidence: 99%

Thermophilic Fungi: Their Physiology and Enzymes

Maheshwari

Bharadwaj

Bhat

2000

Microbiol Mol Biol Rev

634

364

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SUMMARY Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20°C and a maximum temperature of growth extending up to 60 to 62°C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45°C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62°C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.

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“…The thermophilic fungus H. grisea var. thermoidea has attracted attention as a rich source of xylanolytic enzymes (1,13,20). Earlier studies have demonstrated that H. grisea var.…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a new Xylanase from Humicola grisea var. Thermoidea

Lucena-Neto

Ferreira-Filho

2004

Braz. J. Microbiol.

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The thermophilic fungus Humicola grisea var. thermoidea secretes extracellular xylanase when grown on solid and in liquid media containing wheat bran and banana plant residue as substrates, respectively. At 55ºC, xylanase from the culture filtrate of H. grisea var. thermoidea grown on banana stalk retained 50% of its activity after 28 h of incubation. A xylanase (X2) was isolated from solid state cultures with wheat bran as the carbon source. It was purified to apparent homogeneity by ultrafiltration followed by ion-exchange and hydrophobic interaction chromatography on DEAE-Sepharose and Phenyl-Sepharose resins, respectively. The enzyme had an apparent molecular weight of 29 kDa, as determined by SDS-PAGE. The purified enzyme was most active at pH and temperature ranges of 4.5-6.5 and 55-60ºC, respectively. In addition, X2 showed thermostability at 60ºC with a half-life of approx. 5.5 h. The apparent Km values, using soluble and insoluble arabinoxylans as substrates, were 10.87 and 11.20 mg/ml, respectively.

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Purification and biochemical characterization of Î²-xylosidase fromHumicola griseavar. thermoidea

Cited by 44 publications

References 15 publications

Purification and biochemical properties of a thermostable xylose-tolerant β-D-xylosidase from Scytalidium thermophilum

Purification and biochemical properties of a thermostable xylose-tolerant β-D-xylosidase from Scytalidium thermophilum

Thermophilic Fungi: Their Physiology and Enzymes

Purification and characterization of a new Xylanase from Humicola grisea var. Thermoidea

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