Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Thermomyces lanuginosus ATCC 46882

Bennett, Neil A.; Ryan, James W.; Biely, Peter; Vršanská, Mária; Kremnický, Lubomı́r; Macris, Basil J.; Kekos, Dimitris; Christakopoulos, Paul; Katapodis, Petros; Claeyssens, Marc; Nerinckx, Wim; Ntauma, Patricia; Bhat, M. K.

doi:10.1016/s0008-6215(97)10076-3

Cited by 79 publications

(67 citation statements)

References 17 publications

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“…The recombinant and native xylanases showed similar thermostability and were as stable at high temperatures as the xylanases secreted by other T. lanuginosus strains (1,32). Surprisingly, in the culture medium, both enzymes were less thermostable than in the purified form (Fig.…”

Section: Discussionmentioning

confidence: 78%

Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris

Damaso

Almeida

Kurtenbach

et al. 2003

Appl Environ Microbiol

101

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Highly efficient production of a Thermomyces lanuginosus IOC-4145 ␤-1,4-xylanase was achieved in Pichia pastoris under the control of the AOX1 promoter. P. pastoris colonies expressing recombinant xylanase were selected by enzymatic activity plate assay, and their ability to secrete high levels of the enzyme was evaluated in small-scale cultures. Furthermore, an optimization of enzyme production was carried out with a 2 3 factorial design. The influence of initial cell density, methanol, and yeast nitrogen base concentration was evaluated, and initial cell density was found to be the most important parameter. A time course profile of recombinant xylanase production in 1-liter flasks with the optimized conditions was performed and 148 mg of xylanase per liter was achieved. Native and recombinant xylanases were purified by gel filtration and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, matrix-assisted laser desorption ionization-time of flight-mass spectrometry and physicochemical behavior. Three recombinant protein species of 21.9, 22.1, and 22.3 kDa were detected in the mass spectrum due to variability in the amino terminus. The optimum temperature, thermostability, and circular dichroic spectra of the recombinant and native xylanases were identical. For both enzymes, the optimum temperature was 75°C, and they retained 60% of their original activity after 80 min at 70°C or 40 min at 80°C. The high level of fully active recombinant xylanase obtained in P. pastoris makes this expression system attractive for fermentor growth and industrial applications.

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Section: Discussionmentioning

confidence: 78%

Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris

Damaso

Almeida

Kurtenbach

et al. 2003

Appl Environ Microbiol

101

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“…A variety of materials have been used for induction of xylanases: pure xylan (98,142,163,164,205) and xylan-rich natural substrates, such as sawdust (274), corn cob (26,99,205), wheat bran (272), sugar beet pulp (253), and sugarcane bagasse (57,199). Paper of inferior quality was an excellent carbon source and inducer for xylanase in Thermoascus aurantiacus (139), Humicola lanuginosa (13,15), and Paecilomyces varioti (144).…”

Section: Xylanasementioning

confidence: 99%

Thermophilic Fungi: Their Physiology and Enzymes

Maheshwari

Bharadwaj

Bhat

2000

Microbiol Mol Biol Rev

Self Cite

658

380

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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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“…These data were closed to optimal temperature and optimal pH of T. lanuginosus xylanases produced by strain DSM 10635, CAU44, CBS 288.54, SSBP, ATCC 46882, DSM 5826, and RT9 [7][8][9][14][15][16][17] . The thermostability of pure xylanases was analysed at 70 °C in 50 mM phosphate buffer (pH 6.0).…”

Section: Thermal Stability and K M Values Of Pure Xylanasesmentioning

confidence: 94%

Characterization of the thermostability of xylanase produced by new isolates of Thermomyces lanuginosus

Khucharoenphaisan¹,

Tokuyama²,

Kitpreechavanich³

2008

ScienceAsia

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ABSTRACT:Characterization of the thermostability of purified enzymes of low and high thermostable xylanase produced by new isolates of T. lanuginosus THKU-9 and THKU-49 were performed. Half-life at 70 °C of the purified xylanases from T. lanuginosus THKU-9 and THKU-49, in 50 mM phosphate buffer (pH 6.0) was 178 and 336 min, respectively. These enzymes were unstable at pH 5.0 and completely lost their activity after incubation at 70 °C for 30 min. The xylanase produced by THKU-9 retained 87% and 30% activity in 50 mM sodium phosphate buffer (pH 7.0) after 1080 min incubation at 60 °C and 70 °C, respectively, whereas xylanase produced by THKU-49 retained full activity and 41% activity, respectively. The enzymes were more stable in phosphate buffer than in citrate buffer. When the buffer concentration increased, the half-life of the enzymes decreased significantly. Amino acid sequence analysis of low thermostable T. lanuginosus THKU-9 xylanase and high thermostable T. lanuginosus THKU-49 xylanase showed that high thermostable xylanase had a single substitution (V96G), which is a small hydrophobic amino acid of β sheet (B5) of the protein located on the outer surface of the enzyme structure.

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Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Thermomyces lanuginosus ATCC 46882

Cited by 79 publications

References 17 publications

Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris

Optimized Expression of a Thermostable Xylanase from Thermomyces lanuginosus in Pichia pastoris

Thermophilic Fungi: Their Physiology and Enzymes

Characterization of the thermostability of xylanase produced by new isolates of Thermomyces lanuginosus

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