Thermophilic Fungus Thermomyces Lanuginosus Research Articles

Application of Thermomyces lanuginosus polygalacturonase produced in Komagataella phaffii in biomass hydrolysis and textile bioscouring

In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6 U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ˚C) with a 2.83 mg/mL and 0.063 μmol/minute for Km and Vmax, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43 mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7 mg/mL, the time for the fabric to absorb water was 19.77 seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38 seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.

Improved deinking and biobleaching efficiency of enzyme consortium from Thermomyces lanuginosus VAPS25 using genetic Algorithm-Artificial neural network based tools

The present study reports the combined enzymatic production efficiency of thermophilic fungus Thermomyces lanuginosus VAPS25 using a combinatory artificial intelligence-based tool, resulting in 2.7 IU/ml, 5.2 IU/ml, and 18.85 U/ml activity of endoglucanase, amylase, and lipase, respectively with good thermostability at 90 °C (pH 8–10). Interestingly, the metal ions viz. Cu2+ and Mg2+ increased the endoglucanase activity to 5 folds, i.e.,5.6 IU/ml compared to control. Further, the amylase and lipase activity was also enhanced by Fe2+ and Co2+ to 5.4 IU/ml and 19.57 U/ml, respectively. Additionally, the deinking efficiency was improved by 68.9%, 42.7%, and 52.8% by endoglucanase, amylase, and lipase, respectively, while the consortium increased the deinking efficiency to 72.7%. The bio-bleached paper strength parameters such as burst index, breaking length, tear index, and tensile index of sheets were significantly improved by 1.38%, 13.54%, 7.54%, and 20.88%, respectively. These enzymes at an industrial scale would help develop an economical paper recycling process.

Crystal structure of a thermophilic fungal cyanase and its implications on the catalytic mechanism for bioremediation

Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source. Strikingly, the cyanase from the thermophilic fungus Thermomyces lanuginosus (Tl-Cyn) has the highest catalytic efficiency reported among these enzymes. However, its molecular mechanism of action is not clearly understood, because currently there is no structural information available on fungal cyanases. Here we report the crystal structure of Tl-Cyn in complex with inhibitors malonate and formate at 2.2 Å resolution. The structure reveals extensive interactions at the subunit interfaces in a dimer, and a decamer is formed by a pentamer of these dimers. Our biochemical, kinetic and mutagenesis studies confirm the structural observations on the complex and provide further insights into its catalytic mechanism and inhibition. The structure has also aided the creation of a mutant enzyme with enhanced catalytic activity, and such enzymes may have the potential for biotechnological applications, including biotransformation and bioremediation. Moreover, other fungal cyanases with potentially high catalytic activity could also be predicted based on the Tl-Cyn structure, as the active site region among fungal cyanases are highly conserved.

Novel β-glucanases along with xylanase identified in Thermomyces lanuginosus secretome for enhanced saccharification of different lignocellulosics

The LC/MS-based exo-proteome analysis of thermophilic fungus Thermomyces lanuginosus showed 22.59% (40 proteins) of the total identified proteins (177) as CAZymes (carbohydrate-active enzymes). The CAZymes were primarily represented by glycosyl hydrolases (72.5%) belonging to 21 different GH families. Xylanase (GH11) was found to be the major protein (24.3%) in addition to the β-glucanase and another complex polysaccharide-degrading (carbohydrate-active enzymes) CAZymes in the secretome. FPLC-based fractionation of secretome was employed to identify proteins responsible for enhancing the catalytic efficacy of Cellic Ctec2 during hydrolysis of acid pre-treated lignocellulosics and eventually xylanase (GH11) and β-glucanases (GH64 and GH81) were identified and purified. The purified xylanase when supplemented with Cellic Ctec2 resulted in 2.05-, 1.79-, and 1.60-fold increase in the release of glucose from acid-treated bagasse at 10, 15, and 20% substrate loading rate, respectively when compared with the control. Similarly, enhanced hydrolysis of acid pre-treated corn residue and rice straw was observed upon supplementation with xylanase. Spiking benchmark cellulase with purified GH64 and GH81 also resulted in 1.18- and 1.23-fold enhanced hydrolysis of acid pre-treated sugarcane bagasse. The supplementation of xylanase (1000 units/g substrate) was found to reduce the Cellic Ctec2 loading rate from 36 mg/g substrate by 2.70-, 2.88-, and 2.57-fold required for hydrolysis of acid treated bagasse, corn residue, and rice straw, respectively, at high substrate loading rate (20%) and thus, it is an important candidate for economizing the 2G ethanol process.

High-level expression and characterization of the thermostable leucine aminopeptidase Thelap from the thermophilic fungus Thermomyces lanuginosus in Aspergillus niger and its application in soy protein hydrolysis

Leucine aminopeptidase (LAP), an exopeptidase that releases amino acid residues, especially leucine, from the N-terminus of polypeptides, is often applied to debitter protein hydrolysate in the food industry. However, there are no thermostable and high activity enzymes that can be used in the food industry. In this study, we obtained the highly active and thermostable leucine aminopeptidases screened from the thermophilic fungi Thermomyces lanuginosus, Talaromyces thermophilus, and Malbranchea cinnamomea. The activity of the recombinant leucine aminopeptidase Thelap was significantly increased to 2771.5 U/mL, as mediated by the CRISPR/Cas9 tool. The recombinant Thelap was easily purified from fermentation broth by Ni-affinity chromatography, and the specific activity of the purified Thelap was increased to 7449.6 U/mg. The recombinant Thelap showed optimal activity at pH 8.5 and 75 °C and remained above 70% of the maximum activity over a wide temperature range (30–80 °C). With regard to temperature stability, Thelap retained more than 90% activity when it was incubated at 65–75 °C for 2 h. K+ and Co2+ increased the enzyme activity of the recombinant Thelap, while Ba2+, Mn2+, Ni2+, Ca2+, Mg2+ and SDS inhibited its enzyme activity, and the inhibition capacity of Mg2+ was the weakest. Upon application in soy protein hydrolysis, Thelap could significantly increase the degree of hydrolysis and remove more hydrophobic amino acids from the N-terminal region of the polypeptide to decrease the bitterness.

Comparative biocontrol ability of chitinases from bacteria and recombinant chitinases from the thermophilic fungus Thermomyces lanuginosus

Microbial chitinases (EC 3.2.1.14) are known to hydrolyse the chitinous gut epithelium of insects and cell walls of many fungi. In this study, seven chitinases from different bacteria and fungi were produced, characterized and their biocontrol abilities against graminaceous stem borers Eldana saccharina, Chilo partellus and Sesamia calamistis were assessed. All chitinases were stable over broad ranges of pH and temperature, however, recombinant fungal chitinases were more acid-stable than the bacterial counterparts. Chitinases from the thermophilic filamentous fungi Thermomyces lanuginosus SSBP (Chit1) and from Bacillus licheniformis (Chit lic) caused 70% and 80% mortality, respectively, in second instar larvae of E.saccharina. Six of the seven partially-purified microbial chitinases inhibited Aspergillus niger, A. flavus, A. alliaceus, A. ochraceus, Fusarium verticillioides and Mucor sp. Overall, microbial chitinases show promise as biocontrol agents of fungi and stalk-boring lepidopterans.

Microbial synthesis of mammalian metabolites of spironolactone by thermophilic fungus Thermomyces lanuginosus

Mesophilic fungi are well recognized as models of mammalian drug metabolism. Thermophilic fungi remained unexplored despite having a unique mechanism of growing at higher temperatures and performing wide diverse reactions. The present investigation is directed to isolate a promising thermophilic fungal strain capable of biotransformation using spironolactone as a model drug. Two-stage fermentation protocol was followed for the process. The transformation of spironolactone was identified by HPLC and structure elucidation of the metabolites was done with the help of LC-MS/MS analysis and previous reports. A strain of Thermomyces lanuginosus isolated from decomposed banana peel waste was found to be most promising in transforming spironolactone to 4 metabolites viz.7α-thiospironolactone (M1) canrenone (M2), 7α-thiomethylspironolactone (M3) and 6β-OH-7α-thiomethylspironolactone (M4), the major mammalian metabolites reported previously.The synthesis of metabolites of spironolactone by T. lanuginosus similar to mammals clearly states that this fungus possess enzyme system similar to mammals. Hence, this fungus has the potential to use as a model organism for studying drug metabolism.

Expression of a novel recombinant cyanate hydratase (rTl-Cyn) in Pichia pastoris, characteristics and applicability in the detoxification of cyanate

A recombinant Pichia pastoris harbouring the cyanate hydratase gene (rTl-Cyn) from the thermophilic fungus Thermomyces lanuginosus SSBP yielded a high titre of extracellular cyanate hydratase (100±13UmL−1) which was ∼10-fold higher than the native fungal strain. The purified rTl-Cyn had a molecular mass of ∼20kDa on SDS-PAGE, with Km, Vmax, kcat and kcat/Km values of 0.34mM, 2857.14µmolesmg−1min−1, 2.14×104s−1 and 6.3 ×107M−1s−1, respectively. Its properties of thermostability, pH stability, and heavy metals insensitivity, make it a suitable candidate for bioremediation in extreme environments. The rTl-Cyn was able to degrade toxic cyanate completely with the liberation of ammonia, which was confirmed by FTIR analysis. This is the first report of any known cyanate hydratase that has been expressed in P. pastoris, characterized and effectively evaluated for cyanate detoxification.

Thermomyces lanuginosus STm: A source of thermostable hydrolytic enzymes for novel application in extraction of high-quality natural rubber from Taraxacum kok-saghyz (Rubber dandelion)

Hydrolytic enzymes from a newly isolated strain of the thermophilic fungus Thermomyces lanuginosus were used to extract rubber from Taraxacum kok-saghyz commonly known as rubber (or Russian or Kazak(h)) dandelion. The fungus was isolated from garden soil and identified as Thermomyces lanuginosus STm based on 18S rRNA gene sequence analysis. The isolate produced considerable amounts of extracellular hydrolytic enzymes on lignocellulosic substrates at 55°C incubated for 8days in 150mL shake flask experiments. The maximum enzyme activities on wheat straw and guayule bagasse were: xylanase (167.41; 130.1U/mg), inulinase (69.8; 34.1U/mg), cellulase (carboxymethyl cellulase) (16.7; 4.8U/mg), filter paper assay (FPase) (14.2; 5.5FPUg−1) and pectinase (7.2; 3.2U/mg), respectively. In addition, alkali-pretreated roots of Taraxacum kok-saghyz (TK), incubated with crude enzyme extracts from T. lanuginosus STm grown on guayule bagasse, subsequently yielded more natural rubber (90mg/g dry TK root) than previously established protocols, Eskew process (24mg/g) and commercial-enzyme-combination process (45mg/g). Rubber purity in the T. lanuginosus STm treatment was 71.7%, greater than the Eskew process at 37.5%. However, the crude T. lanuginosus STm enzyme treatment at 91.6% rubber purity approached the purity of the commercial-enzyme-combination process at 94.1% purity.

Cloning, expression, and molecular dynamics simulations of a xylosidase obtained from Thermomyces lanuginosus

The aim of this study was to clone, express, and characterize a β-xylosidase (Tlxyn1) from the thermophilic fungus Thermomyces lanuginosus SSBP in Pichia pastoris GS115 as well as analyze optimal activity and stability using computational and experimental methods. The enzyme was constitutively expressed using the GAP promoter and secreted into the medium due to the alpha-mating factor secretion signal present on the expression vector pBGPI. The 1276 bp gene consists of an open reading frame that does not contain introns. A 12% SDS–PAGE gel revealed a major protein band at an estimated molecular mass of 50 kDa which corresponded to zymogram analysis. The three-dimensional structure of β-xylosidase was predicted, and molecular dynamics simulations at different ranges of temperature and pH were performed in order to predict optimal activity and folding energy. The results suggested a strong conformational temperature and pH dependence. The recombinant enzyme exhibited optimal activity at pH 7 and 50°C and retained 80% activity at 50°C, pH 7 for about 45 min. This is the first report of the cloning, functional expression, and simulations study of a β-xylosidase from Thermomyces species in a fungal host.

The multi-chitinolytic enzyme system of the compost-dwelling thermophilic fungus Thermomyces lanuginosus

The recent sequencing of the Thermomyces lanuginosus SSBP genome by our group has revealed four putative family 18 chitinases. In this study, three novel chitinase genes (chit2, chit3 and chit4) and the previously-reported chit1 gene were cloned from T. lanuginosus SSBP. chit1, encoding a 44.1kDa protein, and chit2, encoding a 36.6kDa protein, were successfully expressed in Pichia pastoris. The recombinant Chit1 and Chit2 enzymes exhibited optimum activity at pH 5.0 and pH 4.0, respectively. Chit1 had optimal activity at 50°C and retained 56% of its activity at 60°C after 30min, while Chit2 was optimally active at 40°C and retained 71% of its activity at 50°C after 60min. Both enzymes produced chitobiose as the major product using different substrates. Chit2 displayed antifungal activity against Penicillium verrucosum and Aspergillus niger. These activities could be useful in the environmental degradation of chitinous wastes as well as for biotechnological applications.

Thermophilic esterase from Thermomyces lanuginosus: Molecular cloning, functional expression and biochemical characterization

A novel esterase encoding gene, tle, was cloned from the thermophilic fungus Thermomyces lanuginosus DSM 10635. The tle had an open reading frame of 945bp encoding TLE of 314 amino acids with a theoretical molecular mass of 34.5kDa. The putative catalytic triad of TLE was consisted of Ser151, His279, and Asp249. TLE was heterologously expressed in Escherichia coli in biologically active form and purified to homogeneity. Several biochemical properties of TLE were studied: Among the tested p-nitrophenol esters, TLE showed the highest hydrolytic activity with p-nitrophenyl butyrate (C4) and exhibited the maximum activity at 60°C and pH 8.5. The enzyme was stable at temperatures below 60°C and retained 53% of the maximum activity after treatment at 70°C for 60min. Esterase activity was notably enhanced by addition of Ca2+ and Ba2+, respectively. Furthermore, TLE showed high enantioselectivity (E=95) in the kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE), which produce a valuable chiral intermediate-(3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid for Pregabalin. These unique properties of the esterase indicate that TLE is a potential candidate for industrial application.

High-level soluble expression of a thermostable xylanase from thermophilic fungus Thermomyces lanuginosus in Escherichia coli via fusion with OsmY protein

A thermostable xylanase is encoded by xynA from fungus Thermomyces lanuginosus. The problem emerged from overexpression of xynA in Escherichia coli has been the formation of inclusion bodies. Here we describe the xynA was fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY. The fusion protein OsmY-xynA was expressed as almost all soluble form. The soluble expression level of fusion protein reached 98±6U/ml when cells containing pET-OsmY-xynA were expressed without IPTG induction at 37°C. The induction is probably due to auto-induction due to lactose in the medium (Studier (2005) [21]). The cells harboring pET-OsmY-xynA expressed an activity level about 24 times higher than that expressed from pET-20b-xynA. Xylanase activity was observed in the extracellular (36±1.3U/ml) and the periplasmic (42±4U/ml) when cells containing pET-OsmY-xynA were induced without IPTG addition. After the cold osmotic shock procedure followed by nickel affinity chromatography, the purified fusion protein showed a single band on SDS–PAGE gel with a molecular mass of 44kDa. The purified fusion enzyme exhibited the highest activity at 65°C and pH 6.0.

Secretory expression of a β-xylosidase gene fromThermomyces lanuginosusinEscherichia coliand characterization of its recombinant enzyme

To characterize a β-xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. A gene (designated TlXyl43) encoding β-xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017-bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β-xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6-fold to homogeneity with an estimated mass of 51·6kDa by SDS-PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a Ki value of 63mmol l(-1). In this study, a GH family 43 β-xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. This is the first report of the cloning and functional expression of a β-xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries.

Thermophilic lipase from Thermomyces lanuginosus: Gene cloning, expression and characterization

Abstract An extracellular lipase gene ln1 from thermophilic fungus Thermomyces lanuginosus HSAUP0380006 was cloned through RT-PCR and RACE amplification. Its coding sequence predicted a 292 residues protein with a 17 amino acids signal peptide. The deduced amino acids showed 78.4% similarity to another lipase lgy from T. lanuginosus while shared low similarity with other fungi lipases. Higher frequencies hydrophobic amino acids related to lipase thermal stability, such as Ala, Val, Leu and Gly were observed in this lipase (named LN). The sequence, -Gly-His-Ser-Leu-Gly-, known as a lipase-specific consensus sequence of mould, was also found in LN. High level expression for recombinant lipase was achieved in Pichia pastoris GS115 under the control of strong AOX1 promoter. It was purified to homogeneity through only one step DEAE-Sepharose anion exchange chromatography and got activity of 1328 U/ml. The molecular mass of one single band of this lipase was estimated to be 33 kDa by SDS-PAGE. The enzyme was stable at 60 °C and kept 65% enzyme activity after 30 min incubation at 70 °C. It kept half-activity after incubated for 40 min at 80 °C. The optimum pH for enzyme activity was 9.0 and the lipase was stable from pH 8.0 to 12.0. Lipase activity was enhanced by Ca2+ and inhibited by Fe2+, Zn2+, K+, and Ag+. The cell-free enzyme hydrolyzed and synthesized esters efficiently, and the synthetic efficiency even reached 81.5%. The physicochemical and catalytic properties of the lipase are extensively investigated for its potential industrial applications.

Effect of water activity on the thermal stability of Thermomyces lanuginosus xylanases for process time–temperature integration

Three strains of the thermophilic fungus Thermomyces lanuginosus were used to produce β-xylanases. The thermal stability of these xylanases at low levels of water activity was studied. Isothermal inactivation experiments were performed in the temperature range of 100–130 °C. Reduction of water activity to 0.63 and as low as 0.13 had a drastic effect on the observed D and z-values. At water activity of 0.13 the D 120 °C and z-values of the three xylanases ranged from 20.4 to 37.6 min and from 23.3 to 28.9 °C, respectively. The applicability of the developed kinetic models was tested under time–temperature profiles representative of typical thermal processes. The developed systems can be applied as time–temperature integrators (TTI) at this high thermal processing range. Calculations demonstrated that the use of a triple xylanase TTI system could provide acceptable F-values prediction for z-values lower than the achieved range.

Improvement of xylanase production by thermophilic fungus Thermomyces lanuginosus SDYKY-1 using response surface methodology

Response surface methodology (RSM) was employed to optimize medium composition for the production of a thermostable xylanase from T. lanuginosus SDYKY-1 using economical carbon and nitrogen sources (soybean meal and corncobs, respectively). Plackett-Burman (P-B) design was applied to evaluate the effects of nine variables (powdered corncobs, soybean meal, Tween-80, CaCl(2), MgSO(4)·7H(2)O, FeSO(4), KH(2)PO(4), initial pH and inoculum culture volume). Corncobs, soybean meal and FeSO(4) were found to significantly influence on the xylanase production. The concentrations of these three factors were therefore optimized using central composite design and RSM. Adjusting the concentration of corncobs to 38.7g/L, soybean meal to 17.5g/L and FeSO(4) to 0.26g/L favored maximum xylanase production. Xylanase activity of 3078U/mL was obtained after optimization, which was a 144% increase that obtained before optimization (1264U/mL).

Optimization of Xylanase Production byThermomyces lanuginosusin Solid State Fermentation

Extracellular xylanase production by the thermophilic fungus Thermomyces lanuginosus 195 in solid state fermentation (SSF) was found to be significantly affected by fermentation temperature, duration, and inoculum volume (p < or = 0.001). Optimization of these parameters corresponded to a 21.7% increase in xylanase yield. Maximum activity (2,335 U/g of wheat bran) was obtained when 10 g of wheat bran was inoculated with 10 ml of liquid culture and cultivated at 45 degrees C for 40 h. The influence of supplemental carbon and nitrogen sources (3% w/v) on xylanase production was also assessed. Wheat bran, supplemented with glucose and cellulose, facilitated 10% and 7% increases in relative activity respectively. Ammonium based salts, nitrates, and a number of organic nitrogen sources served only to reduce xylanase production (p < or = 0.005) significantly. The enhanced xylanase titers achieved in the present study emphasize the need for optimizing growth conditions for maximum enzyme production in SSF.

Effects of galactomannan as carbon source on production of α-galactosidase by Thermomyces lanuginosus: Fermentation, purification and partial characterisation

Thermophilic fungus Thermomyces lanuginosus CBS 395.62/b strain is able to grow and synthesise extracellular α-galactosidase in media containing galactomannan such as locust bean gum (LBG) or guar gum (GG). Production of extracellular α-galactosidase was enhanced from 1.2 U/mL to 4–6 U/mL meaning about 3–5 times increase by optimisation of medium composition. This enzyme was purified to homogeneity by partial precipitation with 2-propanol and different liquid chromatographical steps. The developed purification protocol yielded 22% of enzyme activity with 900 purified fold. Molecular mass of the purified α-galactosidase enzyme was estimated to be 53 kDa. Maximal catalytic activity of the enzyme was obtained in the acidic pH range between pH 4.6 and 4.8 and in the temperature range 60–66 °C. More than 95% of enzyme activity was remaining after 1-day incubation at 70 °C and on pH in the range from 4.0 to 7.0. The enzyme activity was significantly stimulated by Mg 2+, Mn 2+ and K + ions, while considerably inhibited by the presence of Ca 2+, Ag + and Hg 2+.

Production, purification and characterization of thermostable phytase from thermophilic fungus Thermomyces lanuginosus TL-7

Ten different strains of Thermomyces lanuginosus, isolated from composting soils were found to produce phytase when grown on PSM medium. The wild type strain CM was found to produce maximum amount ofphytase (4.33 units/g DW substrate). Culturing T. lanuginosus strain CM on medium containing wheat bran and optimizing other culture conditions (carbon source, media type, nitrogen source, level of nitrogen, temperature, pH, inoculum age, inoculum level and moisture), increased the phytase yield to 13.26 units/g substrate. This culture was further subjected to UV mutagenesis for developing phytase hyperproducing mutants. The mutant (TL-7) showed 2.29-fold increase in phytase activity as compared to the parental strain. Employing Box-Behnken factor factorial design of response surface methodology resulted in optimized phytase production (32.19 units/g of substrate) by mutant TL-7. A simple two-step purification (40.75-folds) ofphytase from mutant TL-7 was achieved by anion exchange and gel filtration chromatography. The purified phytase (approximately 54 kDa) was characterized to be optimally active at pH 5.0 and temperature 70 degrees C, though the enzyme showed approximately 70% activity over a wide pH and temperature range (2.0-10.0 and 30-90 degrees C, respectively). The phytase showed broad substrate specificity with activity against sodium phytate, ADP and riboflavin phosphate. The phytase from T. lanuginosus was thermoacidstable as it showed up to 70% residual activity after exposure to 70 degrees C at pH 3.0 for 120 min. The enzyme showed Km 4.55 microM and Vmax 0.833 microM/min/mg against sodium phytate as substrate.