Different strategies to kill the host presented by Metarhizium anisopliae and Beauveria bassiana.

Studies conducted over the last decades have shown the potential of entomopathogenic fungi for the biocontrol of some insect pests. Entomopathogenic fungi infect their host through the cuticle, so they do not need to be ingested to be effective. These fungi also secrete secondary metabolites and proteins that are toxic to insect pests. In this context, we analyzed the pathogenicity of Metarhizium anisopliae (Metschn.) strains IBCB 384 and IBCB 425 and Beauveria bassiana (Bals.-Criv.) Vuill. strains E 1764 and E 3158 against Galleria mellonella (Linn.) larvae, during pre-invasion and post-invasion phases. The results showed M. anisopliae, especially strain IBCB 384, was most virulent in the pre-invasion phase against G. mellonella, whereas B. bassiana, especially strain E 1764, was most virulent in the post-invasion phase. During in vivo development and in the production of toxic serum, B. bassiana E 3158 was the most virulent. Different fungal growth (or toxin) strategies were observed for studied strains. Metarhizium anisopliae IBCB 425 prioritizes the growth strategy, whereas strain IBCB 384 and B. bassiana strains E 1764 and E 3158 have a toxic strategy. All strains have pathogenicity against G. mellonella, indicating their possible use for biocontrol.

Secretome Analysis of Metarhizium anisopliae Under Submerged Conditions Using Bombyx mori Chrysalis to Induce Expression of Virulence-Related Proteins.

The entomopathogenic fungus Metarhizium anisopliae is used to control insect pests. This species is specialized for the secretion of an enzymatic complex consisting of proteases, lipases, and chitinases related to pathogenicity and virulence. In this context, the secretomes of strains IBCB 167 and IBCB 384 of M. anisopliae var. anisopliae, grown under submerged fermentation in the presence of chrysalis as an inducer, were analyzed. Analysis of two-dimensional gels showed qualitative and quantitative differences between secreted proteins in both isolates. Around 102 protein spots were analyzed, and 76 % of the corresponding proteins identified by mass spectrometry were grouped into different classes (hydrolases, oxidases, reductases, isomerases, kinases, WSC domains, and hypothetical proteins). Thirty-three per cent of all the proteins analyzed were found to be common in both strains. Several virulence-related proteins were identified as proteases and mannosidases. Endo-N-acetyl-ß-D-glucosaminidase expression was observed to be 10.14-fold higher for strain IBCB 384 than for strain IBCB 167, which may be an important contributor to the high virulence of IBCB 384 in Diatraea ssaccharalis. These results are important for elucidation of the host-pathogen relationship and the differences in virulence observed between the two strains.

Characterization of the co-purified invertase and ß-glucosidase of a multifunctional extract from Aspergillus terreus.

The filamentous fungus Aspergillus terreus secretes both invertase and ß-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a ß-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0-6.0 and 55-65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0-10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²âº (161 %), Co²âº (68 %) and Mg²âº (61 %) and was inhibited by Al³âº, Ag⁺, Fe²âº and Fe³âº. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K(M) = 22 mM). However, in the presence of Mn²âº, the apparent affinity and V(max) for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V(max) was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and ß-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.

Aspergillus labruscus ITAL 22.223 xylanase - immobilization and application for the obtainment of corncob xylan targeting xylitol production.

Corncob is an agro-residue rich in lignocellulosic material that can be used for the xylitol production, through its enzymatic conversion obtaining fermentable sugars and their subsequent fermentation. In light of the above, this study targeted the immobilization of Aspergillus labruscus xylanase and the use of the derivative to hydrolyze the corncob xylan for the obtainment of xylose, and its subsequent use for the production of xylitol. The extracellular xylanase was immobilized using different supports (sodium alginate, DEAE-Cellulose, DEAE-Sephadex and CM-Sephadex). Among all supports used, the best results were obtained with the DEAE-Cellulose derivative showing an efficiency of immobilization of 97-99%, yield of 93-95% and recovered activity of 81-100%. The sodium alginate derivative showed 3 cycles of reuse, with drop in activity of about 65% in the 3rd cycle using both CaCl2 and MnCl2 as crosslinkers. The best enzymatic activity for the DEAE-Cellulose derivative was observed at 55ºC and pH 5.0. This derivative presented reuse of 10 cycles using commercial xylan as substrate, and 4 cycles using corncob xylan. This derivative was used in an enzymatic reactor to hydrolyze corncob xylan, obtaining 2.7 mg/mL of xylose after 48 h of operation under optimal condition of temperature and pH. The xylose obtained from the corncob was fermented by Candida tropicalis for 96 h with consumption of 60%. The HPLC analyses indicated a production of 1.02 mg/mL of xylitol with 48 h of fermentation. In conclusion, this is the first report on the immobilization of the A. labrucus xylanase as an alternative for the obtainment of xylose from corncob xylan, and the subsequent production of xylitol.

Characterization of a tannase from Emericella nidulans immobilized on ionic and covalent supports for propyl gallate synthesis.

The extracellular tannase from Emericela nidulans was immobilized on different ionic and covalent supports. The derivatives obtained using DEAE-Sepharose and Q-Sepharose were thermally stable from 60 to 75 °C, with a half life (t50) >24 h at 80 °C at pH 5.0. The glyoxyl-agarose and amino-glyoxyl derivatives showed a thermal stability which was lower than that observed for ionic supports. However, when the stability to pH was considered, the derivatives obtained from covalent supports were more stable than those obtained from ionic supports. DEAE-Sepharose and Q-Sepharose derivatives as well as the free enzyme were stable in 30 and 50 % (v/v) 1-propanol. The CNBr-agarose derivative catalyzed complete tannic acid hydrolysis, whereas the Q-Sepharose derivative catalyzed the transesterification reaction to produce propyl gallate (88 % recovery), which is an important antioxidant.

Extracellular ß-fructofuranosidase from Fusarium graminearum: stability of the spray-dried enzyme in the presence of different carbohydrates.

Microbial enzymes have been used for various biotechnological applications; however, enzyme stabilization remains a challenge for industries and needs to be considered. This study describes the effects of spray-drying conditions on the activity and stability of ß-fructofuranosidase from Fusarium graminearum. The extracellular enzyme ß-fructofuranosidase was spray dried in the presence of stabilizers, including starch (Capsul) (SC), microcrystalline cellulose (MC), trehalose (TR), lactose (LC) and ß-cyclodextrin (CD). In the presence of TR (2% w/v), the enzymatic activity was fully retained. After 1 year of storage, 74% of the enzymatic activity was maintained with the CD stabilizer (10% w/v). The residual activity was maintained as high as 80% for 1 h at 70°C when MC, SC and CD (5% w/v) stabilizers were used. Spray drying with carbohydrates was effective in stabilizing the F. graminearum ß-fructofuranosidase, improved enzymatic properties compared to the soluble enzyme and demonstrated a potential use in future biotechnology applications.

Production of a xylose-stimulated ß-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation.

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of ß-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 ± 411.2 U g(-1), while ß-glucosidase production was increased about 2.6-fold, reaching 20.7 ± 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis ß-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for ß-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The ß-glucosidase maintained about 95 % of its activity after 26 h in water at 55 °C, with half-lives of 15.7 h at 60 °C and 5.1 h at 65 °C. The presence of xylose during heat treatment at 65 °C protected ß-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 °C. Xylose stimulated ß-glucosidase activity up to 1.7-fold, at 200 mmol L(-1). The notable features of both xylanase and ß-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues.

The filamentous fungus Paecylomices variotii was able to produce high levels of cell extract and extracellular invertases when grown under submerged fermentation (SbmF) and solid-state fermentation, using agroindustrial products or residues as substrates, mainly soy bran and wheat bran, at 40°C for 72 h and 96 h, respectively. Addition of glucose or fructose (≥1%; w/v) in SbmF inhibited enzyme production, while the addition of 1% (w/v) peptone as organic nitrogen source enhanced the production by 3.7-fold. However, 1% (w/v) (NH(4))(2)HPO(4) inhibited enzyme production around 80%. The extracellular form was purified until electrophoretic homogeneity (10.5-fold with 33% recovery) by DEAE-Fractogel and Sephacryl S-200 chromatography. The enzyme is a monomer with molecular mass of 102 kDa estimated by SDS-PAGE with carbohydrate content of 53.6%. Optima of temperature and pH for both, extracellular and cell extract invertases, were 60°C and 4.0-4.5, respectively. Both invertases were stable for 1 h at 60°C with half-lives of 10 min at 70°C. Mg(2+), Ba(2+) and Mn(2+) activated both extracellular and cell extract invertases from P. variotii. The kinetic parameters K(m) and V(max) for the purified extracellular enzyme corresponded to 2.5 mM and 481 U/mg prot(-1), respectively.

Thermostable saccharogenic amylase produced under submerged fermentation by filamentous fungus Penicillium purpurogenum.

The effect of several nutritional and environmental parameters on Penicillium purpurogenum growth and sacharogenic amylase production was analyzed. High enzyme levels (68.2 U mg(-1)) were obtained with Khanna medium at initial pH 6.0, incubated at 30°C for 144 hours. The optimum pH and temperature activities were 5.0 and 65°C, respectively. The enzyme presented a half-life (t50) of 60 min, at 65°C. Only glucose was detected after 24 hours of reaction using soluble starch as substrate.

Expression of F-actin and ß-tubulin genes in free mycelia and robust biofilms of the filamentous fungus Aspergillus niger.

The morphology and growth of the filamentous fungi are influenced by different factors as the culture conditions and the type of fermentative process. The production and secretion of metabolites by these organisms present a direct relationship with their morphology. The organization of the microtubules and actin in the cytoskeleton is determinant for both the fungal growth and morphology. In this context, this study aimed to analyze the expression of the ß-tubulin, F-actin, and glucan synthase in the A. niger mycelia obtained from submerged fermentation and biofilm fermentation through qPCR, as well as the analysis of the nucleus distribution in the hypha. Herein, we showed that ß-tubulin and the F-actin gene were more expressed in the biofilm condition, while the glucan synthase was in the submerged condition. No significant difference was observed in the nucleus distribution between the mycelia obtained from both the fermentative processes. In conclusion, the different morphologies observed for the mycelia from submerged fermentation and biofilm fermentation might be influenced by the differential modulation of genes that codify cytoskeleton proteins, which seems to be potentially regulated by mechanosensing during fungal contact with solid supports.

Immobilization of the Tannase From Aspergillus fumigatus CAS21: Screening the Best Derivative for the Treatment of Tannery Effluent Using a Packed Bed Reactor.

Enzyme immobilization is an important alternative to stabilize enzyme properties favoring the efficiency of derivatives (enzyme + support/matrix) for different purposes. According to this, the current study aimed to immobilize the Aspergillus fumigatus CAS21 tannase and the use of the derivatives in the treatment of the effluent produced by the tannery industry. The tannase was immobilized on sodium alginate, DEAE-Sephadex, amberlite, and glass pearls as supports. Calcium alginate was the most adequate support for tannase immobilization with 100% yield and 94.3% for both efficiency and activity. The best tannase activity for the calcium alginate derivative was obtained at 50°C-60°C and pH 5.0. Thermal and pH stabilities evaluated for 24 h at 30°C-60°C and pH 4-7, respectively, were improved if compared to the stability of the free enzyme. Considering the reuse of the calcium alginate derivative, 78% of the initial activity was preserved after 10 catalytic cycles, and after the 9-month storage at 4°C, the activity was maintained in 70%. This derivative was applied in a packed bed reactor (PBR) for the treatment of tannin-rich effluents from the tannery industry. The reduction of the tannin content was effective reaching degradation of 74-78% after 48 h of PBR operation. The concentration of total phenolic compounds was also reduced, and the color and clarity of the effluent improved. In conclusion, the calcium alginate derivative is an attractive alternative as biocatalyst for large-scale treatment of the effluents from the tannery industry.

Stabilization and application of spray-dried tannase from Aspergillus fumigatus CAS21 in the presence of different carriers.

The Aspergillus fumigatus CAS21 tannase was spray dried with ß-cyclodextrin, Capsul® starch, soybean meal, lactose, and maltodextrin as adjuvants. The moisture content and water activity of the products ranged from 5.6 to 11.5% and from 0.249 to 0.448, respectively. The maximal tannase activity was achieved at 40-60 ºC and pH 5.0-6.0 for the powders containing ß-cyclodextrin and Capsul® starch, which was stable at 40 ºC and 40-60 ºC for 120 min, respectively. For all the dried products, tannase retained its activity of over 80% for 120 min at pH 5.0 and 6.0. Salts and solvents influenced the activity of the spray-dried tannase. The activity of the spray-dried tannase was maintained when preserved for 1 year at 4 ºC and 28 ºC. Spray-dried tannase reduced the content of tannins and polyphenolic compounds of leather effluent and sorghum flour and catalyzed the transesterification reaction. The spray drying process stabilized the tannase activity, highlighting the potential of dried products for biotechnological applications.

A novel Trichoderma reesei mutant RP698 with enhanced cellulase production.

A new strain of Trichoderma reesei (teleomorph Hypocrea jecorina) with high cellulase production was obtained by exposing the spores from T. reesei QM9414 to an ultraviolet light followed by selecting fast-growing colonies on plates containing CMC (1% w/v) as the carbon source. The mutant T. reesei RP698 reduced cultivation period to 5 days and increased tolerance to the end-products of enzymatic cellulose digestion. Under submerged fermentation conditions, FPase, CMCase, and Avicelase production increased up to 2-fold as compared to the original QM9414 strain. The highest levels of cellulase activity were obtained at 27 °C after 72 h with Avicel®, cellobiose, and sugarcane bagasse as carbon sources. The temperature and pH activity optima of the FPase, CMCase, and Avicelase were approximately 60 °C and 5.0, respectively. The cellulase activity was unaffected by the addition of 140 mM glucose in the enzyme assay. When T. reesei RP698 crude extract was supplemented by the addition of ß-glucosidase from Scytalidium thermophilum, a 2.3-fold increase in glucose release was observed, confirming the low inhibition by the end-product of cellulose hydrolysis. These features indicate the utility of this mutant strain in the production of enzymatic cocktails for biomass degradation.

Production of thermostable invertases by Aspergillus caespitosus under submerged or solid state fermentation using agroindustrial residues as carbon source.

The filamentous fungus Aspergillus caespitosus was a good producer of intracellular and extracellular invertases under submerged (SbmF) or solid-state fermentation (SSF), using agroindustrial residues, such as wheat bran, as carbon source. The production of extracellular enzyme under SSF at 30°C, for 72h, was enhanced using SR salt solution (1:1, w/v) to humidify the substrate. The extracellular activity under SSF using wheat bran was around 5.5-fold higher than that obtained in SbmF (Khanna medium) with the same carbon source. However, the production of enzyme with wheat bran plus oat meal was 2.2-fold higher than wheat bran isolated. The enzymatic production was affected by supplementation with nitrogen and phosphate sources. The addition of glucose in SbmF and SSF promoted the decreasing of extracellular activity, but the intracellular form obtained in SbmF was enhanced 3-5-fold. The invertase produced in SSF exhibited optimum temperature at 50°C while the extra- and intracellular enzymes produced in SbmF exhibited maximal activities at 60°C. All enzymatic forms exhibited maximal activities at pH 4.0-6.0 and were stable up to 1 hour at 50°C.

Production of short-chain fructooligosaccharides (scFOS) using extracellular ß-D-fructofuranosidase produced by Aspergillus thermomutatus.

Aspergillus thermomutatus produces an extracellular ß-D-fructofuranosidase when cultured in Khanna medium with sucrose as additional carbon source at 30°C under agitation for 72 hr. Addition of glucose and fructose in the culture medium affected the production of the enzyme negatively. The optimum hydrolytic activity was achieved at 60°C and pH 5.0, with half-life (T50) of 30 hr at 50°C and 62% of its activity maintained at pH 5.0 for 48 hr. The extracellular extract containing ß-D-fructofuranosidase was effective in producing fructooligosaccharides (FOS), mainly 1-kestose. The highest concentration of FOS was obtained at 30°C and 60°C, indicating the existence of at least two enzymes with transfructosylating activity. At 30°C, the maximal FOS concentration was obtained from 48 to 72 hr, while at 60°C, it was achieved only at 72 hr. The best production of FOS (86.7 g/L) was obtained using 500 g/L sucrose as substrate. PRACTICAL APPLICATION: Fructooligosaccharides (FOS) are linear oligomers of fructose units with important applications in the food industry as sweetening agents and biopreservatives. Due to the presence of ß-glycosidic bonds, they cannot be hydrolyzed by human enzymes, allowing the use of FOS-containing products by diabetics. FOS used in the preparation of dairy products imparts humectancy to soft baked products, lowers the freezing point of frozen desserts, provides crispness to low-fat cookies, and provides many other advantages. Diets containing FOS can reduce the levels of triglycerides and cholesterol and improve the absorption of ions, such as Ca2+ and Mg2+ . FOS also exhibit bifidogenic effect on Bifidobacterium and Lactobacillus strains in the colon. Industrially, FOS is produced during the transfructosylation reaction of sucrose catalyzed by ß-D-fructofuranosidase. Identifying new sources of ß-D-fructofuranosidase is an important challenge to meet its industrial demand.

Extracellular Tannase from Aspergillus ochraceus: Influence of the Culture Conditions on Biofilm Formation, Enzyme Production, and Application.

Aspergillus ochraceus biofilm, developed on an inert support, can produce tannase in Khanna medium containing 1.5% (w/v) tannic acid as the carbon source, at an initial pH of 5.0, for 72 h at 28 °C. Addition of 0.1% yeast extract increased enzyme production. The enzyme in the crude filtrate exhibited the highest activity at 30 °C and pH 6.0. At 50 °C, the half-life was 60 min and 260 min at pH 6.0. In general, addition of detergents and surfactants did not affect tannase activity significantly. Tannase has potential applications in various biotechnological processes such as the production of propyl gallate and in the treatment of tannin-rich effluents. The content of tannins and total phenolic compounds in effluents from leather treatment was reduced by 56-83% and 47-64%, respectively, after 2 h of enzyme treatment. The content of tannins and total phenolic compounds in the sorghum flour treated for 120 h with tannase were reduced by 61% and 17%, respectively. Interestingly, the same A. ochraceus biofilm was able to produce tannase for three sequential fermentative process. In conclusion, fungal biofilm is an interesting alternative to produce high levels of tannase with biotechnological potential to be applied in different industrial sectors.

Characterization of Aspergillus fumigatus CAS-21 tannase with potential for propyl gallate synthesis and treatment of tannery effluent from leather industry.

One of the tannase isoforms produced by the fungus Aspergillus fumigatus CAS-21 under submerged fermentation (SbmF) was purified 4.9-fold with a 10.2% recovery. The glycoprotein (39.1% carbohydrate content) showed an estimated molecular mass of 60 kDa. Optimum temperature and pH for its activity were 30-40 °C and 5.0, respectively. It showed a half-life (t50) of 60 min at 45 and 50 °C, and it was stable at pH 5.0 and 6.0 for 3 h. The tannase activity was insensitive to most salts used, but it reduced in the presence of Fe2(SO4)3 and FeCl3. On contrary, in presence of SDS, Triton-X100, and urea the enzyme activity increased. The Km value indicated high affinity for propyl gallate (3.61 mmol L-1) when compared with tannic acid (6.38 mmol L-1) and methyl gallate (6.28 mmol L-1), but the best Kcat (362.24 s-1) and Kcat/Km (56.78 s-1 mmol-1 L) were obtained for tannic acid. The purified tannase reduced 89 and 25% of tannin content of the leather tannery effluent generated by manual and mechanical processing, respectively, after 2-h treatment. The total phenolic content was also reduced. Additionally, the enzyme produced propyl gallate, indicating its ability to do the transesterification reaction. Thus, A. fumigatus CAS-21 tannase presents interesting properties, especially the ability to degrade tannery effluent, highlighting its potential in biotechnological applications.

Production and characterization of a thermostable antifungal chitinase secreted by the filamentous fungus Aspergillus niveus under submerged fermentation.

The filamentous fungus Aspergillus niveus produced extracellular antifungal chitinase when cultured under submerged fermentation (SbmF) using crab shells as the carbon source. Maximal chitinase production was achieved at 192 h of cultivation using minimal medium containing 1% chitin. The enzyme was purified 1.97-fold with 40% recovery by ammonium sulfate precipitation and Sephadex G-100 gel filtration. The molecular mass was estimated to be 44 kDa by both 12% SDS-PAGE and Sepharose CL-6B gel filtration. Maximal A. niveus chitinase activity was obtained at 65 °C and pH 5.0. The enzyme was fully stable at 60 °C for up to 120 min and the enzymatic activity was increased by Mn2+. In the presence of reducing and denaturing compounds, the enzyme activity was not drastically affected. The chitinase was able to hydrolyze colloidal chitin, azure chitin, and 4-nitrophenyl N-acetyl-ß-D glucosaminide; for the latter, the K0.5 and maximal velocity (Vmax) were 3.51 mM and 9.68 U/mg of protein, respectively. The A. niveus chitinase presented antifungal activity against Aspergillus niger (MIC = 84 µg/mL), A. fumigatus (MIC = 21 µg/mL), A. flavus (MIC = 24 µg/mL), A. phoenicis (MIC = 24 µg/mL), and Paecilomyces variotii (MIC = 21 µg/mL). The fungus A. niveus was able to produce a thermostable and denaturation-resistant chitinase able to inhibit fungal development, signaling its biotechnological potential.

Acid and alkaline phosphatase activities of a fraction isolated from Parawixia bistriata spider venom.

This present study describes the isolation of a high molecular weight fraction (F1) from the venom of the social spider Parawixia bistriata, by gel filtration and also its subfractions by further purification with affinity chromatography on a Concanavalin A-Sepharose column. Acid and an alkaline phosphatase activities were found in fractions. The effects of pH, temperature and metallic ions on these activities were evaluated. Optimal temperature for both enzymes was 55 degrees C and optimal pH was 5.0 and 8.5 for the acid and alkaline phosphatase activities, respectively. As ZnCl(2) inhibited enzymatic activities and the chelating agent ethylenediaminetetracetic acid (EDTA) raised the basal phosphatase activities, it was speculated that the venom itself could contain Zn(+ +); this was confirmed with the use of an atomic absorption flame spectrometer. In conclusion, the high molecular weight components of the spider venom of P. bistriata have acid and alkaline phosphatase activities, which may reflect the presence of at least two different enzymes.

Characterization of a Thermotolerant Phytase Produced by Rhizopus microsporus var. microsporus Biofilm on an Inert Support Using Sugarcane Bagasse as Carbon Source.

The Rhizopus microsporus var. microsporus biofilm was able to produce increased levels of an extracellular thermotolerant phytase using polyethylene and viscose as an inert support in both modified NBRIP medium and modified Khanna medium containing sugarcane bagasse as the carbon source. The enzyme production was strictly regulated by the phosphorus content with optimal production at 0.5 mM of sodium phytate and KH2PO4. The extracellular phytase, RMPhy1, was purified 4.18-fold with 4.78 % recovery using DEAE-cellulose and CM-cellulose. A single protein band with a molecular mass of 35.4 kDa was obtained when the samples were subjected to 10 % SDS-PAGE. The optimum temperature for activity was 55 °C and the optimum pH was 4.5. R. microsporus var. microsporus phytase exhibited high stability at 30 and 40 °C with a half-life of 115 min at 60 °C. The enzyme activity increased in the presence of Ca (2+) and was inhibited by Zn(2+), arsenate, and sodium phosphate. Phytase demonstrated high substrate specificity for sodium phytate with K m = 0.72 mM and V max = 94.55 U/mg of protein and for p-NPP with K m = 0.04 mM and V max = 106.38 U/mg of protein. The enzyme also hydrolyzed ATP, AMPc, glucose 6-phosphate, glucose 1-phosphate, and UDPG. This is the first report on phytase characterization delivered with biofilm technology. The properties of the enzyme account for its high potential for use in biotechnology and the possibility of application in different industrial sectors as feed in the future.