Increase of the phytase production by Aspergillus japonicus and its biocatalyst potential on chicken feed treatment.

Phytase hydrolyzes phytic acid from the plant components of animal feed, releasing inorganic phosphorus. The phytase production by Aspergillus japonicus was optimized using Plackett-Burman designs (PBD), composite central rotational designs (CCRD), and response surface methodology from standard Czapek medium. The enzyme was applied in broiler chicken and laying hen foods. Analysis from PBD showed that KH2 PO2, MgSO4 · 7H2O, and yeast extract had significant influences on phytase secretion (p < 0.05). The best results from the CCRD experiments were obtained using (A) 0.040% KH2 PO4, (B) 0.050% MgSO4 · 7H2O, and (C) 0.040% yeast extract, enhancing in 49-53 U mg(-1) protein. The determination coefficient (R(2)) was 0.92 and Fcalc was 7.48 times greater than Flisted . Thus, the reduced coded model: Y (U mg-1) = 50.29 + 4.30A - 3.35(A)2 - 4.80(B)2 + 5.62C - 4.26(C)2 was considered predictive and statistically significant (p < 0.05). The optimized culture medium increased the phytase yield in 250%. A. japonicus phytase released high levels of Pi from broiler chicken and laying hen food. A. japonicus is an excellent phytase producer in a culture medium using inexpensive components and agricultural wastes. Therefore, these results provide sound arguments for the formulation of a low cost culture medium for phytase production.

Functional properties of a manganese-activated exo-polygalacturonase produced by a thermotolerant fungus Aspergillus niveus.

A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40 °C, presenting protein levels almost twofold higher than at 25 °C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7 % carbohydrate, molecular mass of 102.6 kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50 °C and 4.0-6.5, respectively. The enzyme was stable at pH 3.0 to 9.0 for 24 h. The DEAE-cellulose derivative was about sixfold more stable at 60 °C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232 % activated by Mn(2+). The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K m, V max, K cat, and K cat/K m values were 6.7 mg/ml, 230 U/mg, 393.3/s, and 58.7 mg/ml/s, respectively. The N-terminal amino acid sequence presented 80 % identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.

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.

Biochemical properties of an extracellular trehalase from Malbranchea pulchella var. Sulfurea.

The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680 U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0-5.5 and 55° C, respectively. The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver ions. The apparent K(M) values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70 ± 0.29 and 2.58 ± 0.13 mM, respectively. Manganese ions affected only the apparent V(max), increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also from trehalases from other thermophilic fungi.

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 30ºC for 144 hours. The optimum pH and temperature activities were 5.0 and 65ºC, respectively. The enzyme presented a half-life (t50) of 60 min, at 65ºC. Only glucose was detected after 24 hours of reaction using soluble starch as substrate.

Biotechnological Potential of Agro-Industrial Wastes as a Carbon Source to Thermostable Polygalacturonase Production in Aspergillus niveus.

Agro-industrial wastes are mainly composed of complex polysaccharides that might serve as nutrients for microbial growth and production of enzymes. The aim of this work was to study polygalacturonase (PG) production by Aspergillus niveus cultured on liquid or solid media supplemented with agro-industrial wastes. Submerged fermentation (SbmF) was tested using Czapeck media supplemented with 28 different carbon sources. Among these, orange peel was the best PG inducer. On the other hand, for solid state fermentation (SSF), lemon peel was the best inducer. By comparing SbmF with SSF, both supplemented with lemon peel, it was observed that PG levels were 4.4-fold higher under SSF. Maximum PG activity was observed at 55°C and pH 4.0. The enzyme was stable at 60°C for 90 min and at pH 3.0-5.0. The properties of this enzyme, produced on inexpensive fermentation substrates, were interesting and suggested several biotechnological applications.

Production of xylanolytic enzymes by Aspergillus terricola in stirred tank and airlift tower loop bioreactors.

Fungi producing high xylanase levels have attracted considerable attention because of their potential industrial applications. Batch cultivations of Aspergillus terricola fungus were evaluated in stirred tank and airlift bioreactors, by using wheat bran particles suspended in the cultivation medium as substrate for xylanase and ß-xylosidase production. In the stirred tank bioreactor, in physical conditions of 30°C, 300 rpm, and aeration of 1 vvm (1 l min⁻¹), with direct inoculation of fungal spores, 7,475 U l⁻¹ xylanase was obtained after 36 h of operation, remaining constant after 24 h. In the absence of air injection in the stirred tank reactor, limited xylanase production was observed (final concentration 740 U l⁻¹). When the fermentation process was realized in the airlift bioreactor, xylanase production was higher than that observed in the stirred tank bioreactor, being 9,265 U l⁻¹ at 0.07 vvm (0.4 l min⁻¹) and 12,845 U l⁻¹ at 0.17 vvm (1 l min⁻¹) aeration rate.

Biochemical properties of an extracellular beta-D-fructofuranosidase II produced by Aspergillus phoenicis under Solid-Sate Fermentation using soy bran as substrate

The filamentous fungus A. phoenicis produced high levels of beta-D-fructofuranosidase (FFase) when grown for 72 hrs under Solid-State Fermentation (SSF), using soy bran moistened with tap water (1:0.5 w/v) as substrate/carbon source. Two isoforms (I and II) were obtained, and FFase II was purified 18-fold to apparent homogeneity with 14 percent recovery. The native molecular mass of the glycoprotein (12 percent of carbohydrate content) was 158.5 kDa with two subunits of 85 kDa estimated by SDS-PAGE. Optima of temperature and pH were 55ºC and 4.5. The enzyme was stable for more than 1 hr at 50ºC and was also stable in a pH range from 7.0 to 8.0. FFase II retained 80 percent of activity after storage at 4ºC by 200 hrs. Dichroism analysis showed the presence of random and beta-sheet structure. A. phoenicis FFase II was activated by Mn2+, Mg2+ and Co2+, and inhibited by Cu2+, Hg2+ and EDTA. The enzyme hydrolyzed sucrose, inulin and raffinose. Kd and Vmax values were 18 mM and 189 U/mg protein using sucrose as substrate.

Biotechnological potential of alternative carbon sources for production of pectinases by Rhizopus microsporus var. rhizopodiformis

Fungi collected from Brazilian soil and decomposing plants were screened for pectinase production. R. microsporus var. rhizopodiformis was the best producer and was selected to evaluate the pectic enzyme production under several nutritional and environmental conditions. The pectinase production was studied at 40ºC, under 28 carbon sources-supplemented medium. The inducer effect of several agro-industrial residues such as sugar cane bagasse, wheat flour and corncob on polygalacturonase (PG) activity was 4-, 3- and 2-fold higher than the control (pectin). In glucose-medium, a constitutive pectin lyase (PL) activity was detected. The results demonstrated that R. microsporus produced high levels of PG (57.7 U/mg) and PL (88.6 U/mg) in lemon peel-medium. PG had optimum temperature at 65 ºC and was totally stable at 55 ºC for 90 min. Half-life at 70 ºC was 68 min. These results suggested that the versatility of waste carbon sources utilization by R. microsporus, produce pectic enzymes, which could be useful to reduce production costs and environmental impacts related to the waste disposal.

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed.

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 °C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 °C and 50-55 °C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 °C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu++ and Mn++ increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

cAMP signaling pathway controls glycogen metabolism in Neurospora crassa by regulating the glycogen synthase gene expression and phosphorylation.

The cAMP-PKA signaling pathway plays an important role in many biological processes including glycogen metabolism. In this work we investigated its role in the Neurospora crassa glycogen metabolism control using mutant strains affected in components of the pathway, the cr-1 strain deficient in adenylyl cyclase activity therefore has the PKA pathway not active, and the mcb strain a temperature-sensitive mutant defective in the regulatory subunit of PKA therefore is a strain with constitutively active PKA. We analyzed the expression of the gene encoding glycogen synthase (gsn), the regulatory enzyme in glycogen synthesis as a potential target of the regulation. The cr-1 strain accumulated, during vegetative growth, glycogen levels much higher than the wild type strain indicating a role of the PKA pathway in the glycogen accumulation. The gsn transcript was not increased in this strain but the GSN protein was less phosphorylated "in vitro", and therefore more active, suggesting that the post-translational modification of GSN is likely the main mechanism controlling glycogen accumulation during vegetative growth. Heat shock down-regulates gsn gene transcription in the two mutant strains, as well as in the wild type strain, suggesting that the PKA pathway may not be the only pathway having a direct role in gsn transcription under heat shock. DNA-protein complexes were formed between the STRE motif in the gsn promoter and nuclear proteins from heat-shocked mycelium. However STRE was not able to induce transcription of a reporter gene in Saccharomyces cerevisiae, suggesting that the motif might be involved in a different way of regulation in the N. crassa gene expression under heat shock. The CRE-like DNA elements present in the gsn promoter were shown to be bound by different proteins from the PKA mutant strains. The DNA-protein complexes were observed with proteins from the strains grown under normal condition and under heat shock indicating the functionality of this DNA element. In this work we presented some evidences that the PKA signaling pathway regulates glycogen metabolism in N. crassa in a different way when compared to the well-characterized model of regulation existent in S. cerevisiae.

Purification and partial characterization of an exo-polygalacturonase from Paecilomyces variotii liquid cultures.

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K(m) and V(max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 micromol/min/mg, respectively. PG was found to have temperature optimum at 65 degrees Celsius and was totally stable at 45 degrees Celsius for 90 min. Half-life at 55 degrees Celsius was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

Purification and biochemical characterization of a novel alpha-glucosidase from Aspergillus niveus.

An extracellular a-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS-PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical alpha-glucosidase activity, hydrolyzing p-nitrophenyl alpha-D-glucopyranoside and presented an optimum temperature and pH of 65 degrees C and 6.0, respectively. In the absence of substrate the purified alpha-glucosidase was stable for 60 min at 60 degrees C, presenting t(50) of 90 min at 65 degrees C. Hydrolysis of polysaccharide substrates by alpha-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and beta-ciclodextrin were poor substrates, and sucrose and alpha-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an a-helical content of 31% and a beta-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.

Production of thermostable invertases by Aspergillus caespitosus under submerged or solid state fermentation using agroindustrial residues as carbon source / Produção de invertases termoestáveis por Aspergillus caespitosus em fermentação submersa e em estado sólido usando resíduos agroindustriais como fonte de carbono

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 30ºC, 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 50ºC while the extraand intracellular enzymes produced in SbmF exhibited maximal activities at 60ºC. All enzymatic forms exhibited maximal activities at pH 4.0-6.0 and were stable up to 1 hour at 50ºC.

O fungo filamentoso Aspergillus caespitosus foi um bom produtor de invertases intracelular e extracelular em fermentação submersa (FSbm) ou em estado sólido (FES), usando resíduos agroindustriais como fonte de carbono, sendo que para ambas as condições de cultivo, a maior produtividade foi obtida empregandose farelo de trigo. A produção da forma extracelular em FES mantido a 30ºC, por 72 horas, foi aumentada usandose solução de sais SR (1:1, m/v) para umidificar o substrato, sendo aproximadamente 5,5 vezes maior se comparada a FSbm (Meio Khanna) com a mesma fonte de carbono. Entretanto, a mistura de farelo de trigo e farinha de aveia em FES levou a um aumento de 2,2 vezes na produção enzimática se comparada ao uso isolado do farelo de trigo. A produção enzimática, em ambas as condições de cultivo, foi afetada pela adição suplementar de fontes de nitrogênio e fosfato. A adição de glicose em FSbm e em FES promoveu a diminuição da enzima extracelular, mas favoreceu um acúmulo intracelular de 35 vezes maior. A temperatura ótima de atividade para as invertases produzidas em FES e em FSbm foi de 50ºC e 60ºC, respectivamente, sendo estáveis a 50ºC por mais de 60 minutos. Todas as formas enzimáticas apresentaram atividade máxima em uma faixa de pH de 4.0-6.0.

Properties of a purified thermostable glucoamylase from Aspergillus niveus.

A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40 degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65 degrees C and retained 100% activity after 240 min at 60 degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The K(m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.

Effect of glycosylation on the biochemical properties of beta-xylosidases from Aspergillus versicolor.

Aspergillus versicolor grown on xylan or xylose produces two beta-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these beta-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same R(f). Temperature optimum of xylan-induced and xylose-induced beta-xylosidases was 45 degrees C and 40 degrees C, respectively, and 35 degrees C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55 degrees C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching.

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4-5 days in liquid medium at 40 degrees C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60-70 degrees C. The enzymes were stable for 30 min at 60 degrees C, maintaining 95-98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5-5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0-8.0, while the enzyme from A. niveus was more stable at pH 4.5-6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.

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.

Regulation of xylanase in Aspergillus phoenicis: a physiological and molecular approach.

Microbial xylanolytic enzymes have a promising biotechnological potential, and are extensively applied in industries. In this study, induction of xylanolytic activity was examined in Aspergillus phoenicis. Xylanase activity induced by xylan, xylose or beta-methylxyloside was predominantly extracellular (93-97%). Addition of 1% glucose to media supplemented with xylan or xylose repressed xylanase production. Glucose repression was alleviated by addition of cAMP or dibutyryl-cAMP. These physiological observations were supported by a Northern analysis using part of the xylanase gene ApXLN as a probe. Gene transcription was shown to be induced by xylan, xylose, and beta-methylxyloside, and was repressed by the addition of 1% glucose. Glucose repression was partially relieved by addition of cAMP or dibutyryl cAMP.

Characterisation of an acid trehalase produced by the thermotolerant fungus Rhizopus microsporus var. rhizopodiformis: biochemical properties and immunochemical localisation.

An acid trehalase from Rhizopus microsporus var. rhizopodiformis was purified to apparent homogeneity. The molecular weight by SDS-PAGE (60 kDa) or Sephacryl S-200 filtration (105 kDa) suggested a homodimer. The carbohydrate content was 72%. Endoglycosidase H digestion resulted in one sharp band of 51.5 kDa in SDS-PAGE. pH and temperature optima were 4.5 and 45 degrees C, respectively. The isoelectric point was 6.69 and activation energy was 1.14 kcal mol(-1). The enzyme was stable for 1 h at 50 degrees C and decayed at 60 degrees C (t50 of 1.3 min.). Apparent KM for trealose was 0.2mM. Immunolocalisation studies showed the enzyme tightly packed at the surface of the cells.