Growth and glucoamylase production by the thermophilic fungusThermomyces lanuginosus in a synthetic medium

Summary The production of glucogenic amylase from the thermophilic fungus Thermomyces lanuginosus was studied in shake flasks and laboratory fermentors. As conidia were not able to germinate in media without yeast extract, pregerminated conidia were applied as inoculum. By this procedure it was possible to use different NH _inf4 ^sup+ salts as the sole source of nitrogen for growth and amylase formation in a synthetic medium. In pH-controlled fermentors a fourfold increase in the extracellular glucogenic amylase activity was obtained with (NH₄)H₂PO₄ as the nitrogen source as compared with yeast extract. However, by fractionation of these activities, comparable yields of partially purified glucoamylases were obtained. The glucoamylase preparation from fermentations with either of the nitrogen sources had a temperature optimum at 70° C and showed similar thermal stability. By incubation without substrate at 60° C. 90% of the activity was still present after 5 h. At 70° C, 50% of the activity was retained after 30 min incubation. Offprint requests to: I. Hassum     

Effect of media composition on the production of extracellular amylase from the thermophilic fungusThermomyces lanuginosus

Summary The production of extracellular amylase by the thermophilic fungusThermomyces lanuginosus was studied in shake flask cultures with different carbon sources in the growth medium. Maximum amylase production was obtained with dextran as carbon source. The greatest yield of amylolytic activity was found when low molecular weight dextrans were used as carbon source.     

Amylases of the thermophilic fungusThermomyces lanuginosus: Their purification,properties, action on starch and response to heat

A thermophilic fungus Thermomyces lanuginous strain IISc 91, secreted one form each of α-amylase and glucoamylase during growth. Both enzymes were purified to homogeneity by ion-exchange and gel-filtration chromatography and obtained in mg quantities. α-Amylase was considered to be a dimeric protein of ∼ 42 kDa and contained 5% (by mass) carbohydrate. It was maximally active at pH 5.6 and at 65°C. It had an activation energy of 44 kJ mol^-1. The apparent K_m for soluble starch was 2.5 mg ml^-1. The enzyme produced exceptionally high levels of maltose from raw potato starch. At 50°C, the enzyme was stable for > 7h. At 65°C, α-amylase was nearly 8-times more stable in the presence of calcium. Addition of calcium increaed the melting temperature of α-amylase from 66°C to 73°C. Upon incubation at 94°C, α-amylase was progressively and irreversibly inactivated, and converted into an inactive 72 kDa trimeric species. Glucoamylase was a monomeric glycoprotein of ∼ 45 kDa with a carbohydrate content of 11% (by mass). It effected up to 76% conversion of starch in 24 h producing glucose as the sole product. Its apparent K_m for soluble starch was 0.04 mg ml^-1 and V_max was 660 Mmol glucose min^-1 mg protein^-1. It also hydrolyzed maltose. Its activity on maltooligosaccharides increased with the chain length of the substrates. Glucoamylase was stable at 60°C for over 7h. Its activation energy was 61 kJ mol^-1 Glucoamylase did not show synergistic effect with α-amylase. The properties of α-amylase and glucoamylase of Thermomyces lanuginosus strain IISc 91 suggest their usefulness in the commercial production of maltose and glucose syrups.     

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K _m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C.The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.     

Enhancement in activity of an invertase from the thermophilic fungus Thermomyces lanuginosus by exogenous proteins

A highly purified invertase from a thermophilic fungus Thermomyces lanuginosus showed enhanced activity when incubated with exogenous proteins. These proteins also stabilized the invertase when incubated at 50°C, 4°C and –20°C. However, none of these proteins stabilized the invertase at or above 55°C, the temperature of inactivation. This property was found to be specific for the thermophilic invertase, as no such activation was observed for the mesophilic invertases from yeasts.     

Cloning and expression of kinesins from the thermophilic fungus Thermomyces lanuginosus

The motor domain regions of three novel members of the kinesin superfamily TLKIF1, TLKIFC, and TLBIMC were identified in a thermophilic fungus Thermomyces lanuginosus. Based on sequence similarity, they were classified as members of the known kinesin families Unc104/KIF1, KAR3, and BIMC. TLKIF1 was subsequently expressed in Escherichia coli. The expression level was high, and the protein was mostly soluble, easy to purify, and enzymatically active. TLKIF1 is a monomeric kinesin motor, which in a gliding motility assay displays a robust plus-directed microtubule movement up to 2 microm/s. The discovery of TLKIF1 also demonstrates that a family of kinesin motors not previously found in fungi may in fact be used in this group of organisms.     

Characterization of hygromycin-resistant transformants of thermophilic fungus Thermomyces lanuginosus

Hygromycin-resistant stable transformants of the thermophilic fungus, Thermomyces lanuginosus, were obtained by electroporation of germinating aleurospores with a plasmid pMP6, coding for hygromycin resistance. Southern hybridization analysis revealed that the gene is integrated into the chromosome. The hygromycin-resistant transformants were characterized for morphological changes, growth response towards the presence of antagonistic metabolites (hygromycin, 2-deoxy-D-glucose, cylcoheximide, benlate and acriflavine) on plates and enzyme production (amylases, pectinases and xylanase) in shake flask cultures. A hygromycin-resistant transformant hyg 33 was characterized as non-sporulating, 2-deoxy-D-glucose-resistant, acriflavine-sensitive and xylanase hypo-producer and is being used as parental strain for breeding strains through protoplast fusion.     

Amylase hyperproduction by deregulated mutants of the thermophilic fungus Thermomyces lanuginosus

Thermomyces lanuginosus was subjected to three cycles of mutagenesis (UV/NTG) and a selection procedure to develop amylase-hyperproducing, catabolite-repression-resistant and partially constitutive strains. One of the selected derepressed mutant strain III₅₁, produced ∼7- and 3-fold higher specific activity of α-amylase (190 U/mg protein) and glucoamylase (105 U/mg protein), respectively, compared to a wild-type parental strain. Further, the effect of production parameters on mutant strain III₅₁ was studied using a Box–Behnken design. The regression models computed showed significantly high R ² values of 96 and 97% for α-amylase and glucoamylase activities, respectively, indicating that they are appropriate for predicting relationships between corn flour, soybean meal and pH with α-amylase and glucoamylase production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 70–74 doi:10.1038/sj.jim.7000270 Received 05 July 2001/ Accepted in revised form 16 April 2002     

Enzyme production in continuous cultivation by the thermophilic fungus, Thermomyces lanuginosus

The production of extracellular enzymes by the thermophilic fungus Thermomyces lanuginosus was studied in chemostat cultures at a dilution rate of 0.08 h^–1 in relation to variation in the ammonium concentration in the feed medium. Under steady state conditions, three growth regimes were recognised and the production of several extracellular enzymes from T. lanuginosus was recorded under different nutrient limitations ranging from nitrogen limitation to carbon/energy limitation. The range and the production of carbohydrate hydrolysing enzymes and lipase increased from Regime I (NH₄Cl 600 mg l^–1) to Regime III (NH₄CI 1200 mg l^–1), whereas production of protease was highest in Regime II (600 mg l^–1 < NH₄Cl <1200 mg l^–1).     

Biochemical characterisation of the trehalase of thermophilic fungi: an enzyme with mixed properties of neutral and acid trehalase

The trehalases from some thermophilic fungi, such as Humicola grisea, Scytalidium thermophilum, or Chaetomium thermophilum, possess mixed properties in comparison with those of the two main groups of trehalases: acid and neutral trehalases. Such as acid trehalases these enzymes are highly thermostable extracellular glycoproteins, which act at acidic pH. However, these enzymes are activated by calcium or manganese, and as a result inhibited by chelators and by ATP, properties typical of neutral trehalases. Here we extended the biochemical characterisation of these enzymes, by assaying their activity at acid and neutral pH. The acid activity (25-30% of total) was assayed in McIlvaine buffer at pH 4.5. Under these conditions the enzyme was neither activated by calcium nor inhibited by EDTA or ATP. The neutral activity was estimated in MES buffer at pH 6.5, after subtracting the activity resistant to EDTA inhibition. The neutral activity was activated by calcium and inhibited by ATP. On the other hand, the acid activity was more thermostable than the neutral activity, had a higher temperature optimum, exhibited a lower K(m), and different sensitivity to several ions and other substances. Apparently, these trehalases represent a new class of trehalases. More knowledge is needed about the molecular structure of this protein and its corresponding gene, to clarify the structural and evolutionary relationship of this trehalase to the conventional trehalases.     

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa.

Trehalase (alpha,alpha-Trehalose glucohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50 degrees C in acetate buffer at pH 5.5. It was highly specific for alpha,alpha-trehalose and had an apparent Km = 0.4 mM at 50 degrees C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170 000. Trehalase from mycelium grown at 40 and 50 degrees C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by "protection against heat-inactivation by "protective factor(s)" present in mycelial extracts. The "protective factor(s)" was sensitive to proteolytic digestion. It was not diffusible and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.     

Purification and characterization of alpha-galactosidase from a thermophilic fungus Thermomyces lanuginosus

An extracellular alpha-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl alpha-D-galactosides but efficiently hydrolyzed alpha-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl alpha-galactopyranoside and galactosyl(1)mannotriose, followed by (1)H NMR spectroscopy, pointed out the alpha-anomer of D-galactose was the primary product of hydrolysis from which the beta-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl alpha-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.     

A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus

A thermostable superoxide dismutase (SOD) from a Thermomyces lanuginosus strain (P134) was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography, and gel filtration on Sephacryl S-100. The molecular mass of a single band of the enzyme was estimated to be 22.4 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 89.1 kDa, indicating that this enzyme was composed of four identical subunits of 22.4 kDa each. The SOD was found to be inhibited by NaN₃, but not by KCN or H₂O₂, suggesting that the SOD in T. lanuginosus was of the manganese superoxide dismutase type. The SOD exhibited maximal activity at pH 7.5. The optimum temperature for the activity was 55°C. It was thermostable at 50 and 60°C and retained 55% activity after 60 min at 70°C. The half-life of the SOD at 80°C was approximately 28 min and even retained 20% activity after 20 min at 90°C.     

The glutathione response to salt stress in the thermophilic fungus, Thermomyces lanuginosus

In order to investigate the role of glutathione in response to salt stress in the thermophilic fungus, Thermomyces lanuginosus, the biomass and the intracellular pool of protein and the glutathione + glutathione disulphid (GSH + GSSG) was measured for four days in a medium with NaCl or KCl added and in the basal medium. Due to the osmotic and ionic stress imposed by the salts, the growth of T. lanuginosus was delayed and the inhibitory effect of KCl exceeded that of NaCl. Glutathione seemed to be involved in the response of T. lanuginosus towards high concentrations of salt, as the level of stress was negatively correlated with the amount of total glutathione. Salt stress did not result in an increased intracellular protein production. GSH accumulated while nutrients were abundant and were subsequently degraded later, suggesting that nutrients stored in GSH are used when the medium is depleted.     

嗜热真菌Thermomyces lanuginosus热稳定几丁质酶基因的cDNA克隆及表达

根据Thermomyces lanuginosus热稳定几丁质酶Chit的N-端氨基酸序列和同源保守序列设计简并引物,通过RT-PCR及快速扩增cDNA末端(RACE)的方法,克隆了该几丁质酶的编码基因chit,全长cDNA为1500bp,包含一个由442个氨基酸组成的开放阅读框。该基因已在GenBank中注册,登录号为DQ092332。将成熟肽几丁质酶Chit阅读框与酵母表达载体pPIC9K连接,构建重组质粒pPIC9K/chit,转化毕赤酵母GS115,在甲醇的诱导下,成功地分泌出具生物活性的几丁质酶,诱导6d后酶活性达2.261U/mL,酶蛋白表达量为0.36mg/mL。该酶的最适反应温度和pH值分别为60℃和5.5,该酶在50℃以下稳定;65℃的半衰期为40min。     

疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究

采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8～ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 、Na 、K 、Ba2 对酶有激活作用 ;Ag 、Fe2 、Cu2 、Hg2 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。     

Purification and characterization of an extracellular polygalacturonase from the thermophilic fungus,Thermomyces lanuginosus

A polygalacturonase was purified from the thermophilic fungus, Thermomyces lanuginosus to apparent homogeneity by ultrafiltration, acetone precipitation and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60 °C. The apparent K_M with potassium pectate was 0.67 mg/ml and the V_max was 7.2 × 10⁵ mol/min/mg protein. The apparent molecular weight of the enzyme was 59 kDa and it contained approximately 10% carbohydrate. The enzyme was completely stable at room temperature (32 ± 3 °C) and retained about 50% activity at 50 °C for 6 h. The zymogram of the purified enzyme revealed two activity bands, one of which was a major one. Polyclonal antibodies raised against the enzyme did not show any immunological relatedness with other mesophilic polygalacturonases.     

Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus.

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) was purified from the culture filtrates of the thermophilic fungus Thermomyces lanuginosus and was established to be homogeneous by a number of criteria. The enzyme was a glycoprotein with an average molecular weight of about 57 000 and a carbohydrate content of 10-12%. The enzyme hydrolysed successive glucose residues from the non-reducing ends of the starch molecule. It did not exhibit any glucosyltransferase activity. The enzyme appeared to hydrolyse maltotriose by the multi-chain mechanism. The enzyme was unable to hydrolyse 1,6-alpha-D-glucosidic linkages of isomaltose and dextran. It was optimally active at 70 degrees C. The enzyme exhibited increase in the Vmax. and decreased in Km values with increasing chain length of the substrate molecule. The enzyme was inhibited by the substrate analogue D-glucono-delta-lactone in a non-competitive manner. The enzyme inhibited remarkable resistance towards chemical and thermal denaturation.