Thermomyces lanuginosus lipase-catalyzed regioselective acylation of nucleosides: Enzyme substrate recognition

Substrate recognition of Thermomyces lanuginosus lipase in the acylation of nucleosides was revealed through rational substrate engineering for the first time. T. lanuginosus lipase displayed higher catalytic activities and excellent 5′-regioselectivities (94–>99%) in the acylation of ribonucleosides 1f–1j as compared to those in the acylation of 2′-deoxynucleosides 1a–1e. The higher reaction rates and excellent 5′-regioselectivities might derive from a favorable hydrogen bonding between the 2′-hydroxyl group of 1f–1j and phenolic hydroxyl group of Tyr21 present in the hydrophilic region of the lipase.     

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.     

Production of high level of cellulase-free and thermostable xylanase by a wild strain of Thermomyces lanuginosus using beechwood xylan

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was studied for production of high level of cellulase-free thermostable xylanase at 50°C using xylan. Optimization of the medium composition was carried out on shake-flask level using Graeco-Latin square technique. This increased xylanase production from 527 nkat ml⁻¹ in the original medium to 9168–9502 nkat ml⁻¹ in the optimized medium under optimized culture conditions e.g. initial medium pH (6.0–6.5), culture temperature (50°C) and time (5–6 d). The lag phase was very much shorter in the laboratory reactor compared to which existed in the shake cultures and 7111 nkat of xylanase activity were obtained per ml of culture filtrate at 60 h of cultivation. With a 15 min reaction time, the optimal pH and temperature for the xylanase activity were at 6.5 and 65°C, respectively. The enzyme was almost stable over a broad range of pH 3–9 at 20°C, with an optimum stability at pH 6.5. After 51 h heating at 50°C the enzyme retained 60%, 100% and 90% activity at pH 5.0, 6.5 and 8.0, respectively. The crude enzyme could hydrolyse xylan effectively and in only 6 h 67.3%, 54.0% and 49.2% saccharifications were achieved for 2%, 5% and 10% substrate levels, respectively. The principal product of hydrolysis was xylobiose together with smaller amounts of xylooligosaccharides (degree of polymerization 3–7) and xylose.     

Positive effects of the multipoint covalent immobilization in the reactivation of partially inactivated derivatives of lipase from Thermomyces lanuginosus

Different immobilized preparations of lipase from Thermomyces lanuginosus (TLL) have been inactivated by exposure to high temperatures, guanidine or 95% of dioxane. The studied preparations were: non-stabilized cyanogen bromide (CNBr-TLL), aminated CNBr-TLL (CNBr-TLL-A), and two stabilized preparations of aminated TLL by immobilization on glyoxyl support, Gx(9/10)-TLL-A (TLL-A immobilized at pH 9 and later incubated at pH 10) or Gx(10)-TLL-A (directly immobilized at pH 10). The reactivation of the partially inactivated immobilized enzymes under mild conditions by incubation in aqueous buffer, allowed recovery of some of the original activity, which was improved when it was pre-incubated in guanidine. Amination produced a fairly negative effect on the reactivation of the enzyme, but the multipoint covalent attachment of this aminated enzyme reversed the effect (e.g., recovered activity increased from 20% for CNBr-TLL to 80% for Gx(9/10)-TLL-A). The negative effect of the amination was clearer when the inactivation was caused by exposure to high temperatures, although the multipoint attachment of aminated enzyme was able to improve the recovered activity. The determination of enzyme activity in the presence of hexadecyltrimethylammonium bromide slowed the inactivation rates of all preparations and improved the recovery of activity after incubation under mild conditions, suggesting that the opening mechanism of the lipase could be a critical step in the TLL inactivation/reactivation. The use of multipoint attached TLL preparations did not only improve enzyme stability, but it also increased activity recovery when the preparation was incubated under mild conditions.     

Phytase production by the thermophilic fungus Rhizomucor pusillus

A thermophilic fungus, Rhizomucor pusillus, isolated from composting soil, was studied for phytase production using solid-state fermentation. The optimization of phytase production was carried out by Box–Behnken design of experiments, using three independent variables (pH of medium, culture age and incubation period), resulting in a maximal level of phytase (9.18 units/g substrate). The partially purified phytase was optimally active at 70 °C and pH 5.4, though the enzyme showed 80% activity over a wide pH range, 3.0–8.0. The phytase was found to have broad substrate specificity.     

Enzymology of the thermophilic ascomycetous fungus Thermoascus aurantiacus

Different strains of the thermophilic ascomycetous fungus Thermoascus aurantiacus have been reported in the literature to produce high levels of a variety of industrial interest enzymes (i.e. amylases, cellulases, pectinases and xylanases), which have been shown to be remarkably stable over a wide range of temperatures and appear to have tremendous commercial potential. Most studies on enzyme production by T. aurantiacus are carried out in chemically defined liquid medium, under conditions suitable for induction of a particular enzyme. A few studies have investigated the production of some enzymes by T. aurantiacus by solid-state fermentation, using lignocellulosic materials. The present review focuses on the enzymes produced by T. aurantiacus, their main kinetic parameters, and the effect of different culture conditions on production and enzyme activity. It also provides a view of the possible applications of T. aurantiacus enzymes, considering that this thermophilic fungus could comprise a potential source of thermostable enzymes.     

Protease production by the ontjom fungus,Neurospora sitophila

Summary The production of protease and mycelium byNeurospora sitophila cultured on solid and liquid potato dextrose media was studied. Maximal activity of protease extracted from 4-day-old cultures occurred at pH 6.5 when an unfractionated peanut (groundnut) protein substrate was used. The greatest protease activity and mycelium production occurred during the first day of the 4-day test period. Potato dextrose media containing more than 0.2 M NaCl resulted in decreased protease and mycelium production, while tapioca starch was without affect at concentrations up to 1.4%. Addition of up to 0.3 M sucrose to growth media greatly stimulated protease production and mycelial growth. Maximal proteolytic activity was observed in extracts from mycelium cultured in potato dextrose media adjusted from pH 6.0 to 7.5. Activity was greater when soluble peanut protein was used as a substrate, compared to unfractionated or globulin protein substrates.     

Xylanase,CM-cellulase and Avicelase production by the thermophilic fungus Sporotrichum thermophile

Biotechnology Letters - When wheat straw was used as carbon source, Sporotrichum thermophile produced large amounts of xylanase extracellularly in addition to CM-cellulase and Avicelase. These...     

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).     

Biodegradation of indole at high concentration by persolvent fermentation with the thermophilic fungus Sporotrichum thermophile

Indole and its derivatives form a class of toxic recalcitrant environmental pollulants. Sporotrichum thermophile was grown in a persolvent fermentation system containing a large amount of indole. The medium contained up to 20% by volume soybean oil and up to 2 g L⁻¹ indole. Most of the indole was partitioned in the organic solvent layer. When the organism was grown in the medium containing indole at 1 g L⁻¹, indole was totally consumed after 6 days. Under a fed–batch fermentation process where daily batches of indole (1 g L⁻¹) supplemented the microbial culture for 4 days, the biodegradation level was 3.0 g L⁻¹. These values make this process promising and worthy of further investigation for the microbial degradation of other toxic compounds.     

Avicel-adsorbable endoglucanase production by the thermophilic fungus Scytalidium thermophilum type culture Torula thermophila

Scytalidium thermophilum type culture Torula thermophila was isolated from mushroom compost and the total cellulase, endoglucanase, Avicel-adsorbable endoglucanase activities, as well as the fungal biomass generation and cellulose utilisation were analyzed in shake flask cultures with Avicel (microcrystalline cellulose) as the carbon source. Results were compared with an industrial strain of Scytalidium thermophilum type culture Humicola insolens. The pH and temperature optima for endoglucanase activities during enzyme assays were also analyzed for both organisms and determined to be pH 6.0 and 65 degrees C for type culture Torula thermophila, and pH 6.5 and 60 degrees C for type culture Humicola insolens. Analysis of the effect of growth temperature showed that type culture T. thermophila can grow and produce cellulases in the range of 35 to 55 degrees C although 40 to 50 degrees C seemed to favor growth and cellulase production. Although 45 degrees C was found optimal for fungal growth, both the specific endoglucanase and Avicel-adsorbable endoglucanase activities (U/mg protein) as well as the percentage of Avicel-adsorbable endoglucanase activity reached maxima at 50 degrees C and were higher as compared to type culture H. insolens. Results indicate that type culture T. thermophila, with further optimisations, is of potential use in the industrial production of cellulases.     

Purification and characterization of an endocellulase from the thermophilic fungus Chaetomium thermophilum CT2

Chaetomium thermophilum CT2 produced endocellulases at 50 °C, when grown on 2% microcrystalline cellulose, 1% soluble starch, and 0.4% yeast extract medium. A major endocellulase component was purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography and gel filtration on Sephacryl S-100. The molecular weight of the enzyme was estimated to be 67.8 kDa and the enzyme was found to be a glycoprotein containing 18.9% carbohydrate. The K_m of the purified enzyme for carboxymethyl cellulose, sodium salt (CMC), was 4.6 mg ml⁻¹. The enzyme displayed highest activity towards CMC and significantly lower activities towards phosphoric acid swollen cellulose and filter paper. The activity was enhanced in the presence of Na⁺, K⁺ and Ca²⁺ but inhibited by Hg²⁺, Zn²⁺, Ag⁺, Mn²⁺, Ba²⁺, Fe²⁺, Cu²⁺, Mg²⁺ and NH₄⁺. Optimum activity was at 60 °C and pH 4.0. The enzyme was stable over 60 min incubation at 60 °C and half-life at 70, 80 and 90 °C was approximately 45, 24 and 7 min, respectively.     

基于半理性设计提高疏棉状嗜热丝孢菌脂肪酶催化特异性

甘油二酯(diacylglycerol,DAG)是油脂代谢的中间产物,在人体中具有重要的生理功能,主要通过脂肪酶水解油脂制备。但对1,2-甘油二酯(1,2-diacylglycerol,1,2-DAG)、1,3-甘油二酯(1,3-diacylglycerol,1,3-DAG)的检测分离方法及脂肪酶的催化特异性的研究较少,限制了其广泛应用。基于以上问题,本研究首先通过超临界流体色谱与蒸发光散射检测器联用,优化检测分析参数,建立了1,2-DAG(0.025–0.200 g/L)和1,3-DAG(0.025–0.150 g/L)的高效定量检测方法。进一步基于疏棉状嗜热丝孢菌脂肪酶(Thermomyces lanuginosus lipase,TLL)与三油酸甘油酯的分子对接,挑选了5个潜在底物结合位点并通过定点饱和突变构建了突变体库,利用超临界流体色谱方法对突变体催化特异性进行筛选。突变文库中I202V表现出最高的1,3-位置选择特异性,较野生型TLL提高了11.7%。本文基于半理性设计方法实现了对TLL位置催化特异性的改造,并建立了一种能够高效分离检测DAG位置异构体的方法,为脂肪酶催化特异性研究提供了参考。     

An unusual ring—A opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila

A series of steroids (progesterone, testosterone acetate, 17β-acetoxy-5α-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17β side-chain.This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17β-acetoxy-5α-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17β-alcohol of testosterone.All steroidal metabolites were isolated by column chromatography and were identified by ¹H, ¹³C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.