Xylanase production by Thermomyces lanuginosus wild and mutant strains

Thermomyces lanuginosus strains from different culture collections, namely ATCC 26909, ATCC 22083, DEN 1457, IMI 84400 and BS1 were compared for xylanase production, and isozyme profile. Of all the strains of T. lanuginosus, BS1 a soil isolate produced the largest amount of xylanase. All strains were found to produce two forms of xylanase (I & II) with molecular mass corresponding to 25.0 and 54.0 KDa. The u.v/NTG mutagenesis of T. lanuginosus BS1 aleurospores/protoplasts resulted in xylanase-hyperproducing mutants. A morphological colour mutant RB 524 produced approximately 2.5-fold higher xylanase (2506.0 units/ml) as compared to the parent strain (1018.1 units/ml).     

The lipolytic activity of Thermomyces lanuginosus strains isolated from different natural sources

The ability of 144 Thermomyces lanuginosus wild strains isolated from biohumus, mushroom and garden composts, decayed leaves, hazelnuts, and raw coffee beans to hydrolyze synthetic (tributyrin, Tween 20, Tween 40, Tween 60, and Tween 80) and natural fatty substrates (sunflower, soybean, rapeseed and corn oil) was evaluated, and whether the lipolytic activity depended on the isolation source determined. All strains incubated at 55 °C on solid media containing 1% synthetic and 15% natural fatty substrates hydrolyzed both types of substrate. Mean lipolytic activity on natural substrates was significantly higher than on synthetic substrates. The highest mean activity index was noted after growth on sunflower oil, followed by soybean oil and tributyrin; indices on other fatty substrates were low. Strains isolated from raw coffee beans showed the highest mean index, followed by those from biohumus and garden compost; the lowest index being for strains isolated from hazelnuts. Thus, the lipolytic activity index depended on the specific fatty substrate and the source of the isolates.     

Relatedness of Thermomyces lanuginosus strains producing a thermostable xylanase

Properties of an endo-beta-xylanase produced by a locally isolated Thermomyces lanuginosus strain SSBP was compared to seven other T. lanuginosus strains isolated from different geographical regions. Strain SSBP produced the highest xylanase activity of 59600 nkat ml(-1) when cultivated on corn cobs (maize) medium, whereas the seven other strains produced xylanase activities ranging from 6000 to 32000 nkat ml(-1). No cellulase activity was produced by the strains. Despite the variability in the production of xylanase, little difference in the other characteristics of the strains could be found. The optimal temperature and pH for xylanase production by the strains was either 40 or 50 degrees C and between pH 6 and 7, respectively. Optimal xylanase activity of the strains was observed at 70 degrees C and at pH 6 or 6.5. Culture supernatant analysis by SDS-PAGE and isoelectric focusing PAGE of all strains revealed the presence of a single 24.7 kDa and pI 3.9 xylanase. Phylogenetic analysis by PCR amplification and sequencing of the internal transcribed spacer of nuclear rRNA repeat units and 5.8S rDNA revealed no strain diversity. However, random amplified polymorphic DNA analysis pointed to greater diversity and with one primer (5'-GCCCGACGCG-3'), a relationship was established between xylanase levels and the RAPD pattern.     

Thermomyces lanuginosus: properties of strains and their hemicellulases

The non-cellulolytic Thermomyces lanuginosus is a widespread and frequently isolated thermophilic fungus. Several strains of this fungus have been reported to produce high levels of cellulase-free beta-xylanase both in shake-flask and bioreactor cultivations but intraspecies variability in terms of beta-xylanase production is apparent. Furthermore all strains produce low extracellular levels of other hemicellulases involved in hemicellulose hydrolysis. Crude and purified hemicellulases from this fungus are stable at high temperatures in the range of 50-80 degrees C and over a broad pH range (3-12). Various strains are reported to produce a single xylanase with molecular masses varying between 23 and 29 kDa and pI values between 3.7 and 4.1. The gene encoding the T. lanuginosus xylanase has been cloned and sequenced and is shown to be a member of family 11 glycosyl hydrolases. The crystal structure of the xylanase indicates that the enzyme consists of two beta-sheets and one alpha-helix and forms a rigid complex with the three central sugars of xyloheptaose whereas the peripheral sugars might assume different configurations thereby allowing branched xylan chains to be accepted. The presence of an extra disulfide bridge between the beta-strand and the alpha-helix, as well as to an increase in the density of charged residues throughout the xylanase might contribute to the thermostability. The ability of T. lanuginosus to produce high levels of cellulase-free thermostable xylanase has made the fungus an attractive source of thermostable xylanase with potential as a bleach-boosting agent in the pulp and paper industry and as an additive in the baking industry.     

Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion

Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of alpha-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).     

疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计提高其活性和温度稳定性

目的:通过对疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计,获得高酶活与耐高温的脂肪酶品种,为脂肪酶在饲料、油脂加工和生物柴油等领域的应用奠定基础.方法:对脂肪酶典型结构域lid和loop区域的系统发育分析,找到候选的位点,理性设计并通过实验验证,获得脂肪酶活性和耐高温特性显著提高的...     

Cellulase-free xylanase by thermophilic fungi: a comparison of xylanase production by two Thermomyces lanuginosus strains

Thermomyces lanuginosus strains RT9 and MH4 were studied to find favourable cultivation conditions and to compare their abilities to produce xylanolytic enzymes in three media on different substrates at 50° C or 55° C under shake-culture conditions. Both organisms produced xylanases free of cellulase at widely different levels in all cultivation conditions employed. Wheat bran, corn cobs and xylan induced xylanases in increasing order of producing with both cultures. T. lanuginosus RT9 demonstrated the highest xylanase production in all cultivation conditions but with lower soluble protein, reducing sugar, -xylosidase and debranching enzymes levels (arabinosidase, acetylxylanesterase, mannanase) when compared to T. lanuginosus MH4. The study reveals that xylanase production was highly influenced by nitrogen sources and their concentrations and by the initial pH in the cultures. The two strains may therefore be unique, when technical application is considered in terms of the quantity and quality of the xylanolytic enzymes produced.     

疏棉状嗜热丝孢菌脂肪酶基因在毕赤酵母工程菌中的遗传稳定性

【背景】重组工程菌的传代稳定性是保证外源蛋白高效稳定表达的前提,是决定工业化生产能力的关键因素之一。【目的】对异源的疏棉状嗜热丝孢菌脂肪酶基因在毕赤酵母重组工程菌中的遗传稳定性进行研究。【方法】将工程菌连续传代15次,取第1、5、10、15代作为受检代次,结合重组菌的菌落及菌体形态、水解酶活、目的基因片段、外源基因拷贝数等指标综合评价其遗传稳定性。【结果】传代过程中重组菌的菌落和菌体形态、目的蛋白分子量、目的基因序列均保持一致。目的基因的整合拷贝数经5次传代后发生一定损失,但随后稳定为7左右,而脂肪酶的相对酶活则提高至90%以上。【结论】适量的整合拷贝数更有利于该脂肪酶基因在毕赤酵母重组菌中的表达,经综合评价此工程菌的遗传稳定性良好,应用于工业化大规模生产是可行的。     

嗜热真菌DSM10635生产耐热木聚糖酶的小试研究

应用嗜热真菌Thermomyces lanuginosus DSM10635,采用固体发酵的方法探索耐热木聚糖酶的优化生产条件。在研究玉米芯,玉米皮,玉米秆,麸皮,松树屑,桦树屑等不同底物,在不同温度、玉米芯颗粒大小以及料水比条件下培养比较酶产量后,发现该嗜热真菌产耐热木聚糖酶的最佳底物为玉米芯或玉米皮,最佳培养温度为50℃--55℃,在加水量为1份玉米芯：2．8份水,玉米芯的颗粒直径大约为1mm时产酶量最高。实验结果显示,嗜热真菌DSM10635在优化后的培养条件下木聚糖酶产量可达到12525．80IU／g玉米芯。     

Optimized butyl butyrate synthesis catalyzed by Thermomyces lanuginosus lipase

Butyl butyrate is an ester present in pineapple flavor, which is very important for the food and beverages industries. In this work, the optimization of the reaction of butyl butyrate synthesis catalyzed by the immobilized lipase Lipozyme TL‐IM was performed. n‐Hexane was selected as the most appropriate solvent. Other reaction parameters such as temperature, substrate molar ratio, biocatalyst content and added water, and their responses measured as yield, were evaluated using a fractional factorial design, followed by a central composite design (CCD) and response surface methodology. In the fractional design 2^4–1, the four variables were tested and temperature and biocatalyst content were statistically significant and then used for optimization on CCD. The optimal conditions for butyl butyrate synthesis were found to be 48°C; substrate molar ratio 3:1 (butanol:butyric acid); biocatalyst content of 40% of acid mass. Under these conditions, over 90% of yield was obtained in 2 h. Enzyme reuse was tested by washing the biocatalyst with n‐hexane or by direct reuse. The direct reuse produced a rapid decrease on enzyme activity, while washing with n‐hexane allowed reusing the enzyme for five reactions cycles keeping approximately 85% of its activity. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1416–1421, 2013     

Ionic strength-dependent denaturation of Thermomyces lanuginosus lipase induced by SDS

Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10 mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS–lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.     

Production and properties of hemicellulases by a Thermomyces lanuginosus strain

Thermophilic fungi producing extremely high beta-xylanase and their associated hemicellulases have attracted considerable attention because of potential industrial applications. Thermomyces lanuginosus strain SSBP isolated from soil, produced beta-xylanase activity of 59 600 nkat ml-1 when cultivated on a medium containing corn cobs as substrate and yeast extract as nitrogen source. Lower beta-xylanase activities were produced after growth on other xylan substrates, sugars and soluble starch. Other hemicellulases were produced extracellularly at significantly lower levels than the beta-xylanase activity produced on corn cobs. No cellulase activity was observed. The optimal conditions for beta-xylanase production were 50 degrees C and pH 6.5, whereas 70 degrees C and between pH 5. 5 and 9.5 were optimal for beta-xylanase activity. The temperature optima for other hemicellulases were less than the xylanase with the exception of beta-mannosidase. The pH optima of the other hemicellulases were between 5.0 and 6.5. Xylanase was stable up to 70 degrees C and between pH 5.5 and 9.0 for 30 min whereas the other hemicellulase were less stable. These results suggest that the most suitable conditions for hydrolysis of hemicellulose by these enzymes would be at 50 degrees C and pH 6.0.     

Reactivation of covalently immobilized lipase from Thermomyces lanuginosus

Lipase from Thermomyces lanuginosus (TLL) immobilized on cyanogen bromide agarose (CNBr) may be fully inactivated when incubated in saturated solutions of guanidine. When this inactivated enzyme is re-incubated in aqueous medium, 20% of the activity may be recovered for several cycles. However, if the activity was determined in the presence of a detergent (CTAB, an activator of this enzyme), 100% of the initial activity in the presence of detergent was recovered. The enzyme was also inactivated in the presence of organic solvents and at high temperatures. Inactivations were more rapid when the activity was determined in absence of detergent. In both cases, some activity could be recovered just by incubation under mild conditions, and this increase was higher if the activity measurements were performed in the presence of CTAB. These results suggested that the opening of the lipase could be a critical step in the inactivation or reactivation of immobilized TLL. In inactivations in the presence of solvents, 100% of activity could be recovered during several cycles, while in thermal inactivations, the recovered activity decreased in each inactivation–reactivation cycle. The incubation of the enzyme inactivated by temperature in guanidine improved the results, but still 100% could not be achieved during several cycles even measured in the presence of CTAB.Thus, the simple incubation of the partially or fully inactivated enzyme under mild conditions permitted to recover some activity (enhancing the half life of the biocatalysts), even in thermal inactivations.     

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     

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.     

Structure of ribosomes from Thermomyces lanuginosus by electron microscopy and image processing

Multivariate statistical analysis and hierarchical ascendant classification techniques have been used to sort electron images of ribosomes from the thermophilic fungus Thermomyces lanuginosus into their characteristic views. Three predominant views were elucidated, called here overlap, non-overlap and top, showing reproducible detail approaching 1.8 nm resolution. The overlap and non-overlap forms of the fungal ribosomes appeared to be similar to those from the eubacterium Escherichia coli, despite differences in rRNA composition. The non-overlap projection predominated for the fungal complexes, suggesting different adsorption properties for ribosomes from the two species. Additionally, the top view has not been previously described for eubacteria. No major morphological differences could be detected between the fungal and eubacterial ribosomes at the resolution achieved in this study, suggesting a strong conservation of tertiary structure of this macromolecular complex despite the evolutionary gap between these two organisms.     

Activation, inhibition, and destabilization of Thermomyces lanuginosus lipase by detergents

Lipases catalyze the hydrolysis of triglycerides and are activated at the water-lipid interface. Thus, their interaction with amphiphiles such as detergents is relevant for an understanding of their enzymatic mechanism. In this study, we have characterized the effect of nonionic, anionic, cationic, and zwitterionic detergents on the enzymatic activity and thermal stability of Thermomyces lanuginosus lipase (TlL). For all detergents, low concentrations enhance the activity of TlL toward p-nitrophenyl butyrate by more than an order of magnitude; at higher detergent concentrations, the activity declines, leveling off close to the value measured in the absence of detergent. Surprisingly, these phenomena mainly involve monomeric detergent, as activation and inhibition occur well below the cmc for the nonionic and zwitterionic detergents. For anionic and cationic detergents, activation straddles the monomer-micelle transition. The data can be fitted to a three state interaction model, comprising free TlL in the absence of detergent, an activated complex with TlL at low detergent concentrations, and an enzyme-inhibiting complex at higher concentrations. For detergents with the same headgroup, there is an excellent correspondence between carbon chain length and ability to activate and inhibit TlL. However, the headgroup and number of chains also modulate these effects, dividing the detergents overall into three broad groups with rising activation and inhibition ability, namely, anionic and cationic detergents, nonionic and single-chain zwitterionic detergents, and double-chain zwitterionic detergents. As expected, only anionic and cationic detergents lead to a significant decrease in lipase thermal stability. Since nonionic detergents activate TlL without destabilizing the protein, activation/inhibition and destabilization must be independent processes. We conclude that lipase-detergent interactions occur at many independent levels and are governed by a combination of general and structurally specific interactions. Furthermore, activation of TlL by detergents apparently does not involve the classical interfacial activation phenomenon as monomeric detergent molecules are in most cases responsible for the observed increase in activity.     

Directed evolution of the thermostable xylanase from Thermomyces lanuginosus

The thermostability of the endo-beta-1,4-xylanase from Thermomyces lanuginosus (xynA) was improved by directed evolution using error-prone PCR. Transformants expressing the variant xylanases were first selected on 0.4% Remazol Brilliant Blue-xylan and then exposed to 80 degrees C. Whereas the wild type XynA lost 90% activity after 10 min at 80 degrees C, five mutants displayed both higher stabilities and activities than XynA. Four mutants were subjected to further mutagenesis to improve the stability and activity of the xylanase. Subsequent screening revealed three mutants with enhanced thermostability. Mutant 2B7-10 retained 71% of its activity after treatment at 80 degrees C for 60 min and had a half-life of 215 min at 70 degrees C, which is higher than that attained by XynA. Sequence analysis of second generation mutants revealed that mutations were not concentrated in any particular region of the protein and exhibited much variation. The best mutant obtained from this study was variant 2B7-10, which had a single substitution (Y58F) in beta-sheet A of the protein, which is the hydrophilic, solvent-accessible outer surface of the enzyme. Most of the mutants obtained in this study displayed a compromise between stability and activity, the only exception being mutant 2B7-10. This variant showed increased activity and thermostability.