A mass rearing method for the linyphiid spider species Erigone atra (Blackwall) (Araneae: Linyphiidae)

Abstract:  A mass rearing method for Erigone atra (Blackwall) (Araneae: Linyphiidae) allowing continuous laboratory rearing is described. Twenty 1–2-day old spiderlings were kept together in plastic boxes, which were filled with soil containing a culture of the Collembola species, Lepidocyrtus lanuginosus (Gmelin) (Entomobryidae), and serving the spiders as a continuous available prey source. Once per week vestigial-wing fruit flies of Drosophila melanogaster Meigen (Diptera: Drosophilidae) were provided as additional prey. In addition, the rearing boxes were filled with wood-wool serving spiders as points of contact for their webs. After 3–5 weeks most of the spiderlings developed to adults, which were separated individually into glass tubes filled with soil and Collembola until all of them became adult. To produce a new generation of spiders 20–40 adult/subadult spiders originating from different mass rearing boxes were brought together and kept and fed in the same way as the spiderlings. Within a few days females started to produce eggsacs. The eggsacs were transferred into glass tubes filled with a layer of moist plaster of Paris until the spiderlings hatched, which were then bred as described above. Erigone atra was bred over 12 generations within a period of 2 years. The mean rearing success (from 1 to 2-day-old spiderlings to adults) was 59.3%. Decreasing rearing success, decrease of fecundity or decrease of adult spider size were not observed. Advantages and use of the mass rearing method are discussed in relation to rearing methods for other spiders.     

Scale-up of flat-plate reactors for enzymatic hydrolysis of fats and oils

To determine the feasibility of continuous enzymatic fat-splitting, immobilized lipase reactors were constructed from alternating layers of enzyme support material and separator screens. Partially purified lipase from Thermomyces lanuginosus was loaded onto the support material at pH 5.5 by irreversible adsorption. Melted edible tallow at 51°C was pumped through the immobilized enzyme layers and swept from the downstream separator screens by buffer recycled from a continuous oil/water separator. Results from continuous operation of 10-layer reactors were compared with data from single-layer reactors. The activity per square centimeter of 10-layer reactors was nearly as much as that of single-layer reactors at the same enzyme loading and oil feed rate. Data were fitted to an empirical mathematical model.     

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.     

Transcriptional analysis of the cyclophilin A gene (cypA) of Streptomyces chrysomallus

In the thermophilic fungus Thermomyces lanuginosus, invertase displays an unusual pattern of development: the induced activity begins to diminish even before any substantial quantity of sucrose has been utilized or an appreciable amount of biomass has been produced. Despite this pattern of invertase activity, neither the growth rate nor the final mycelial yield is affected adversely. T. lanuginosus invertase is a thiol protein and the enzyme is active when specific sulfhydryl group(s) is in the reduced state. Measurements of reduced coenzyme and glutathione pools in sucrose-growth mycelia excluded oxidative stress as the primary reason for the observed decline in invertase activity. Rather, this unusual pattern of invertase is considered to be due to its localization in the hyphal tips. At the early stage of growth, the number of hyphal tips per unit mass of mycelium is maximum, whereas at later times their numbers do not increase in proportion to the biomass. As a result invertase activity shows an apparent inverse relationship with biomass. The enzyme activity disappears when the inducing carbon source is consumed and growth is completed.     

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).     

一种耐热几丁质酶的产生及其稳定性研究

疏绵状嗜热丝孢菌(Thermomyces lanuginosus)SY2 在以胶状几丁质为唯一碳源的诱导培养基中产生了胞外几丁质酶.该酶在50℃保温1 h,酶活稳定;65℃时半衰期为25 min;酶液在室温下保存到12周,残余酶活性为45%左右.该酶有较宽的pH范围,3.0～9.0之间保持稳定,pH值为2.5时,仍具有70%的剩余酶活性.Ca2 对几丁质酶的活性有显著的激活作用;高浓度变性剂对酶有抑制作用.结果表明该酶是一种热稳定性高且耐酸碱的新型几丁质酶,能在酸性和高温环境中发挥作用,这些特性赋予了T.lanuginosus几丁质酶在几丁质的生物转化及其它生物技术中极大的应用优势.     

Unexpected reversal of the regioselectivity in <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> lipase-catalyzed acylation of floxuridine

Unexpected inversion of the 3′:5′-regioselectivity was observed in the enzymatic methacryloylation, crotonylation and cinnamoylation of floxuridine (1.5:1, 2.3:1 and 4.4:1, respectively), where Thermomyces lanuginosus lipase preferentially catalyzed the acylation of 3′-hydroxyl rather than that of 5′-hydroxyl group. The possible reason might be the presence of a remote interaction between the unsaturated bond in the acyl group and the aromatic ring of amino acid residue Trp89 in the lid of the lipase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inducible character of β‐xylanase in a hyperproducing mutant of Thermomyces lanuginosus

Ten strains of Thermomyces lanuginosus from various culture collections were evaluated for extracellular endo‐β‐1,4‐xylanase production. The best xylanase producer (5771±173 nkat/mL) T. lanuginosus SK, was subjected to UV and N‐methyl‐N‐nitro‐N‐nitrosoguanidine mutagenesis. A mutant strain T. lanuginosus MC134, that showed on oatspelts xylan a 1.5 fold higher xylanase production than the parent strain SK, was subjected to a study of the regulation of xylanase synthesis during growth on various carbohydrates and during induction in glucose‐grown cells. In the growth experiments the highest production of xylanase was observed in the presence of xylans, however, an appreciable amount of the enzyme, about 10%, was also produced during growth on xylose. Xylobiose was found to be the most efficient xylanase inducer in the glucose‐grown cells. Its induction efficiency was followed by xylose, beechwood and birchwood xylan. Xylanase induction by polysaccharides started several hours later but proceeded for a longer time than that induced by the low molecular mass inducers, indicating that the polysaccharides serve as more sustainable source of inducers and that they have to be first hydrolyzed by the low level of constitutively synthesized xylanase. The repression of the induction of xylanase by glucose confirmed that the xylanase synthesis in the mutant strain is similar to the parent strain and exhibits an induction‐repression regulation mechanism.     

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.     

A lipase‐catalyzed process for green synthesis of temsirolimus

Temsirolimus is an intravenous drug for the treatment of renal cell carcinoma that can be prepared using enol acyl donors, which is not favorable in process development. An improved enzymatic process to prepare temsirolimus has been developed employing lipase‐catalyzed regioselective acylation of rapamycin with environmentally friendly acyl donors. After screening of common commercial lipases and none‐enol acyl donors, it was found that p‐nitrophenyl 2,2,5‐trimethyl‐1,3‐dioxane‐5‐carboxylate reacted as efficient acyl donor when catalyzed by immobilized Thermomyces lanuginose lipase. By optimizing the process conditions (i.e., reaction temperature, solvents, and additives), the reaction time was significantly shortened while the reaction conversion reached 95.4% in methyl tert‐butyl ether after 48 h at 50°C using the new acyl donors. This work demonstrated a cost‐effective, efficient, and scalable process to synthesize temsirolimus.