Simple, low-cost molecular assays for TR34/L98H mutations in the cyp51A gene for rapid detection of triazole-resistant Aspergillus fumigatus isolates.

Simple, low-cost PCR/PCR-restriction fragment length polymorphism (RFLP) assays targeting cyp51A promoter and codon 98 regions were developed for the detection of triazole-resistant Aspergillus fumigatus strains carrying TR34/L98H mutations. The assays were evaluated using 40 itraconazole-susceptible isolates and 35 itraconazole-resistant isolates. The prevalence of TR34/L98H mutations in clinical/environmental A. fumigatus isolates may now be determined easily from resource-poor settings.

Laccase production optimization by response surface methodology with Aspergillus fumigatus AF1 in unique inexpensive medium and decolorization of different dyes with the crude enzyme or fungal pellets.

In the present study, alkaline pretreatment was applied for the enhanced laccase production from rice straw. Various process parameters including sodium hydroxide concentration, pH and fermentation temperature were optimized via response surface methodology (RSM) with a Box-Behnken design (BBD). Through regression analysis, it was found that laccase activity was well fitted by a quadratic polynomial equation (R(2)=0.998, Adj R(2)=0.995), and the fermentation temperature was the most significant factor influencing laccase activity. The optimized process conditions found were NaOH concentration of 0.39 mol L(-1), pH 3.12 and temperature 25.43 °C, under which laccase activity reached 142,198 ± 3586 U L(-1). Further studies were carried out to probe different dyes decolorization ability of laccase produced by Aspergillus fumigatus, A. fumigatus pellets and whole fermentation broth (WFB) using sodium hydroxide pretreated rice straw as sole carbon source. Results showed that pure laccase demonstrate limited decolorization ability to all the studied dyes, while crude laccase, A. fumigatus pellets and WFB exhibit significant decolorization ability to all the studied dyes with WFB being the most excellent one. Effectiveness of degradation was confirmed by uv-vis and phytotoxicity studies, which indicated that A. fumigatus transformed the dyes into non-toxic metabolites.

Low cost single-step purification of endoglucanase from Aspergillus fumigatus ABK-9.

Low cost agro-waste was used as adsorption support for single-step purification of endoglucanase from the culture filtrate of A. fumigatus ABK-9. Among various agro-waste substrates, 1% NaOH pretreated rice bran was proved to be the best for adsorbing about 74.8 and 71.1% of endoglucanase at 4 degrees C and 10 degrees C respectively. Langmuir type adsorption isotherm at 4 degrees C showed maximum adsorption of enzyme at pH 5.0, which was in the range of optimum pH of the enzyme. The rice bran column bound enzyme was maximally eluted by a mixture of acetate buffer (0.05 M, pH 5.5) and ethanol (40%, v/v) at a ratio of 3:2 and a flow rate of 1 mL/min. A 5.52-fold purification of the enzyme was achieved from culture supernatant. The specific activity and recovery yield after purification were 294.0 U/mg and 40.15%, respectively, which were comparable with other contemporary protocols. The homogeneity of the enzyme was tested through sodium dodecyl sulphate polyacrylamide gel electrophoresis and a single band of 56.3 kDa was observed. Zymogram analysis finally confirmed the occurrence of endoglucanase in the single band.

Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus.

A putative dimethylallyltryptophan synthase gene, fgaPT2, was identified in the genome sequence of Aspergillus fumigatus. fgaPT2 was cloned and overexpressed in Saccharomyces cerevisiae. The protein FgaPT2 was purified to near homogeneity and characterized biochemically. This enzyme was found to convert L-tryptophan to 4-dimethylallyltryptophan, a reaction known to be the first step in ergot alkaloid biosynthesis. FgaPT2 is a soluble, dimeric protein with a subunit size of 52 kDa, and contains no putative prenyl diphosphate binding site (N/D)DXXD. Km values for L-tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 microM, respectively. Metal ions, such as Mg2+ and Ca2+, enhance the reaction velocity, but are not essential for the enzymic reaction. FgaPT2 showed a relatively strict substrate specificity for both tryptophan and DMAPP. FgaPT2 is the first enzyme in the biosynthesis of ergot alkaloids to be purified and characterized in homogeneous form after heterologous overproduction.

An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha.

An efficient process was developed for the low-cost production of phytases using Hansenula polymorpha. Glucose or glucose syrups, previously reported as repressive substrates, were used as main carbon sources during fermentation. Glucose was even the most productive substrate for high-level production of phytases. Compared with the process using glycerol, the standard carbon source used for this process until now, the use of glucose led to a reduction of more than 80% in the raw materials costs. In addition, exceptionally high concentrations of active enzyme (up to 13.5 g/L) were obtained in the medium, with phytase representing over 97% of the total accumulated protein. These levels greatly exceed those reported so far for any yeast-based expression system. Very efficient downstream processing procedures were developed with product recovery yields over 90%. Both the fermentation and downstream processing were successfully tested in pilot scale up to 2000 L. As a result, H. polymorpha can be used as a highly competitive system for low-cost phytase production.