ABSTRACT
Aspergillus terreus is a ubiquitous fungus in our environment. It is an opportunistic human pathogen and economically important as the main producer of lovastatin, a cholesterol lowering drug. Our aim was to examine the genetic variability of A. terreus and closely related species using molecular and analytical techniques. Lovastatin production was examined by HPLC. Lovastatin was produced by seven isolates belonging to the species A. terreus. RAPD analyses were carried out using 25 different random primers. Neighbor-joining analysis of RAPD data (120 characters) resulted in clustering of the A. terreus isolates into distinct groups. Some correlation was observed between lovastatin producing abilities of the isolates and their position on the dendrogram based on RAPD profiles. The internal transcribed spacer region and the 5.8S rRNA gene of A. terreus and related isolates was also sequenced. Phylogenetic analysis of sequence data let us classify the isolates into different clades which mostly correspond to the species Aspergillus terreus, Aspergillus flavipes, Aspergillus niveus, Aspergillus carneus and Aspergillus janus/A. janus var. brevis. Aspergillus allahabadii, A. terreus var. aureus and A. niveus var. indicus belonged to the A. niveus clade, while an Aspergillus isolate previously classified as A. niveus was most closely related to A. flavipes isolates. Aspergillus anthodesmis formed a distinct branch on the tree. Although it was previously suggested based on 28S rDNA sequence data that Aspergillus section Terrei should include A. carneus and A. niveus isolates, phylogenetic analysis of ITS sequences indicate that A. flavipes isolates are more closely related to A. terreus than A. carneus isolates. Our data suggest that sections Terrei and Flavipedes should be merged. However, further loci should be analysed to draw more definite conclusions.
Subject(s)
Aspergillus/classification , Aspergillus/genetics , Evolution, Molecular , Animals , Anticholesteremic Agents/metabolism , Aspergillus/isolation & purification , Aspergillus/metabolism , DNA, Fungal/analysis , DNA, Fungal/isolation & purification , DNA, Ribosomal Spacer , Genes, rRNA , Humans , Lovastatin/metabolism , Molecular Sequence Data , Mycological Typing Techniques , Phylogeny , RNA, Ribosomal, 5.8S , Random Amplified Polymorphic DNA Technique , Sequence Analysis, DNAABSTRACT
Thirty Trichoderma strains representing 15 species within the genus were screened for extracellular production of chitinolytic enzymes in solid substrate fermentation. Trichoderma longibrachiatum IMI 92027 (ATCC 36838) gave the highest yield (5.0 IU/g of dry matter of substrate) after 3 d of fermentation on wheat bran-crude chitin (9:1 mixture) medium. The optimal moisture content (66.7%), chitin content (20%), initial pH of the medium (2.0-5.0), and time course (5 d) of solid substrate fermentation were determined for strain IMI 92027. Cellulase, xylanase, alpha-amylase, and beta-xylosidase activities were also detected. The pH and temperature optima of the chitinase complex of T. longibrachiatum IMI 92027 were 4.5 and 55 degrees C, respectively. The enzyme totally lost its activity at 70 degrees C in 5 min in the absence of the substrate but retained about 15% of its initial activity even at 70 degrees C after a 60-min incubation in the presence of solid substrate fermentation solids. Purification of protein extract from the solid substrate fermentation material revealed high chitinolytic activities between pI 5.9 and 4.8, where N-acetyl-beta-D-hexosaminidase and chitinase peaks have been found in the same pI range. Two chitinases of 43.5 and 30 kDa were purified at acidic pI.
