An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates.

Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl ß-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.

Comparison of stem cell properties of cells isolated from normal and inflamed dental pulps.

AIM: To compare cells from normal and inflamed human dental pulps regarding the presence of stem cells, their proliferation and differentiation potential. METHODOLOGY: Human dental pulp stem cells (hDPSCs) were isolated from normal (DPSC-N) and inflamed dental pulps (DPSC-I). They were compared in respect to proliferation (MTT assay), morphology and STRO-1 expression. STRO-1-positive cells were subject to proliferation (MTT and CFU counting) and morphological analyses and then submitted to odonto-osteogenic, adipogenic and condrogenic differentiation. Differentiated cells were evaluated concerning morphology and the expression, by qRT-PCR, of BSP, LPL and SOX-9 genes. The amount of mineralized matrix produced after odonto-osteogenic differentiation was compared with quantitative Alizarin Red staining. RESULTS: No difference was observed in the morphology and in the proliferation rate of DPSC-N and DPSC-I either before or after separation of STRO-1-positive cells. These cells represented 0.46% (±0.14) and 0.43% (±0.19) of the cell population from normal and inflamed dental pulps, respectively. Both DPSC-N and DPSC-I were capable of differentiating under the three assayed conditions and presented similar patterns for BSP, LPL and SOX-9 expression. Mineralized matrix production was also compatible. In all the quantitative experiments, differences were found between cells from each patient, either from normal or from inflamed pulps. Nonetheless, there was no statistical difference between these two groups. CONCLUSION: The morphology, proliferation rate and differentiation potential of DPSC-I were similar to the observed in DPSC-N, thus demonstrating that the inflammatory process did not affect the stem cell properties that were assessed.

Cloning, characterization and heterologous expression of the first Penicillium echinulatum cellulase gene.

AIMS: Penicillium echinulatum is effective for bioconversion processes. However, nothing is known about the molecular biology of its cellulolytic system. We describe for the first time the isolation, cloning and expression of a P. echinulatum cellulase cDNA (Pe-egl1) encoding a putative endoglucanase. METHODS AND RESULTS: Pe-egl1 cDNA was identified from random sequencing of a P. echinulatum cDNA library. The deduced EGL1 protein possibly belongs to the glycosyl hydrolase family 5A, with 387 amino acid residues and strong similarity with other fungal endoglucanases. The cDNA was heterologously expressed in Pichia pastoris. The recombinant EGL1 secreted by a Pic. pastoris recombinant strain revealed the characteristics of particular interest: an optimal activity over a broad pH range (5.0-9.0), and an optimal temperature of 60 degrees C. The recombinant EGL1 also showed high thermostability (84% of residual activity after 1 h of pre-incubation at 70 degrees C). Calcium exerted a strong stimulatory effect over EGL1 activity. CONCLUSIONS: Altogether, these results point to the potential application of this P. echinulatum endoglucanase in cellulose processing industries, particularly the textile one because of its biochemical properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization and heterologous expression of the first P. echinulatun cDNA inaugurates the exploitation of this potential industrial micro-organism.

Cell cycle, DNA replication, repair, and recombination in the dimorphic human pathogenic fungus Paracoccidioides brasiliensis

DNA replication, together with repair mechanisms and cell cycle control, are the most important cellular processes necessary to maintain correct transfer of genetic information to the progeny. These processes are well conserved throughout the Eukarya, and the genes that are involved provide essential information for understanding the life cycle of an organism. We used computational tools for data mining of genes involved in these processes in the pathogenic fungus Paracoccidiodes brasiliensis. Data derived from transcriptome analysis revealed that the cell cycle of this fungus, as well as DNA replication and repair, and the recombination machineries, are highly similar to those of the yeast Saccharomyces cerevisiae. Among orthologs detected in both species, there are genes related to cytoskeleton structure and assembly, chromosome segregation, and cell cycle control genes. We identified at least one representative gene from each step of the initiation of DNA replication. Major players in the process of DNA damage and repair were also identified.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.

Substrate-dependent differential expression of Humicola grisea var. thermoidea cellobiohydrolase genes.

Transcription of fungal cellulase genes may be affected by substrate induction. We studied the expression of Humicola grisea var. thermoidea cellobiohydrolase genes (cbh1.1 and cbh1.2) under induction by several soluble and insoluble carbon sources. Using the RT-PCR technique, the cbh1.2 transcript was detected in all the conditions assayed along the growth curve. Catabolite repression, which frequently occurs in other fungal celluloytic systems, was not observed. On the other hand, cbh1.1 transcription was shown to be driven by insoluble and complex lignocellulosic substrates. In summary, the cbh1.2 gene product is constitutively produced, while cbh1.1 seems to respond to a distinct regulatory mechanism.