Process optimization of xylanase production using cheap solid substrate by Trichoderma reesei SAF3 and study on the alteration of behavioral properties of enzyme obtained from SSF and SmF.

This study aimed to assess the variability in respect of titer and properties of xylanase from Trichoderma reesei SAF3 under both solid-state and submerged fermentation. SSF was initially optimized with different agro-residues and among them wheat bran was found to be the best substrate that favored maximum xylanase production of 219 U (gws)(-1) at 96 h of incubation. The mycelial stage of the fungi and intracellular accumulation of Ca(++) and Mg(++) induced maximum enzyme synthesis. Inoculum level of 10 × 10(6) spores 5 g(-1) of dry solid substrate and water activity of 0.6 were found to be optimum for xylanase production under SSF. Further optimization was made using a Box-Behnken design under response surface methodology. The optimal cultivation conditions predicted from canonical analysis of this model were incubation time (A) = 96-99 h, inoculum concentration (B) = 10 × 10(6) spores 5 g(-1) of dry substrate, solid substrate concentration (C) = 10-12 g flask(-1), initial moisture level (D) = 10 mL flask(-1) (equivalent to a(w) = 0.55) and the level of xylanase was 299.7 U (gws)(-1). Subsequent verification of these levels agreed (97 % similar) with model predictions. Maximum amount of xylanase was recovered with water (6:1, v/w) and under shaking condition (125 rpm). Purified xylanase from SSF showed better stability in salt and pH, was catalytically and thermodynamically more efficient over enzyme from SmF, though molecular weight of both enzymes was identical (53.8 kDa).

Characterization of tannase protein sequences of bacteria and fungi: an in silico study.

The tannase protein sequences of 149 bacteria and 36 fungi were retrieved from NCBI database. Among them only 77 bacterial and 31 fungal tannase sequences were taken which have different amino acid compositions. These sequences were analysed for different physical and chemical properties, superfamily search, multiple sequence alignment, phylogenetic tree construction and motif finding to find out the functional motif and the evolutionary relationship among them. The superfamily search for these tannase exposed the occurrence of proline iminopeptidase-like, biotin biosynthesis protein BioH, O-acetyltransferase, carboxylesterase/thioesterase 1, carbon-carbon bond hydrolase, haloperoxidase, prolyl oligopeptidase, C-terminal domain and mycobacterial antigens families and alpha/beta hydrolase superfamily. Some bacterial and fungal sequence showed similarity with different families individually. The multiple sequence alignment of these tannase protein sequences showed conserved regions at different stretches with maximum homology from amino acid residues 389-469 and 482-523 which could be used for designing degenerate primers or probes specific for tannase producing bacterial and fungal species. Phylogenetic tree showed two different clusters; one has only bacteria and another have both fungi and bacteria showing some relationship between these different genera. Although in second cluster near about all fungal species were found together in a corner which indicates the sequence level similarity among fungal genera. The distributions of fourteen motifs analysis revealed Motif 1 with a signature amino acid sequence of 29 amino acids, i.e. GCSTGGREALKQAQRWPHDYDGIIANNPA, was uniformly observed in 83.3 % of studied tannase sequences representing its participation with the structure and enzymatic function.

Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads.

In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12+/-0.18 U ml(-1) at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36+/-0.2 U ml(-1)).

Production of cellulase-free xylanase by Trichoderma reesei SAF3

A xylanase producing fungi has been isolated from soil and identified as Trichoderma reesei SAF3. Maximum growth of the organism was found at 48 h under submerged condition in xylan containing enriched medium, whereas highest enzyme production (4.75U/mL) was recorded at 72 h. No detectable cellulase activity was noted during whole cultivation period. The partially purified enzyme hydrolyzed xylan into xylopentose and xylose. All these properties of xylanase highlighten its promising uses in industrial scale.

A partir de solo, isolou-se uma cepa de fungos produtos de xilanase, posteriormente identificado como Trichoderma reesei SAF3. O crescimento máximo do fungo foi obtido após 48h em condições submersas em meio de cultura contendo xilano, enquanto produção máxima de enzima (4,75U/mL) ocorreu em 72h. Durante o período de cultivo, não foi detectada atividade celulásica. A enzima parcialmente purificada hidrolizou xilano a xilopentose e xilose. Essas propriedades da xilanase destacam seu uso promissor em escala industrial.