Solid-state production of polygalacturonase by Aspergillus sojae ATCC 20235
Introduction
Enzyme production which is a growing field of biotechnology has an annual world sales close to a billion dollars together with increasing number of patents and research articles (Layman, 1990, Gonzales et al., 2003). Even though most of the enzyme manufactures produce enzymes by means of submerged fermentation (SmF) techniques, there is recently a significant interest in using solid-state fermentation (SSF) instead (Pandey et al., 1999, Gonzales et al., 2003). The reason behind this, is cited by the advantages that this technique (SSF) has over SmF. These are listed as higher productivity per reactor volume, lower capital costs, less space requirements, simpler equipment and easier downstream processing (Pandey et al., 2000, Martins et al., 2002). Another very important advantage is that, it permits the use of agricultural and agro-industrial residues as substrates which are converted into bulk chemicals and into products with high commercial value like organic acids, proteins, alcohol and enzymes (Manonmani and Sreekantiah, 1987, Spangnuolo et al., 1997, Martins et al., 2002). Furthermore, the utilization of these compounds helps in solving pollution, which otherwise cause their disposal (Couto and Sanroman, 2005, Pandey et al., 1999).
Large numbers of microorganisms, including bacteria, yeast and fungi can be used in SSF systems. Selection of a particular strain, however, remains a tedious task depending upon a number of factors, like the nature of the substrate, environmental conditions and the desired final product. Filamentous fungi are the best-adapted species of SSF and dominate in research and practical application around the world. This can be attributed to the advantage of the hyphal mode of growth with the colonization on solid substrates and utilization of variable substrates (Knapp and Howell, 1980).
Pectinases or pectinolytic enzymes (polygalacturonase, pectin methylesterase, pectin lyase) which are well known with their commercial significance among the enzymes, are mainly produced using SSF technique. Pectinases which hold a share of 25% in the global sales of food enzymes (Jayani et al., 2005), are widely used in the beverage industry due to their ability to improve pressing and clarification of concentrated fruit juices. Besides, they are used in processing of fruits and vegetables, in the production of wine, in the extraction of olive oil and fermentation of tea and coffee (Castilho et al., 2000, Silva et al., 2005). Furthermore, they find applications in paper and pulp industry, waste management, animal feed and textile industry (Silva et al., 2002).
Among the pectinases used, polygalacturonase produced by Aspergillus niger is the most studied one with commercial significance (Castilho et al., 2000, Couto and Sanroman, 2005). However, to best of our knowledge there is no available literature on the production of this enzyme (polygalacturonase) by Aspergillus sojae in either mode (SSF or SmF), which makes this study extra novel.
In order to determine the potential of this strain, and contribute to the literature by filling this gap, a study was initiated using A. sojae ATCC 20235 in the production of the polygalacturonase from here on PG, which has a highly commercial value. With our previous study on submerged fermentation using this organism we proved that this organism has a good potential for PG synthesis with the desired pellet morphology (Gogus et al., 2006). Therefore, it was worth wide to determine if this potential could be increased using SSF. Together with the high enzyme productivity in SSF, the production of high spore counts obtained through this technique, is of great significance for industrial submerged fermentations requiring spore inoculations.
With this perspective, using statistical tools (RSM), these responses were optimized, first independently and later together with respect to the factors of source of solid substrate, inoculum and incubation time. As it is well known these factors are highly significant in SSF (Sangeetha et al., 2004, Couto and Sanroman, 2005) and very specific to every single strain. In solid-state fermentations, the selection of a suitable solid substrate for a fermentation process is a critical factor. The selection of suitable substrates for pectinase production has mainly been centered on tropical agro-industrial crops and residues. Among these are wheat bran, rice bran, apple pomace, orange bagasse, sugar cane bagasse, the most studied ones (Maldonado and Strasser de Saad, 1998, Bai et al., 2004). However, there is no study on the interaction of these substrates with factors like inoculum and incubation time, which are of primary importance in solid-state fermentations. With this perspective, inoculum (X1) and incubation time (X2) as quantitative variables, corncob, maize meal and crushed maize which are produced in surplus quantities worldwide (Kunamneni and Singh, 2005), as qualitative variables (X3) were taken for the optimization, using RSM for maximum PG activity and total spore generation.
Although the main focus of this paper is on SSF, a brief comparison on enzyme titers between the optimized conditions of SmF and SSF are given at the end.
The results of this study by introducing a strain with a new application mode in the production of a highly valuable enzyme, is thought to be beneficial to the enzyme industry, food industry and the microbiology area.
Section snippets
Microorganism
A. sojae ATCC 20235 (from here on A. sojae only for simplicity) was purchased in the lyophilized form, from Procochem Inc., an international distributor of ATCC (American Type of Culture Collection) in Europe. The propagation of this culture was done on YME agar slant medium containing, malt extract (10 g/l), yeast extract (4 g/l), glucose (4 g/l) and agar (20 g/l), incubated at 30 °C until well sporulation (1 week). Stock cultures of these strains were prepared with 20% glycerol water and stored at
Solid-state fermentation
The optimization procedure in solid-state fermentation was done in two steps. First, the selection of the suitable solid substrate for maximum PG activity and total spore count was done using D-optimal design (Table 1). In the second step, the best substrate which resulted into maximum enzyme activity (from step 1) was kept constant and two independent variables (X1 and X2) were optimised using 22 face centered CCD design as mentioned in Section 2.8.
Conclusion
Overall this study demonstrated that A. sojae which has not been considered so far, can be a good candidate for the production of polygalacturonase, an enzyme with highly commercial value, in SSF. Using a statistical tool (RSM) it was possible to point out, the interactive effect of solid substrate, inoculum and incubation time which are highly significant in SSF, on the response variables of PG activity and total spore formation. The optimized regions obtained through this technique will give
Acknowledgement
Financial support of Izmir Institute of Technology through the project IYTE 2004, 04 is gratefully acknowledged.
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