The abfB gene from A. niger was overexpressed in the A. niger D15 protease-deficient strain under the transcriptional control of glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP) and glucoamylase terminator (glaAT). The recombinant A. niger D15
secreted extracellularly recombinant AbfB in both the standard 2xMM media and 2xMM media enriched with 2% CCSL, which was free of xylanase activity. Therefore, the transformed A. niger D15 strain presents an improved microbial system for production of the recombinant AbfB enzyme that can be used in crude form for selective hydrolysis of water soluble xylans substituted with arabinose side chains. The silver stained 10% SDS-PAGE clearly displayed prominent single protein species bands of the recombinant AbfB, which was produced in shake flasks on both standard 2xMM and 2xMM with 2% CCSL enriched medium (Fig. 4.8a). Although, multiple proteins species bands were visible in the silver stained 10% SDS-PAGE of the recombinant AbfB produced in the 10 L bioreactor on 2xMM media that was enriched with the 2% CCSL, the protein band for the recombinant AbfB was the most prominent (Fig. 4.8b). It is possible that the observed multiple protein species bands on the silver stained 10% SDS-PAGE originated from intracellular proteins or other protein compounds released upon breakage or lysing of the mycelium in the bioreactor (Fig. 4.6d (iv)) as the biomass was aging (Fig. 4.6c). These results suggest that the modification of the transcriptional control for the constitutive expression of the abfB gene in the A. niger D15 protease-deficient strain was effective in enabling selective extracellular secretion of recombinant AbfB free of xylanase activity.. The recombinant AbfB was secreted in media in the shake flasks (Fig. 4.6a) and bioreactor (Fig. 4.6b) cultures with volumetric activities against pNPA substrate of more than 20 times the volumetric activities of recombinant AbfB of 0.020 U mL-1 when it was previously expressed in S. cerevisiae (Crous et al., 1996). The A. niger microbial systems are considered prolific producers of functional industrial enzymes because of the inherent higher capacity for protein secretion compared to the S.
cerevisae based system. Such characteristic is probably because of highly efficient
protein posttranscriptional modification processes (Verdoes et al., 1997; Conesa et al., 2001; Punt et al., 2002). The specific activity of the recombinant AbfB against pNPA substrate was 2.9 U mg-1, which was higher than the specific activity of 2.7 U mg-1
97 observed in its partially purified state (Table 4.3). The results showed that the recombinant A. niger microbial system had increased capacity to express the recombinant AbfB extracellulary compared to wild type A. niger strains and other microbial expression systems reported in literature (Table 4.3). The results showed that the specific activity of the recombinant AbfB against the pNPA substrate both in crude and partially purified form, were higher than the specific activities of 2.6 U mg- 1
of crude A. niger B arabinofuranosidase reported by De Ioannes et al. (2000) and that of 1.07 Umg-1 for the recombinant AbfB expressed by Fusarium oxysporum f. sp.
dianthi (Carlos et al., 2004) (Table 4.3). The enhanced secretion could be attributed to
the expression of the recombinant AbfB that was under the transcriptional control of a strong gpdP promoter. A similar effect was observed with the expression of homologous feruloyl esterase B in protease deficient A. niger strain microbial system when expressed under a similar promoter (Levasseur et al., 2004). However, the recombinant AbfB protein yields obtained in this study (0.12-0.45 gL-1) were less than the potential protein yields of up to 30 g L-1 reported for A. niger industrial expression systems for production of other proteins (Dunn-Coleman et al., 1992; Kinghorn and Unkles, 1994; Conesa et al., 2001; Punt et al., 2001). Therefore, further improvements of the A. niger microbial systems might be necessary to increase the protein yields. The secretion of the recombinant AbfB by the A. niger D15 protease-deficient strain was growth associated (Fig. 4.6c). The results showed that the increase in the recombinant AbfB volumetric activity coincided with the increase in the biomass growth such that the maximum recombinant AbfB activity of 8.0 nkat mL-1 was achieved immediately after attaining the maximum biomass growth of 33.44 g L-1 between 36 and 48 h of cultivation. Furthermore, in the shake flask cultures, the recombinant AbfB protein concentration almost doubled (from 0.12 mg mL-1 to 0.2 mg mL-1) with enrichment of the standard 2xMM fermentation media with 2% (w/v) CCSL as an additional source of nitrogen (Table 4.2). Growth associated production of enzymes is characteristic of enzymes produced constitutively in A. niger (Gweyn, 1992; Shuler and Kargi, 2002). Such behaviour was also observed during production of α-glucoamylase by A. niger (Pedersen et al., 2000). Therefore, the production of the recombinant AbfB can be enhanced by maintaining high biomass production. The results show that enrichment of the standard 2xMM cultivation medium with the CCSL accelerated to the secretion of the recombinant AbfB by the transformed A.
98
niger D15 protease-deficient strain. The transformed A. niger D15 protease-deficient
strain cultivated in 1%, 2% and 10% CCSL enriched media were by the second day of incubation, secreting recombinant AbfB with volumetric activities against pNPA that were 1.8, 2.2 and 2.6 times respectively, the volumetric activity of the recombinant AbfB secreted in the standard 2xMM media (Fig. 4.6c). The recombinant AbfB activity in 2 and 10% CCSL enriched media had already reached the maximum volumetric activity of 10 nkat mL-1 on the 5th day of incubation, which was one day earlier than in the 2xMM media (Fig. 4.6c). Therefore, the addition of the CCSL to the 2xMM standard cultivation media was a viable strategy for promoting both the biomass growth and the volumetric activity of the recombinant AbfB. The CCSL is an industrial waste and as such presents cost effective way of supplying additional nitrogen for accelerated and increased production of the recombinant AbfB. The recombinant AbfB protein yield per unit biomass (DW) for bioreactor fermentation performed in 2xMM cultivation media enriched with 2% (w/v) CCSL was 366.30 nkat g-1 (DW) which was higher than that of 207.5 nkat g-1 (DW) for fermentation performed in shake flasks in similar media (Table 4.2). Gurlal et al. (2006) reported similar association of increased biomass growth (35 g L-1) with increase in recombinant mannase secretion by A. niger D15 protease deficient strain of up to 32% by enriching the standard 2xMM cultivation media with the CCSL under transcriptional control of gpd promoter. However, extreme biomass growth could present a challenging task in downstream processing (Dun-Colemann, 1992; Harvey and McNeil, 1994).