Mediadores liberados por los mastocitos caninos aislados de la piel: la histamina y el TNF-α

To test the hypothesis that the phylogenetic grouping of fungal sucrolytic GH32 enzymes was related to FOS synthesis activity, two β-fructofuranosidases whose protein sequences were proposed to represent low- and high-level FOS synthesis activities were functionally characterised. The prediction was based on the presence of either the WMNDPNG and ECP or the GQIGDPC and FET sequence motifs in motifs A and E, respectively. The β-fructofuranosidase from A. niger (Suc1 and SucA) was predicted to be a high-level FOS producer while the β-fructofuranosidase from A. pullulans (ApINV) was predicted to be a low- level FOS producer. Their positions in the phylogenetic tree of functionally characterised enzymes are highlighted in grey (Fig. 6.1). The activities of these β-fructofuranosidases were

compared to the A. japonicus FopA, the enzyme used for commercial production of FOS and used in this study as a benchmark for high-level FOS synthesis (Nishizawa et al. 2001).

6.3.2.1 A. niger β-fructofuranosidases

Two versions of the A. niger β-fructofuranosidase were heterologously expressed in K. pastoris. The reasoning for this follows. The original report by Boddy et al. (1993) on the characterisation of the purified enzyme and its gene sequence indicated that the suc1 gene contained a single exon. Furthermore, they did not investigate FOS synthesis by the purified enzyme. Yuan et al. (2006) predicted the presence of an intron one base pair upstream of the termination codon. They verified the presence of the intron by reverse transcriptase PCR and hence predicted an additional 39 extra amino acids on the C-terminal end of Suc1. The presence of the intron was also previously proposed by Yanai et al. (2001) but neither groups examined the enzyme activity experimentally. Based on the report by Yanai et al. (2001), Yuan et al. (2006) speculated that SucA (comprised of two exons) lacked fructosyltransferase activity and that fopA displayed much higher fructosyltransferase activity than Suc1 from A. niger strain B60. Zuccaro et al. (2008) produced oligosaccharides using Suc1, although it was deduced from their cloning strategy that they were using the two exon enzyme (SucA). Furthermore, they claimed that Suc1 possessed superior FOS synthesis activity to FopA even though they did not directly compare the activities of the two enzymes. To obtain clarity on the activity of the A. niger extracellular β-fructofuranosidase, which the bioinformatic analysis predicted to have high-level FOS synthesis abilities, both Suc1 and SucA were produced in K. pastoris. To facilitate a direct comparison FopA was also produced in K. pastoris. Finally, a truncated version of FopA (∆FopA598) was produced such that it resembled Suc1 to allow for investigation of the role of the C-terminal region (encoded by the second exon in sucA) in enzyme activity. The full length

fopA coding sequence did not contain an intron (Yanai et al. 2001).

Fig. 6.4 shows the results of the SDS-PAGE (A) and zymogram analysis (B) of the culture supernatants of K. pastoris expressing the aforementioned enzymes. On the SDS-PAGE gel photograph bands were visible in the region of 150 kDa that corresponded to the enzymes. A band at that size was absent for the yeast transformed with the empty pGAPZ vector. The bands were larger than the theoretical predicted weights for the mature proteins of 66 kDa (SucA) and 69 kDa (FopA), however de-N-glycosylation with PNGase F yielded the expected band sizes (data not shown). The bands for Suc1 and ∆FopA598 were slightly lower than SucA and FopA, owing to the C-terminal truncations. Hydrolytic activity was evident on the zymogram between 130 and 170 kDa for Suc1, SucA and FopA. The ∆FopA598 enzyme was deemed

inactive as no band was visible on the zymogram. In addition, the K. pastoris pGAPZ∆FopA598

transformants did not display growth on solid media with sucrose as sole carbon source.

Fig. 6.4 SDS-PAGE and zymogram gels of Suc1, SucA, FopA and ∆FopA598. A Silver-stained 8%

polyacrylamide gel of culture supernatants of K. pastoris expressing the four β-fructofuranosidases. Lane 2 shows Suc1, Lane 3 SucA, Lane 4 FopA, Lane 5 ∆FopA598 and Lane 6 contains the control strain

transformed with the empty pGAPZ vector. B Zymogram gel of the four β-fructofuranosidases loaded in

the same order as for SDS-PAGE. In both gels, Lanes 1 contained the protein molecular weight markers. The PageRuler unstained protein ladder and the PageRuler prestained protein ladder (Thermo Scientific) were used in A and B, respectively

6.3.2.2 A. pullulans β-fructofuranosidase

A putative β-fructofuranosidase was cloned from A. pullulans and expressed in S. cerevisiae. As in the section above, fopA was also expressed in S. cerevisiae to facilitate a direct comparison of enzyme activities. The sequence of the cloned ApINV was 99.9% identical to jgi|Aurpu_var_sub1|31127|fgenesh1_pg.16_#_95 on the DNA level. Translation of the cloned gene sequence revealed two amino acid substitutions, T61I and T170A relative to the JGI database protein sequence.

The results of the SDS-PAGE and zymogram analyses are shown in Fig. 6.5. Bands corresponding to the FopA and ApINV enzymes were visible between 100 and 130 kDa. The band was absent in the control strain supernatant. Similarly to the β-fructofuranosidases expressed in K. pastoris, the band sizes were larger than the expected 69 and 66.8 kDa for FopA and ApINV, respectively. De-N-glycosylation yielded bands of the expected sizes (data not shown). Both enzymes displayed hydrolytic activity on sucrose as evidenced by bands between 100 and 130 kDa visible on the zymogram gel.

Fig. 6.5 SDS-PAGE and zymogram gels of ApINV and FopA. A Silver-stained 8% polyacrylamide gel of

culture supernatants of S. cerevisiae expressing the two β-fructofuranosidases. Lane 1 contained the Spectra Multicolor High Range Protein Ladder (Thermo Scientific), Lane 2 shows FopA, Lane 4 contains the control strain transformed with the empty pJC1 vector and Lane 7 shows ApINV. B Zymogram gel of the two β-fructofuranosidases loaded in the same order as for SDS-PAGE