Table 3 Characteristics of the purified recombinant aspartic prot

Table 3 Characteristics of the purified recombinant aspartic proteinase

(MCAP) Molecular mass* (kDa) Optimum temperature (°C) Optimum pH Thermostability ** (%) 33 & 37 60 3.6 40 *Enzyme having (2.5 kDa) the additional amino acids of the C-terminal polyhistidine tag. **Thermal stability of the enzymes at 55°C, for 30 min. Proteolytic activity of purified MCAP The ratio of milk clotting activity to proteolytic activity (MCA/PA) of MCAP was compared to the value observed for commercial rennet preparation. The higher the MCA/PA ratio the more desirable the enzyme is during cheese making. Table 4 shows the MCAP ratio of about 20, which is below the calculated ratio for chymosin preparation. Table 4 Clotting and proteolytic activities of P. pastoris and R. miehei aspartic protease #click here randurls[1|1|,|CHEM1|]# Sample Milk clotting activity MCA (U/μg) Proteolytic activity PA (U/μg) Ratio buy Compound C MCA/PA MCAP 137 7.02 ± 0.28 19.5 ± 0.79 R. miehei 311 11.11 ± 0.27 28.0 ± 0.68 Results shown are the means of three sets of experiments. Conclusion The expression of MCAP under the control of the constitutive GAP promoter was investigated. P. pastoris was shown to be a good host for the production of MCAP protein and the novel MCAP was efficiently secreted into the medium to concentrations exceeding 180 mg L-1. Similar

results were obtained by Yamashita and coworkers who cloned the M. pusillus Rennin gene in S. cerevisiae cells [21]. P. pastoris secreted two forms of MCAPs where one form was glycosylated while the other was non-glycosylated and similar to the authentic

aspartic proteinase of the M. circinelloides. The observation was correlated to the presence of an N-glycosylation site Asn-Phe-Thr at position Asn331 of the amino acid sequence of MCAP. Previous reports show that aspartic proteinases expressed in S. cerevisiae[21, 22] and Aspergillus nidulans were secreted as single protein bands and in most cases glycosylated [23]. However, previous observations have shown that a mutant strain defective in N–glycosylation process of M. pusillus excreted three glycoforms of M. pusillus proteins [24]. P. pastoris strain next SMD1168 transformed with pGAPZα+MCAP-5 also excreted two forms of MCAPs (unpublished data). Interestingly, the MCAP protein contains the sequon located towards the C-terminus (Asn331 according to the MCAP of 394 amino acid residues). Therefore, P. pastoris can possibly excrete two forms of MCAPs. Results obtained by Shakin-Eshleman et al., suggest that a particular amino acid at the X position of an Asn-X-Ser sequon is critical for Core-Glycosylation Efficiency (CGE) [25]. They found that the substitution of the amino acid X with Phe, increases the efficiency of core glycosylation. In fact, MCAP contains Phe at the X position of the sequon. The result showed that the density of the band representing glycosylated recombinant protein was more intense than the recombinant non-glycosylated protein.

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