Several advantages of using P. pastoris as biofactory for the production of the recombinant protein have already been mentioned. An additional reason to choose this system to express the O. piceae sterol esterase was that other fungal lipases have been successfully expressed in this yeast, such as the different isoenzymes (Lip1-Lip5) from C. rugosa[8, 10, 40–44] and G. candidum[45, 46], as well as the sterol esterase from M. albomyces.
The screening to select the clones of P. pastoris with sterol esterase activity was carried out by using a simple plate activity assay  based on the hydrolysis of tributyrin in MM medium, which showed clear halos in the positive transformants. Initially, five positive clones were selected for the production of the sterol esterase in 1L Erlenmeyer flask containing different volumes of BMMY liquid medium. In any case, all clones secreted higher activity levels than those attained with O. piceae. However, the highest levels (up to 18 U/mL) were obtained when a lower volume of culture medium was used  because of the increase in the oxygen transfer rate. This is one of the different strategies previously proposed to facilitate oxygen transfer in Erlenmeyer flask cultures [28, 31].
On the other hand, since the production of recombinant proteins in P. pastoris is closely connected to growth yields, the use of different carbon sources on biomass production, and so on the expression of the recombinant protein was studied. As methanol is a poor energetic substrate yielding in theory only 6 ATP molecules, both in assimilation and dissimilation pathways [48, 49], the biomass production in media with methanol as the sole carbon source was lower than those obtained in media with sorbitol. In accordance with this, the biomass was lower for the selected P. pastoris Mut+ transformant growing in media with only methanol (MM, BMM, and BMMY) than in a medium with an alternative carbon source like sorbitol (YEPS), which do not repress AOX1 promoter . Consequently, the greatest activity levels were obtained in YEPS medium, being 3- and 32-fold higher than in BMMY and BMM media, respectively in terms of maximal activity. The performance of the production in YEPS medium, according to generated biomass, was higher than in BMMY and BMM (2- and 15- fold, respectively) and much higher than with MM (127-fold). The use of either complex or unbuffered media could contribute to decrease the effect of proteases during growing of P. pastoris. In the first case, yeast extract and peptone not only improve the growth of the yeast, but also can be substrates for proteases and could suppress their expression when nitrogen is limited [28, 51]. In the second case, the growth of the yeast causes a fall in pH, favouring protease inactivation . All these facts could explain the better activity levels found in BMMY medium as compared with BMM medium, and the very low activity detected in MM media. These results agree with previous reports describing higher activity levels of the recombinant cinnamoyl esterase from Aspergillus niger expressed in the yeast when buffered complex media were used . Nevertheless, low activity levels of recombinant M. albomyces sterol esterase have been obtained in these culture conditions . On the other hand, and also agreeing with our data, G. candidum lipase activity was not detected in cultures in MM medium , probably due to a pronounced decrease in pH with time (around 2), which would cause the denaturalization of the enzyme.
The recombinant enzyme was purified from YEPS medium in a single chromatographic step, with a purification factor higher than that obtained for the native enzyme . LIP3 from C. rugosa was purified with a similar procedure, but yielding lower amounts of protein .
The purified enzyme can work in a wide pH range keeping more than 50% of its initial activity, as has also been reported for recombinant LIP3 , and it is thermostable at 4°C and 30°C for 24 hours in the assayed conditions. In any case, comparing the native and recombinant proteins, OPE* showed higher stability at very alkaline pHs and lower optimum temperature than OPE, which could be advantageous for its industrial application.
The existence of different post-translational modifications was considered in order to explain the observed changes, not only in the optimum temperature but also in the kinetic parameters of the recombinant enzyme. Regarding glycosylation, it has been described that long outer chains can potentially interfere with the folding and function of a foreign protein . However, dichroism spectroscopy experiments (Figure 4) indicated that the recombinant protein was not misfolded, and an identical secondary structure was deduced for both sterol esterases. In addition, N-linked carbohydrates did not seem to be needed for maintaining the hydrolytic activity of these proteins, as deduced from deglycosylation experiments (Figure 3). Similarly, lipase B from Candida antarctica and LIP4 from C. rugosa maintained comparable kinetic properties after their expression in E. coli, although the glycosylated form of LIP4 produced in P. pastoris had higher thermal stability.
On the other hand, the partial oxidation of methionine residues during heterologous expression of proteins in P. pastoris has been reported [38, 39]. Peroxisome environment in P. pastoris is highly oxidative and oxidation of sensitive residues could occur when hydrogen peroxide, produced during methanol metabolism, is released from peroxisomes to the culture medium after minimal cell lysis . The sequence of the O. piceae sterol esterase contains 5 oxidizable methionine residues, one of them located in the surroundings of the substrate binding site . However, amino acid analysis of the recombinant O. piceae sterol esterase suggested that this is not the reason for its improved catalytic properties, since no methionine sulfone residues were found (Figure 5).
Secretion is the preferred approach for heterologous protein production due to the ease of product recovery . Furthermore, the secreted recombinant protein in P. pastoris constitutes the vast majority of total protein in the medium because the yeast secretes low levels of endogenous proteins . However, the high level of expression from P
AOX1 may overwhelm the post-translational machinery of the cell causing an unprocessed foreign protein . The bad processing of the pre-propeptide of the α-mating factor can be explained by the formation of tertiary structures during the expression of a foreign protein that could protect cleavage sites from KEX2 and STE13 proteases . In addition STE13, which cleaves EA repetitions, is a minor protein in the cell and it would not be able to process correctly an overexpressed protein . Sequencing of the N-terminal region of the recombinant O. piceae esterase disclosed a wrong processing of the protein, since its N-terminus contained 6 or 8 additional residues from the secretion signal and the vector. This modification at the N-terminal end seems to influence some properties of the recombinant protein such as its aggregation state, as shown by analytical ultracentrifugation (Figure 6). While the native enzyme forms big aggregates in water solution , as reported for the M. albomyces sterol esterase , the recombinant enzyme remained as a mixture of monomeric and dimeric forms (even at 200 μg/mL). This behaviour could be the ultimate reason responsible for the improved catalytic properties of the recombinant enzyme. A wrong processing of the α-mating factor pre-propeptide has also been described in other proteins expressed in P. pastoris, such as the feruloyl esterase from Talaromyces stipitatus, the xylanase from Thermomyces lanuginosus, as well as the lipases from C. antarctica and Candida parasilopsis, but the catalytic properties of these recombinant proteins were not affected. This wrong processing has been reported even in S. cerevisiae.
Esterases and lipases form pseudo-quaternary structures easily in aqueous solution. Multimeric forms have been described for the M. albomyces sterol esterase in the absence of detergent  although, at low concentrations, tetrameric forms have been reported for the native protein, and dimeric structures for the recombinant variant [15, 60]. This tendency to form multimolecular aggregates has also been reported in the lipase from C. parapsilosis and in the sterol esterases from Streptomyces species . In addition, recombinant C. rugosa LIP2, expressed in P. pastoris, resulted in an aggregated, inactivated form of the protein, and only after diaultrafiltration lipolytic activity was recovered . Monomolecular forms from C. rugosa, Humicola lanuginosa (synonym T. lanuginosus), and Mucor miehei lipases were found only at low enzyme concentrations and in the presence of detergents. So it is difficult to find only the monomolecular form of lipase-type enzymes since it can only be achieved by mixing the enzyme solution with a detergent .
Lipases display different functional properties in their monomeric or aggregated forms . In general, it seems that multimolecular forms exhibit lower specific activity and higher stability to pH and temperature than the monomeric proteins, although controversial data have been published for C. rugosa lipase . For instance, enzymes from M. albomyces and Streptomyces sp. increased their activity in the presence of a detergent, where proteins probably tend to be in their monomolecular form [6, 21]. However, in the case of O. piceae sterol esterase, neither activation nor inactivation of the enzyme (native or recombinant) has been reported in the presence of 0.2% Triton X-100 (used in the purification of these proteins), although above this concentration a decrease in their activity was observed (data not shown) as reported for the lipase BTL2 from Bacillus thermocatenulatus. The use of Genapol X-100, which is indispensable in reactions involving long-chain triglycerides or fattyacid cholesterol esters in order to solubilise them, is detrimental for enzyme activity on pNPB since it acts as a competitive inhibitor for this short chain substrate . In any case, as we report here, the use of detergents favour the monomeric form of the protein.
In accordance with previous works , when a concentrated aqueous solution of the recombinant enzyme was maintained during 16 h at 37°C no significant loss of activity was found. On the contrary, if this solution is diluted and the resulting solution incubated under the same conditions, an appreciable amount of activity was lost.
The high overall content in hydrophobic amino acid residues (38%) of the native enzyme could explain its tendency to form aggregates, as has been suggested for lipase BTL1 from B. thermocatenulatus. However, the modification at the N-terminal end of the recombinant protein expressed in P. pastoris, by the addition of 6-8 extra amino acid residues from pPIC9 vector, used for protein expression, and the inefficient processing of the α-mating factor pre-propeptide, used for secretion, affected the aggregation state of the protein, as was confirmed by analytical ultracentrifugation experiments with deglycosylated recombinant enzyme.
Usually, a bad processing has no effect on recombinant protein activity, such as in G. candidum, Yarrowia lipolytica, and C. parasilopsis lipases . An improvement of catalytic properties [65–69] and stability [8, 42] of some recombinant and native enzymes has been previously reported, speculating on the basis of different glycosylation degree, N-terminal modification or aminoacid substitution (due to a preferential codon use in P. pastoris respect to natural host) . However, to the best of our knowledge, the results presented in this paper constitute the first experimental report of an improvement of the solubility and kinetic constants of the enzyme, as a consequence of its N-terminal modification.