Comparison of the biochemical properties of a recombinant lipase extract from Rhizopus oryzae expressed in Pichia pastoris with a native extract

Biochemical Engineering Journal 54 (2011) 117–123

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Comparison of the biochemical properties of a recombinant lipase extract from Rhizopus oryzae expressed in Pichia pastoris with a native extract Marina Guillén, Maria Dolors Benaiges, Francisco Valero ∗ Departament d’Enginyeria Química, EE, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

a r t i c l e

i n f o

Article history: Received 5 October 2010 Received in revised form 30 December 2010 Accepted 4 February 2011 Available online 12 February 2011 Keywords: Pichia pastoris Rhizopus oryzae Recombinant lipase Biochemical properties Protein extract Specificity

a b s t r a c t Extracts containing mature Rhizopus oryzae lipase overexpressed in Pichia pastoris (rROL) and commercial ones from the native microorganism (nROL) have been characterized. The specific activity of rROL extract was more than 40-fold higher than nROL. The presence of multiple bands of lipases around 34 kDa was detected by western blot and zymogram analysis in both extracts. Nevertheless, rROL showed a slightly lower molecular weight than nROL. The presence of hydrolytic activity not recognised as derived from a lipase was also detected in nROL at higher molecular weights. The influence of the ionic strength on lipase activity was assayed and there was an effect both on pH and optimal temperature. Some differences were found between the two extracts. The specificity against triacylglycerol esters and p-nitrophenol substrates was also analyzed. Similar behaviour was noted towards triacylglycerol esters; however rROL showed opposite behaviour compared to nROL towards p-nitrophenol esters with preferences for long chain derivatives. The properties of rROL were partially different from those reported for nROL showing the influence of the pre-pro-sequence of ROL, the post-translational modifications of Pichia pastoris and the effect of an esterase in nROL powder. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Lipases (glycerol ester hydrolases, EC 3.1.1.3) belong to a group of hydrolytic enzymes whose function is to catalyze the hydrolysis of esters. Nevertheless, in non-aqueous solvents these enzymes are able to catalyze synthesis, transesterification and interesterification reactions. Lipases are widely used in industry, especially in the pharmaceutical sector [1–4]. Lipases can be used to carry out resolutions of racemic mixtures, due to their high selectivity and specificity [5–8]. Other applications of these enzymes are in biosurfactant production for the food, cosmetic and pharmaceutical industries [9,10], biodiesel production [11], synthesis of antitumorals, antioxidants and fluororganic compounds, biosensor construction [12], and a great number of other interesting applications. Initially lipases were obtained from the pancreas of certain kinds of mammals, but nowadays lipases are mainly obtained by fermentation processes using native or recombinant microorganisms such as bacteria, fungus or yeasts. Rhizopus oryzae is a filamentous and lipolytic fungus whose lipase is studied in the present work. This microorganism only produces one form of extracellular lipase which has a high biotech-

∗ Corresponding author. Tel.: +34 93 5811809; fax: +34 93 5812013. E-mail address: [email protected] (F. Valero). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.02.008

nological potential as a catalyst for lipid modification, due to its high regiospecificity. This enzyme acts only at the sn-1 and sn-3 locations because it belongs to a group of lipases that are active against esters of primary alcohols [5,13–16]. The native structure of the enzyme is a 392 amino acids protein. The first 26 belong to a signal sequence (pre-region), the following 97 amino acids belong to the pro-region and the last 269 form the mature protein sequence [17]. The deduced polypeptide sequence of the secreted form was found to be made up of 297 amino acids of which 269 belong to the mature protein sequence and the remaining 28 amino acids to the last part of the pro-region [18]. The molecular weight of this enzyme is 32 kDa, determined by means of a SDS–PAGE [13,18] and the isoelectric point is 6.85 [14]. It has four potential sites of N-linked glycosylation and three disulphide bonds, between the amino acids 152 and 391, 163 and 166, and the 358 and 367. It is known that an effective strategy to increase the productivity of an enzyme bioprocess production and sometimes to simplify the downstream process is through the cloning and expression of its corresponding gene into a foreign host. Thus, the R. oryzae lipase has been cloned and expressed in Escherichia coli [19–21], Saccharomyces cerevisiae [22,23] as well as in the methylotrophic yeast Pichia pastoris [20,24,25]. The production of this lipase in E. coli in its active form entails purification and refolding processes because it is found as inclusion bodies [19,20], except when it is expressed using the host strain E. coli Origami (DE3) which produces the active enzyme correctly folded [21]. When S. cerevisiae was the selected

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host, different gene constructions were used involving the fusion of the alpha-mating factor presequence to the proform of the R. oryzae lipase or directly to the mature encoding gene of the enzyme [22,23]. With the last construction the activity was detected neither in the culture medium nor in the cells. P. pastoris, used as a host for the production of R. oryzae lipase, offers advantages compared to the other tested hosts because it presents a tightly regulated promoter of the alcohol oxidase (PAOX), high productivity, the capacity to grow in a minimal medium at high cell densities, low levels of endogenous protein secretion and the ability to efficiently secrete heterologous protein [26]. Also it performs many of the higher eukaryotic post-transductional modifications. The mature sequence of ROL has been expressed in P. pastoris under the PAOX and secreted into the culture medium using the S. cerevisiae alpha-mating factor pre-prosequence obtaining up to 60 mg L−1 of active enzyme [20]; moreover the productivity of the enzyme was increased more than 5-fold by using a feeding strategy based on the monitoring and control of methanol concentration in a fed-batch cultivation [27–29]. In the present work the biochemical characterization of two protein extracts, rROL and nROL was carried out with the aim of comparing their properties. rROL was a protein extract containing a recombinant ROL produced in P. pastoris and nROL was a commercial extract containing the native lipase. In many biocatalytic processes the use of enzyme extracts is useful because of the reduction of the cost of the process avoiding the purification of the biocatalyst, so that the knowledge of the properties of the extracts is important for their implementation in large-scale processes. 2. Materials and methods 2.1. Lipases nROL was a generous gift from AMANO Pharmaceuticals Co., Ltd. which is referred as F-AP15. rROL is produced by the Bioprocess Engineering and Applied Biocatalysis group of Universitat Autònoma de Barcelona (UAB). This lipase is obtained by a fed-batch cultivation of a recombinant P. pastoris strain using methanol as inductor [28]. The culture broth was centrifuged and microfiltered to remove the biomass. The supernatant was concentrated by ultrafiltration with a Centrasette® Pall Filtron system equipped with an Omega membrane of 10 kDa cut-off, and subsequently dialyzed against 10 mM Tris–HCl buffer pH 7.5 and thereafter lyophilized [30]. Two other commercial protein extracts from R. oryzae supplied by Sigma were used; ROL-Sigma (ref. F-AP 15) and Esterase-Sigma (ref. 9016-18-6). All the extracts were used as lyophilized powders. 2.2. Chemicals The p-nitrophenol esters, triacylglycerol esters and 4methylumbelliferone butyrate (MUF-butyrate) were purchased from Sigma–Aldrich (St. Louis, MO). The lipase colorimetric kit used for the activity assays was obtained from Roche (Roche kit 11821792). Endo-␤-N-acetylglucosaminindase H enzyme was also purchased from Roche. 2.3. SDS–PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE), 12% was carried out in a Mini-PROTEAN II (BioRad) apparatus following the standard procedures recommended by the manufacturer and according to the protocol of Laemmli [31]. Low range protein markers (BioRad) were used for molecular weight determination. Gels were stained using a Coomassie G250 colloidal

stain solution (34% (v/v) ethanol, 2% (v/v) H3 PO4 , 17% (w/v) NH4 SO4 and 0.066% Coomassie G250). 2.4. Western blot Western blots were carried out after protein transference from SDS–PAGE to a nitrocellulose membrane using a Mini TransBlot Electrophoretic Transfer Cell (BioRad) following the manufacturer’s instructions. Once the transference was complete the membranes were incubated in blocking buffer and with the primary antibody (polyclonal anti-mouse antibody) (1:100) according to the procedure described in a previous work [32], changing the incubation time with the primary antibody to 90 min instead of 1 h. After that, the incubation with the secondary antibody (anti-mouse IgG whole molecule alkaline-phosphatase produced in goat, Sigma–Aldrich) diluted 1:5000 in blocking buffer, with a 3% (w/v) of skimmed milk powder was carried out for 1 h at room temperature. Finally the membranes were washed three times with 25 mL washing buffer and the signals were shown using the alkaline phosphatase Conjugate Substrate Kit (BioRad). The membrane was incubated in the freshly prepared solution (2 mL 25× AP colour development buffer, 48 mL water, 500 ␮L AP colour reagent A and 500 ␮L AP colour reagent B) until colour development. The reaction was stopped by washing the membrane with distilled water. 2.5. Zymogram The zymogram method was carried out starting from the SDS–PAGE gel before staining it [33]. The SDS of the SDS–PAGE gel was removed by impregnating it with a 2.5% Triton X-100 solution for 1 h at room temperature. Then the gel was washed twice with Tris–HCl 20 mM at pH 7 for 15 min and incubated in a 100 ␮M MUF-butyrate solution at the same pH and Tris–HCl concentration for 30 s. The gel was then exposed to UV illumination to detect the fluorescent bands. After that the gel was stained using Coomassie G250 colloidal stain procedure, as mentioned before, to detect the molecular weights of the active proteins. 2.6. Determination of the N-terminal sequence After SDS–PAGE the samples were electrotransferred onto a polyvinylidene fluoride (PVDF) membrane with the Mini Trans Blot apparatus (BioRad) according to the manufacturer’s instructions. The PVDF membrane was rinsed with 50 mL MilliQ water and stained with Coomassie Brilliant Blue R-250. The stained components were used for the N-terminal sequencing which were determined by automated Edman’s degradation, using an Applied Biosystems Procise 494 sequencer [34]. The obtained sequences were checked in the National Center for Biotechnology Information (NCBI) database. 2.7. Substrate specificity The substrate specificity was studied using p-nitrophenol esters (C2-C12) and triacylglycerol esters (Tri-C2-C10, Tri-C18, Tri-C18:1). The activity of lipase towards each p-nitrophenol ester was followed spectrophotometrically in a Cary 300 spectrophotometer at 30 ◦ C in 50 mM phosphate buffer at pH 7.0; 0.32% (w/v) Triton X100, 4% (v/v) acetone and 1 mM p-nitrophenol ester concentration (for the C12 and C10 2.5%, w/v Triton X-100 was used because of the low solubility of these compounds). The activity assay took 15 min, adding 30 ␮L of enzyme solution after 5 min of initializing the continuous absorbance measurement. The measurement of the absorbance was done at 348 nm and the slope of the linear part of the activity curve was taken. Under these conditions the molar extinction coefficient of the hydrolytic product, p-nitrophenol, is

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Fig. 1. (A) Western blot analysis, (1) nROL, (2) rROL. (B) Zymogram carried out with MUF-butyrate, (3) nROL, (4) rROL. (C) SDS–PAGE, (5) nROL, (6) rROL, (7) BioRad Low Range marker.

4.93 mM−1 cm−1 . Lipolytic activity was expressed as units. One activity unit was defined as the quantity of enzyme necessary to release 1 ␮mol of product per minute under the described conditions. For the specificity studies towards triacylglycerol esters, the activity was followed with a pH-Stat (Methrom Switzerland) at 30 ◦ C and pH 7 according to the procedure described previously [35]. 2.8. Endo-ˇ-N-acetylglucosaminindase H digestion Protein samples were incubated for 24 h with endo-␤-Nacetylglucosaminindase H (40 AU mg−1 protein), at 37 ◦ C in a 50 mM potassium acetate buffer, pH 5.5, SDS 0.02%, ␤mercaptoethanol 0.8% (v/v), Triton X-100 1% (v/v). For the deglycosylation of denatured samples, the protein was fist incubated without the endo-␤-N-acetylglucosaminindase at 95 ◦ C for 15 min. 2.9. Lipolytic activity assay Lipolytic activity assay was carried out spectrophotometrically in a Cary Varian 300 spectrophotometer at 30 ◦ C in 200 mM Tris–HCl + 5 mM CaCl2 buffer at pH 7.25 using the Roche lipase colorimetric kit. The measurement was done at 580 nm and all analyses were carried out in duplicate. 2.10. Total protein determination Protein concentration was determined by the method of Bradford using bovine serum albumin as standard [36]. 3. Results and discussion 3.1. Molecular weight determination and N-terminal sequencing An immunodetection of lipase was carried out by means of a western blot analysis (Fig. 1A). The molecular weight of the active nROL and rROL was determined by means of a zymogram using MUF-butyrate as substrate (Fig. 1B) and a SDS–PAGE (Fig 1C). Although it is known that R. oryzae produces only one form of lipase of 32 kDa [13,15] made up of the mature sequence of the protein attached to the last 28 amino acids of the pro-peptide sequence [18], the results obtained for the nROL by the western blot analysis (Fig. 1A) showed three positive bands. The molecular weight of these bands was around 34 kDa (Fig. 1C), the predominant band being over 34 kDa, slightly higher compared to the 32 kDa described in the literature [13,15]. The presence of three recognised proteins

may be explained by a different degree of glycosylation. In order to check this hypothesis a treatment with endoglycosidase H was carried out but no significant modification in the molecular weight was observed as it has been reported (data not shown) [15,23]. Nevertheless, other authors have previously described that these proteins could differ in the amino acid sequence due to a proteolytic cleavage of the mature pro- or pre-pro-enzyme [18,37] or to other kinds of post-translational modification of secreted proteins such as O-glycosylation or lipidation [25]. Three bands were also recognised by the antibody in rROL (Fig. 1A) but, in contrast to the nROL, only two bands were around 34 kDa, (the predominant band having a molecular weight slightly lower than 34 kDa), and a third weak band was detected at 45.5 kDa (marked with a square in Fig. 1A). The differences in molecular weight may be explained by a difference in the number of glycosylated residues, but endoglycosydase H treatment did not result in proteins with different molecular weights (data not shown). To assay the hypothesis of different proteolytic processing sites, these three proteins were treated in order to obtain their N-terminal sequence. The two forms with a molecular weight around 34 kDa have the same N-terminal sequence: EAEFSDGGKVVAA. The first two amino acids belong to the final sequence of the alpha-factor from S. cerevisiae and the two next to the restriction site where the ROL gene was cloned in pPICZ␣ [20]. The last nine amino acids left come from the first part of the mature sequence of ROL. On the basis of these results, it cannot be excluded that other kinds of post-translational modification affect the enzymes [25]. In the case of the protein detected at 45.5 kDa the sequencing of its N-terminus was not successful, because the band was not completely purified and the amino acids were not clearly sequenced, but the fact that this protein had been detected in the western blot could indicate that it could be a native protein from P. pastoris linked with the lipase. The presence of an extracellular Pichia protein with lipolytic activity has to be rejected because, in wild-type Pichia cultures, no extracellular lipolytic activity was detected. In spite of the western blot results, the zymogram (Fig. 1B) showed four active proteins in the nROL and three active bands in the rROL. In the nROL, three of these bands were around 34 kDa. These belong to the three proteins detected in the western blot, and the other one has a molecular weight of 40 kDa which was not detected by the immunoassay. Although this protein is not ROL, it has hydrolytic activity which could be associated with an esterase present in the native extract. On the other hand, the rROL showed two active bands located around 34 kDa and the third protein detected by antibodies in the western blot at 45.5 kDa showed only a weak fluorescence when the exposure time to UV was increased (data not shown).

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Fig. 2. Effect of ionic strength on lipolytic activity expressed as percentage of relative activity. The activity value using the 400 mM Tris–HCl buffer concentration for the rROL and nROL at 30 ◦ C and pH 7.25 was taken as 100%.

3.2. Effect of ionic strength, temperature and pH on lipolytic activity Enzyme activity is strongly dependent on ionic strength, temperature and pH. Therefore, it is important to carry out a characterization of the enzyme in order to establish the optimum values of these variables which provide the highest activity. Preliminary experiments showed an important influence of ionic strength on lipolytic activity. Thus, the effect of this variable was tested for a series of Tris–HCl buffer concentrations between 50 mM and 2 M, at pH 7.25, with nROL and rROL (Fig. 2). The results revealed that the patterns of both extracts were similar. The specific activity (AU mg total protein−1 ) reached the highest value at 200 mM Tris–HCl analysis buffer concentration with a pronounced decrease at higher ionic strength. nROL was more affected by ionic strength, in terms of percentage of relative activity, than rROL. Due to the important effect of ionic strength on lipolytic activity, the study of the optimum values of temperature and pH was made at two different ionic strengths, the optimum value corresponding to a buffer concentration of 200 mM, and 400 mM which is the buffer concentration used to monitor the lipolytic activity profile in the recombinant P. pastoris cultures [38]. The optimum values of temperature, pH and lipolytic activity for both extracts are presented in Table 1. The optimum temperature was obtained by maintaining a constant pH of 7.25 and the optimum pH was determined by maintaining a constant temperature of 30 ◦ C with both buffer concentrations. Independently of the ionic strength tested, the specific activity of rROL was more than 40-fold higher than nROL. In Fig. 3 the behaviour of lipolytic activity with temperature for the studied ionic strengths is shown. It can be seen that the optimum value of temperature obtained for nROL was 40 ◦ C, independently from the buffer concentration used. For the rROL, the optimum temperature changed from 40 ◦ C to 30 ◦ C when the buffer concentration increased from 200 mM to 400 mM. The value of 40 ◦ C has been also previously described as the optimum temperature for the pure native lipase [13], although 35 ◦ C is suggested for this enzyme by other authors [15]. On the other hand the value of 30 ◦ C has been shown as the optimum temperature for the pure recombinant ROL in P. pastoris, E. coli [20] and for the intracellular lipase of R. oryzae [39]. The fact that the optimum temperature of the nROL does not change when the buffer concentration is modified, in contrast to the

Fig. 3. Effect of temperature on lipolytic activity for rROL and nROL at pH 7.25, buffer concentration 400 mM and 200 mM. The maximum activity of each experimental condition was taken as 100%.

rROL, could be explained by the effect of stabilizing agents present in the commercial product or by different post-translational modifications compared with the rROL which bring about a more favourable conformational stabilization with regard to temperature and ionic strength changes. Another fact that could explain this behaviour is the difference in the N-terminal sequence of enzymes. As the native enzyme has a part of the pro-region sequence, which is not present in the recombinant product, it could provide different properties such as modified specific activity, specificity and stereoselectivity compared with the mature protein [18,19,23]. Besides, the specific activity of both enzymes decreased (more than 2-fold in the rROL and more than 4-fold in the nROL) for each analyzed temperature, using the 400 mM instead of the 200 mM Tris–HCl buffer concentration. Regarding the pH results, it was noted that the dependence of activity on pH for both lipases had a similar behaviour (Fig. 4). The optimum pH changed for both enzymes from 8 to 7.25 when the ionic strength was modified from 200 mM to 400 mM buffer concentration. This might be explained by the effect of the ionic strength on the three-dimensional protein conformation altering the acid–base equilibria of the amino acids that form the protein. This alteration can affect the enzymatic activity because some of these amino acids could belong to the active centre. Some authors have also described an optimum pH value of 8 for the pure native lipase [13] as for a pure recombinant ROL produced

Fig. 4. Effect of pH on lipolytic activity for rROL and nROL at temperature of 30 ◦ C, buffer concentration 400 mM and 200 mM. The maximum activity of each experimental condition was taken as 100%.

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Table 1 Values of optimum pH, temperature and specific activity for rROL and nROL at two different Tris–HCl analysis buffer concentrations. Optimum values

Temperature (◦ C) pH Specific activity (AU/mg total protein) at optimum temperature and pH 7.25 Specific activity (AU/mg total protein) at optimum pH and 30 ◦ C

nROL

rROL

200 mM

400 mM

40 8 604 593

40 7.25 142 115

200 mM 40 8 21,791 35,281

400 mM 30 7.25 11,111 11,111

in E. coli and in P. pastoris [20]. A value of 7.5 was also referenced for the pure native enzyme [15] and a pH of 8.5 was the optimum value reported for the intracellular lipase from R. oryzae [39]. Moreover, it can be seen in Fig. 4 that in both cases the curve of the dependence of activity on pH is widened at 200 mM Tris–HCl buffer, so that, a variation of the pH around the optimum value entails a loss of activity lower than the loss obtained for the same pH variation at 400 mM analysis buffer concentration. As for the temperature studies, the specific activities reached by both products for every tested pH were higher at low buffer concentration (Table 1). Thus, the maximum value of specific activity was 5.1-fold higher for the nROL and 3.2-fold for the rROL, when the buffer concentration decreased (Table 1). 3.3. Substrate specificity with triacylglycerol esters The specificity towards triacylglycerol esters has been determined for nROL and rROL extracts. Fig. 5 shows the results obtained. The substrate preferably hydrolyzed by both extracts was the TriC8, and also the Tri-C10 in the case of rROL. Moreover, neither the rROL nor the nROL showed activity towards the Tri-C2 (20 mM as well as for 1.2 M) as well as for Tri-C18:0. The highest differences between both products were detected for the specificity towards Tri-C10 and Tri-C18:1 where the specificity of rROL towards these esters was increased more than 20% compared with nROL. The specificity of pure native ROL [15] as well as of pure recombinant ROL [20,22] has been studied previously by other authors; in all cases they found a preference towards Tri-C10 and Tri-C8. 3.4. Substrate specificity with p-nitrophenol esters When the specificity against p-nitrophenol esters was evaluated a different behaviour between both extracts was observed (Fig. 6).

Fig. 5. The specificity of triacylglycerol for nROL and rROL. The maximum activity of each experimental condition was taken as 100%.

nROL hydrolyzed preferably the p-nitrophenol esters with a short carbon chain whereas the rROL showed a preference to hydrolyze esters with a longer carbon chain. For nROL, C4-C12 esters achieved activities that did not reach the 20% of the activity obtained for the C2 which was the compound with the highest hydrolysis rate.

Fig. 6. p-Nitrophenol ester specificity for nROL (A) and rROL (B). The maximum activity of each experimental condition was taken as 100%.

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Fig. 7. (A) SDS–PAGE, (1) BioRad Low Range Marker, (2) rROL, (3) ROL-Sigma, (4) Esterase-Sigma, (5) nROL. (B) Western blot analysis, (1) rROL, (2) ROL-Sigma, (3) EsteraseSigma, (4) nROL. (C) Zymogram carried out with MUF-butyrate, (1) rROL, (2) ROL-Sigma, (3) Esterase-Sigma, (4) nROL.

In contrast to the native extract, the recombinant product is more active when the chain length is increased. This difference of specificity towards p-nitrophenol esters might be explained by the presence of an esterase of R. oryzae in the nROL extract as has been previously suggested. The activity of this esterase could explain the high specificity of the nROL towards p-nitrophenol esters of short chain length compared with rROL. In order to check this hypothesis, commercial R. oryzae protein extracts, Esterase-Sigma and ROL-Sigma, were characterized by SDS–PAGE, western blot and a zymogram (Fig. 7) and compared with nROL and rROL. Results showed that Esterase-Sigma is a crude extract, similar to the one commercialized by AMANO (nROL). However, ROL-Sigma is a purified extract of Esterase-Sigma. It is important to remark that the presence of lipase was detected in the three commercial powders by a western blot analysis. These results could indicate that nROL and Esterase-Sigma extracts could be obtained by similar procedures. This fact could explain the presence of esterase as well as lipase in both protein extracts and this result could explain the higher specificity of nROL extract towards p-nitrophenol esters of short chain length. After this analysis, the specificity towards esters of pnitrophenol was studied for the Esterase-Sigma and ROL-Sigma and their results were compared with nROL as well as with the rROL (Fig. 8). As can be seen, all the extracts obtained from the native microorganism showed a higher similarity in specificity while rROL showed a clearly opposite behaviour towards this kind of esters.

Fig. 8. Comparison of the specificity of p-nitrophenol esters for recombinant and commercial extracts. The maximum activity of each experimental condition was taken as 100%.

Another fact that could explain the difference of specificity could be the difference in the N-terminal sequence of the studied lipases. The different specificity towards p-nitrophenol esters of two recombinant lipases of R. oryzae (produced in two different S. cerevisiae strains) having different N-terminal sequences, has been described [23]. One of them contained the whole pro-sequence and the other one only the last 28 amino acids of the pro-sequence. Another hypothesis that could explain these results is the difference in the amino acid primary sequence of the lipases. Some authors found substitutions in four positions of the amino acid sequence depending on the strain used to produce the enzyme [40]. So, even though the whole sequence of ROL cloned in P. pastoris [20] is known, the commercial product could be obtained from a R. oryzae strain that produces a lipase with a slight different in its amino acid sequence compared with the recombinant lipase one. These differences might produce changes in the enzymatic affinity [25,41]. 4. Conclusions Important differences were detected when rROL and nROL extracts were compared. First of all the specific activity of rROL was more than 40-fold higher than n-ROL. In both extracts multiple bands of molecular weight around 34 kDa were detected, slightly higher than previously reported from pure mature recombinant ROL, also expressed in P. pastoris [20] and pure native ROL [15]. N-terminal analysis revealed that the two bands detected around 34 kDa in rROL have the same sequence. Thus, the differences observed might be caused by post-translational modifications. Interestingly, nROL presented another band with a molecular weight of 40 kDa with activity in zymogram test but not detected by the immunoassay. This band could be an esterase; in fact Sigma also markets a product, named R. oryzae esterase, which shows the same band. Enzyme activity was strongly dependent on ionic strength. Both extracts showed the same behaviour with a maximum of lipolytic activity in 200 mM Tris–HCl buffer. The optimum of temperature of nROL was not affected by the ionic strength and it was similar to rROL at 200 mM Tris–HCl buffer (40 ◦ C). However, when the ionic strength was increased at 400 mM buffer concentration the optimal temperature of rROL decreased to 30 ◦ C. At around the optimum pH (7.25 at 400 mM and 8 at 200 mM), both extracts showed the same behaviour. Similar substrate specificity towards various triacylglycerols was observed for the extracts tested. They preferred the middle chain triacylglycerols, Tri-C8 and Tri-C10. When substrate specificity towards p-nitrophenol esters was studied, rROL specificity increased as carbon chain length was increased, opposite to nROL behaviour and other commercially available extracts.

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