Biochemistry of lipolytic enzymes secreted by Penicillium solitum and Cladosporium cladosporioides

Abstract

Two distinct extracellular lipases were obtained from Penicillium solitum 194A, isolated from domestic compost, and Cladosporium cladosporioides 194B, isolated from dairy wastewater. These alkaline enzymes had molecular masses of 42 and 30 kDa, respectively. The P. solitum 194A lipase differed in mass from previously reported enzyme, indicating that it is a novel lipase, and indicating that penicillia can secrete lipase isoenzymes. The C. cladosporioides lipase was more active on esters of medium-chain acids, whereas the P. solitum lipase was more active on longer chained substrates. The C. cladosporioides enzyme displayed higher thermal stability than the P. solitum lipase, preserving full activity up to 48 °C and showing a T₅₀ (10 min) of 60 °C. Their different catalytic properties and good protein stability should make these enzymes suitable for biotechnological applications. Furthermore, the combined use of these two fungal strains may prove to be valuable in lipid-rich waste management.

Key words:

• Penicillium solitum
• Cladosporium cladosporioides
• lipolytic activity
• alkaline lipase
• lipase isoenzymes

Lipolytic enzymes are important biocatalysts due to their numerous applications in industries, such as food, dairy, pharmaceuticals, cosmetics, leather, fine chemicals, detergents, textiles, and paper.^1–4) Although they can be obtained from different sources, the most widely exploited enzymes are produced by micro-organisms.

Lipolytic enzymes act on carboxyl esters and include lipases (EC 3.1.1.3) and carboxylesterases (EC 3.1.1.1). Lipases, commonly called true lipases, are more active on the carboxyl ester bonds present in triacylglycerols, liberating long-chain fatty acids (>10 carbons) and glycerol, whereas carboxylesterases preferentially hydrolyze esters with relatively short fatty acid chains (<10 carbons).⁵⁾ Many lipases are stable in organic solvents, and under certain conditions they can also perform esterification, transesterification, acidolysis, alcoholysis, and aminolysis.^6–8) Their reactions usually involve high regio- and/or enantio-selectivity, making these enzymes potential tools for organic chemists.

In spite of their distinctive substrate specificities, lipases and carboxylesterases show similar catalytic mechanisms and molecular structures. They are serine hydrolases endowed with both the canonical catalytic triad (i.e. Ser-His-Asp/Glu) in the active site, and the characteristic α/β-hydrolase fold in their three-dimensional structures.⁹⁾ Apart from their structural and catalytic features, bacterial and fungal lipases widely differ in their pH and temperature optima, substrate specificity, effector and inhibitor sensitivity, and cellular localization.^8,10) Hence, they have various applications as biocatalysts in several industrial fields. In addition to the extensive use of lipases in industry, a promising field for the application of lipolytic enzymes and lipolytic micro-organisms lies in environmental management, in particular in the treatment of waste that is rich in fats, oils, and greases (FOGs).²⁾ FOGs are among the main components of organic matter in wastewater and solid waste of municipal and industrial origin. Unless biologically treated, FOGs are detrimental to the environment, causing land and water pollution. Moreover, they tend to clog drainpipes and corrode sewer pipes.^11,12) Thus, pretreating FOG-rich waste with lipolytic biocatalysts can minimize its negative effects by improving removal efficiency and accelerating the degradation process. Several strategies have been developed for this purpose, consisting mainly in the use of hydrolytic microbial enzymes or viable lipase-secreting micro-organisms.^11,13,14) One way of enhancing biodegradation is to select micro-organisms with high degradation activity for FOGs and apply them to the removal of pollutants.¹⁵⁾

The purpose of this study was to characterize the extracellular lipases of Penicillium solitum 194A and Cladosporium cladosporioides 194B, two mesophilic microfungi isolated from domestic compost and dairy wastewater samples and selected for their lipolytic activity. Here we report the production, purification, and a biochemical characterization of these two enzymes, which we found to be attractive for biotechnological applications. The enzyme from P. solitum 194A is an additional lipase secreted by the same species, confirming the hypothesis that penicillia can produce extracellular lipase isoforms.

Materials and methods

Reagents

Tween 20 (polyethylene glycol sorbitan monolaurate), Tween 80 (polyethylene glycol sorbitan monooleate), tributyrin (glycerol tributyrate), p-nitrophenol, p-nitrophenyl octanoate (pNPO, C8), p-nitrophenyl decanoate (pNPD, C10), p-nitrophenyl dodecanoate (pNPDD, C12), p-nitrophenyl myristate (pNPM, C14), p-nitrophenyl palmitate (pNPP, C16), and p-nitrophenyl stearate (pNPS, C18) were purchased from Sigma (Schnelldorf, Germany). The olive oil used in the study was purchased from a local market (Pavia, Italy). All other chemicals used were of reagent grade.

Isolation of microfungi

The microfungal strains used in this study, P. solitum 194A (MUT 3450) and C. cladosporioides 194B (MUT 3447), were selected as good lipase producers based on their lipolytic activity on solid media, starting from a strain pool isolated from various sources, including industrial and domestic compost, oleaginous seeds, food, dairy wastewater, and hydrocarbon-contaminated soils.

P. solitum 194A and C. cladosporioides 194B were isolated from domestic compost and dairy industry wastewater (Pavia, Italy), respectively, by the dilution plate method. The final dilutions of the compost (5000-fold)¹⁶⁾ and of the dairy wastewater (10-fold and 100-fold) were poured into 140 mm Petri dishes containing potato dextrose agar (PDA, Biokar Diagnostics, Beauvais, France), a generic culture medium used to isolate microfungi, and Tween 20-Agar, a medium that is effective in detecting and isolating lipolytic microfungi.¹⁷⁾ Antibiotics (penicillin 70 mg/L and chloramphenicol 50 mg/L) were added to the media to limit bacterial growth. The fungi isolated from the plates were maintained on PDA slants at room temperature.

Taxonomic characterization of fungi

Identification of the isolated strains was performed on the basis of their micro- and macro-morphological features, as evaluated on agar plates containing specific diagnostic media, following Pitt¹⁸⁾ and Domsch et al.¹⁹⁾ Specific identifications were confirmed by molecular biology approaches: β-tubulin gene sequence analysis was used to identify the Penicillium strain, and the translation elongation factor 1-α (tef1) gene and internal transcribed spacer region (ITS) sequencing were used for the Cladosporium strain (BCCM/MUCL, (Agro) Industrial Fungi and Yeasts Collection, Louvain-la-Neuve, Belgium; GenBank accession nos. KF444170, KF444171, and KF444172).

Determination of lipolytic activity on solid media

A semi-quantitative evaluation of the lipolytic activity of the strains was carried out on solid media containing the following substrates: Tween 20, Tween 80, tributyrin, and olive oil. Culture basal medium was prepared for both Tweens following Sierra,²⁰⁾ and for tributyrin and olive oil following Anderson²¹⁾ and Ko et al.²²⁾ Tributyrin and olive oil were added to the basal medium to final concentrations of 1 and 0.5% v/v, respectively, and this was homogenized in a Waring blender. Tributyrin and olive oil were better emulsified in these media by adding Tween 80 (0.1 and 0.05% v/v, respectively). The media containing fatty substrates were poured into Petri dishes and inoculated at the center with conidial suspensions (10⁶ spores/mL) of each fungal strain. Lipolytic activity on the Tween media was recognized by the appearance of a precipitation zone around the colony,²⁰⁾ while on tributyrin and olive oil media it was recognized by the presence of a clearing zone. Three replicates were made for each strain, and the plate cultures were incubated at 25 °C. Colony diameter and precipitation/clearing zones were measured on the 7th and 14th incubation days. Yarrowia lipolytica CBS 6124, a known lipolytic yeast,²³⁾ was adopted as positive control. Enzyme activity was calculated from the ratio between the diameter of the hydrolysis zone and the colony diameter, and was expressed as the lipolytic activity index.²⁴⁾

The effect of carbon source on lipase production

The effect of the carbon source on lipase production was examined in submerged culture in media containing 5.0 g/L of peptone, 3.0 g/L of yeast extract, and Tween 80, tributyrin, or olive oil at a final concentration of 1% v/v. The basal medium with the addition of glucose (2% w/v) was used as control. For inoculum preparation, fungi were grown at 25 °C for 72 h in 2 mL of potato dextrose broth. The cultures obtained were inoculated in 300 mL Erlenmeyer flasks containing 100 mL of the fatty media, and were maintained at 25 °C for 7 or 14 d in a rotatory shaker. Then they were filtered through a filter paper (Albet 412, ALBET-Hahnemuehle, Barcelona, Spain) to remove the mycelium, and then passed through sterile 8 μm filters. The culture broth filtrates were used as crude enzyme preparations to determine lipase activity.

Culture conditions and lipase purification

In order to obtain extracellular lipases, each strain was grown at 25 °C for 14 d in two 2 L Erlenmeyer flasks containing 500 mL of broth, with the addition of 1% olive oil in the case of P. solitum 194A, and with 1% Tween 80 in the case of C. cladosporioides 194B. The broth for P. solitum 194A consisted of basal medium (5.0 g/L of peptone and 3.0 g/L of yeast extract). The broth for C. cladosporioides 194B was the medium of Cho et al.²⁵⁾ One liter of solution contained 6.0 g of KH₂PO₄, 1.0 g of MgSO₄x7H₂O, 2.0 g of (NH₄)₂SO₄, and 4.0 g of peptone. This allowed us to obtain large quantities of enzymes. At the end of incubation, the mycelium was removed by filtration as previously described, and the culture broth filtrate was centrifuged at 23,000 × g for 90 min, concentrated by ultrafiltration to nearly 100 mL with a 10 kDa cut-off membrane (Amicon—Millipore, Billerica, MA, USA), adjusted to pH 8.5 with 1 M Tris, subdivided into two aliquots, and applied separately to two settled-in-series HiTrap DEAE FF columns (5 mL, GE Healthcare), equilibrated in 50 mM Tris-HCl, pH 8.5. After they were washed with the equilibration buffer, the bound proteins were eluted with a discontinuous KCl gradient (0.0–1.0 M). The flow rate was 2 mL/min. Fractions of 1.5 mL were collected, and those containing lipase activity were pooled, concentrated to 5 mL by ultrafiltration, and loaded onto a HiLoad 16/60 Superdex 75 column (120 mL, GE Healthcare) equilibrated in 50 mM Na-phosphate buffer, pH 7.5. Protein elution was performed with the equilibration buffer at a flow rate of 1.0 mL min⁻¹. Fractions of 2 mL were collected, and those containing lipase activity were pooled together, concentrated by ultrafiltration, and tested for purity on 12% SDS-PAGE. The C. cladosporioides 194B lipase was then run on a HiTrap DEAE FF column (1 mL, GE Healthcare) equilibrated in 50 mM Tris-HCl, pH 8.5, at 1.0 mL min⁻¹. Protein elution was performed with a discontinuous gradient of KCl (0.0–1.0 M). Fractions of 0.3 mL were collected. Fractions with lipase activity were pooled, desalted, and concentrated. The protein concentration was determined following Lowry et al.²⁶⁾

Measurement of lipase activity

Unless otherwise indicated, lipase activity was determined spectrophotometrically at 37 °C with pNPP as substrate for the P. solitum 194A enzyme and with pNPO as a substrate for the C. cladosporioides 194B enzyme, and by monitoring the p-nitrophenol released at 405 nm.^27,28) All substrates were dissolved in 2-propanol. The standard reaction mixture contained 50 mM Na-carbonate, pH 9.5 and 0.5 mM substrate in a final volume of 0.5 mL. The reaction mixture for C. cladosporioides 194B also contained 50 mM Na-phosphate. The reaction was started by adding enzyme solution (0.1–0.5 μg). The reference cuvette contained all the components of the standard reaction mixture with the exception of the enzyme. Under these assay conditions, the molar extinction coefficient of p-nitrophenol was 18,200 M⁻¹ cm⁻¹. One unit was defined as the amount of enzyme that catalyzes the formation of 1 μmol of p-nitrophenol/min under the above conditions.

Determination of the optimum pH value

To determine the optimum pH value, enzyme activity was tested at 0.5 mM substrate while varying the pH through a range 7.0–10.5 with the following buffers (50 mM): PIPES pH 7.0–7.5; HEPES pH 7.5–8.5; Na-phosphate pH 7.0–9.0; and Na-carbonate pH 9.0–10.5. The reaction mixtures for C. cladosporioides 194B also contained 50 mM Na-phosphate. The pH-dependent activity was evaluated with six different substrates (C8–C18). The molar extinction coefficients of p-nitrophenol were determined prior to measurement under each reaction condition.

Thermal stability assays

For thermal stability assays, the enzyme (0.1–0.2 mg/mL) was incubated at given temperatures (30–75 °C) in 50 mM Na-phosphate buffer, pH 7.5. At regular intervals, samples were removed, chilled on ice, and assayed for enzyme activity. Relative activity was calculated as the percentage of enzyme activity before incubation. T₅₀ is defined as the temperature at which the enzyme loses 50% of its activity in 10 min.

Kinetic analysis

For steady-state kinetic analysis, unless otherwise indicated, enzymatic activity was assayed at 37 °C with various concentrations (0.05–0.5 mM) of the various substrates in 50 mM Na-carbonate, pH 9.5 under conditions identical to those described above, except for the substrates used. In this case, the substrates were p-nitrophenyl esters with various acyl chains (C8–C18) as follows: pNPO (C8), pNPD (C10), pNPDD (C12), pNPM (C14), pNPP (C16), and pNPS (C18).

In all cases, the reaction was started by adding the enzyme solution (0.1–0.5 μg). All measurements were taken in triplicate with a Jasco V-550 UV–VIS spectrophotometer (Jasco-Europe, Cremalla, Italy). Kinetic parameters were calculated with Sigma Plot software (SPSS Inc, Chicago, IL). k_cat, or turnover number, is the number of catalytic events per second. K_m is the substrate concentration at which the reaction velocity is half-maximal. k_cat/K_m is a measure of how efficiently an enzyme converts substrate to product at sub-saturating substrate concentrations.

Effects of metal ions on enzyme activity

To determine the effects of different ions on enzyme activity, both lipases were assayed as previously reported, but in the presence of various ions at a final concentration of 2 mM. The activity of the enzymes without metallic ions was taken to be 100%.

Results

Lipolytic activity on solid media

Two fungal strains, 194A and 194B, isolated from domestic compost and dairy wastewater, were selected from a strain pool after an evaluation of lipolytic activity on solid media supplemented with four fatty substrates, tributyrin, Tween 20, Tween 80, and olive oil. In all cases, they developed sizeable halos around the fungal colony. On the basis of their micro- and macro-morphological features and distinctive gene sequence analysis (see “Materials and methods”), the two strains were identified as P. solitum 194A and C. cladosporioides 194B. The resulting sequence of the P. solitum 194A β-tubulin gene (459 bp, GenBank acc. no. KF444170) showed the highest identity (99%) and coverage (98%) to sequences of P. solitum strains deposited in the GenBank database. As for the C. cladosporioides 194B analysis, the greatest similarity for the tef1 gene sequence (246 bp, GenBank acc. no. KF444171) was 99% (coverage 99%) with C. cladosporioides deposited sequences, and 100% (coverage 100%) for the ITS sequence (541 bp, GenBank acc. no. KF444172).

To evaluate the trend of enzyme production over time, the two strains were cultured after the first selection for up to 14 d on the same fatty substances. Their lipolytic activities were measured at 7 and 14 d of growth with the lipolytic activity index (hydrolysis zone diameter/colony diameter). The results were then compared with those of the positive control, Y. lipolytica CBS 6124 (Table 1).

Table 1. Lipolytic activity index for P. solitum 194A and C. cladosporioides 194B on solid media on various fatty substrates.

Substrate	Lipolytic Activity Index
	Y. lipolytica CBS 6124		P. solitum 194A		C. cladosporioides 194B
	Day 7	Day 14	Day 7	Day 14	Day 7	Day 14
Tributyrin	1.14 ± 0.04	1.11 ± 0.02	1.37 ± 0.06	1.53 ± 0.06	1.63 ± 0.13	1.92 ± 0.27
Tween 20	3.09 ± 0.04	3.20 ± 0.13	1.49 ± 0.05	1.34 ± 0.02	2.14 ± 0.08	3.29 ± 0.15
Tween 80	1.65 ± 0.12	2.00 ± 0.10	1.55 ± 0.29	1.64 ± 0.30	2.91 ± 0.29	3.79 ± 0.18
Olive oil	–	–	1.10 ± 0.03	1.14 ± 0.01	–	1.08 ± 0.03

Note: Data are means (SE) of three replicates.

The lipolytic activities exhibited by C. cladosporioides 194B increased with growth time, and were generally higher than the activities exhibited by P. solitum 194A and Y. lipolytica CBS 6124 (Table 1). The highest activities were displayed towards the synthetic substrates (Tweens and tributyrin). The activity towards the natural substrate (olive oil) was similar to the activity exhibited by P. solitum 194A, although it was detectable only on the 14th day. The lipolytic activities of P. solitum 194A on tributyrin and olive oil were higher than the activities exhibited by control strain, Y. lipolytica CBS 6124. Overall, the lipolytic activities exhibited by P. solitum 194A towards all substrates reached a maximum level after almost 7 d of growth, and were slightly higher with synthetic substrates than with natural ones.

Lipase production in liquid media

The extracellular lipase activity of the two strains was also evaluated spectrophotometrically at 7 and 14 d of culture in liquid media supplemented with various carbon sources (see “Materials and methods”). Both strains exhibited lipase activity only in the presence of olive oil and Tween 80. No activity was recorded in the presence of glucose (control) or tributyrin (Table 2). In addition, growth time affected the level of activity: both strains showed maximum lipase activity on the 14th day.

Table 2. Lipase activities of P. solitum 194A and C. cladosporioides 194B in liquid media containing various carbon sources.

Substrate	P. solitum 194A				C. cladosporioides 194B
	Day 7		Day 14		Day 7		Day 14
	Lipase activity (U/mL)	Biomass (mg/mL)	Lipase activity (U/mL)	Biomass (mg/mL)	Lipase activity (U/mL)	Biomass (mg/mL)	Lipase activity (U/mL)	Biomass (mg/mL)
Glucose	0.000	2.270	0.000	3.130	0.000	2.860	0.000	7.100
Tributyrin	0.000	0.340	0.000	0.390	0.000	0.550	0.000	0.720
Tween 80	0.009	0.490	0.071	1.720	0.004	0.820	0.044	1.505
Olive oil	0.010	5.470	0.110	8.790	0.004	3.810	0.038	7.340

Note: Fungal biomass, expressed as mg dry weight/mL, is also reported.

As shown in Table 2, olive oil appeared to be the best substrate for lipase production by P. solitum 194A (0.11 U/mL). This was not a finding: some Penicillium species have been reported to display similar behavior.²⁹⁾ The amount of lipase expressed was generally lower for C. cladosporioides 194B than for P. solitum 194A, both with Tween 80 (0.044 U/mL vs. 0.071 U/mL) and olive oil (0.038 U/mL vs. 0.11 U/mL).

Based on these findings, in order to obtain the enzyme, P. solitum 194A was grown in the presence of olive oil, and C. cladosporioides 194B in the presence of Tween 80.

Lipase purification

The extracellular lipases from P. solitum 194A and C. cladosporioides 194B were purified by the procedures described above in “Materials and methods”. Shortly afterwards, at the end of incubation, culture broth filtrate was applied to a HiTrap DEAE FF column (pH 8.5) (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The elution profiles of the proteins are reported in Fig. 1(A) and (B). Both lipases eluted at KCl concentrations between 0.2 and 0.4 M. After concentration, the lipase solutions were subjected to size exclusion chromatography on a HiLoad 16/60 Superdex 75 column (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The elution profiles of lipase activity for P. solitum 194A and C. cladosporioides 194B are reported in Fig. 1(C) and (D), respectively. The major peak of P. solitum 194A lipase activity (Fig. 1(C)) was just before the major peak of C. cladosporioides 194B (Fig. 1(D)), indicating that the former had a higher molecular mass. Based on 12% SDS-PAGE, the P. solitum 194A lipase was found to be a pure protein with a molecular mass of nearly 42 kDa (Fig. 1(E)). The specific activity of the purified enzymes was 44 U/mg.

Fig. 1. Purification of P. solitum 194A and C. cladosporioides 194B lipases.

Note: Elution profiles of P. solitum 194A and C. cladosporioides 194B enzymes from a HiTrap DEAE FF column (panels (A) and (B), respectively) and from a HiLoad 16/60 Superdex 75 column (panels (C) and (D) respectively). Circles represent lipase activity, the continuous line represents the absorbance at 280 nm, and the broken line represents the KCl gradient used for elution from the anionic exchange columns. Silver-stained 12% SDS-PAGE of the two purified lipases is represented in panel (E). Lane M, molecular weight markers; lane P, P. solitum 194A lipase; lane C, C. cladosporioides 194B lipase. Panel (F) represents the final chromatography performed for the C. cladosporioides 194B lipase on a HiTrap DEAE FF column.

As for the C. cladosporioides 194B lipase, 12% SDS-PAGE of the enzyme obtained by size exclusion chromatography indicated the presence of additional proteins (data not shown). To remove these proteins, the C. cladosporioides 194B lipase was subjected to a further chromatographic step on the HiTrap DEAE FF column. The enzyme eluted at 0.3 M KCl (Fig. 1(F)). Its apparent molecular mass, as judged by 12% SDS-PAGE (Fig. 1(E)), approximated 30 kDa. The specific activity of the C. cladosporioides 194B lipase was 12 U/mg. The enzymes were stored at 4 °C in 50 mM Na-phosphate buffer, pH 7.5.

Lipases from both strains were inhibited by serine hydrolase inhibitor PMSF (IC₅₀, 6.5 and 10.5 mM for P. solitum 194A and C. cladosporioides 194B, respectively; data not shown), indicating that they are classic serine hydrolases.⁸⁾ Moreover, the enzyme from C. cladosporioides 194B required phosphate for enzyme activity and stability (data not shown).

Dependence of activity on pH

The effect of pH on the enzymatic activity of the lipases from P. solitum 194A and C. cladosporioides 194B was investigated with p-nitrophenyl esters of various fatty acids (C8–C18) in a pH range of 7.0–10.5. All substrates were used at a fixed concentration of 0.5 mM. As shown in Fig. 2, both enzymes were active at alkaline pH, and their maximal activities varied according to the type of substrate used.

Fig. 2. pH activity profiles of the lipases.

Note: The effects of pH on the activity of P. solitum 194A (panel (A)) and C. cladosporioides 194B (panel (B)) lipases were determined at 0.5 mM of p-nitro derivative substrates, with the following buffers (50 mM): PIPES (7.0–7.5), HEPES (7.5–8.5), Na-phosphate (7.0–9.0), and Na-bicarbonate (9.0–10.5). For the assays of the C. cladosporioides 194B lipase, all the buffers contained 50 mM Pi. ● pNPO; ▼ pNPD; ■ pNPDD; ♦ pNPM ▲ pNPP;

pNPS.

The pH activity profiles fitted the bell-shaped model with plateau, and optimum pH values ranged between 8.5 and 9.7. The P. solitum 194A lipase exhibited higher activities with all substrates than the C. cladosporioides 194B enzyme, with a maximum towards esters of long-chain fatty acids (Fig. 2(A)). The C. cladosporioides 194B enzyme was more active with esters of medium-chain fatty acids (Fig. 2(B)), and it failed to show any activity with esters of fatty acids as long as C16 and C18 at any pH value tested.

Thermal stability properties

To evaluate heat stability, the enzymes were incubated at various temperatures for 10 min and assayed for residual activities (Fig. 3). The P. solitum 194A lipase was found to be less stable than the C. cladosporioides 194B lipase, showing a steep decline in activity when heated to over 40 °C. Its T₅₀ value was calculated to be 42.4 °C, 18 °C lower than the value for the C. cladosporioides 194B enzyme. The C. cladosporioides 194B lipase preserved full activity up to an incubation temperature of 48 °C, after which it showed gradual inactivation as the temperature increased, until it was completely inactive after 10 min of incubation at 72 °C.

Fig. 3. Thermal stability of the lipases.

Note: The thermal stability of P. solitum 194A (●) and C. cladosporioides 194B (▼) lipases was assessed by subjecting each to heat treatment ranging between 30 and 75 °C. After 10 min of incubation at a given temperature, the enzyme sample was chilled and residual activity was measured. Residual activity was expressed as a percentage of the enzyme activity before incubation.

Catalytic properties

Activity as a function of substrate concentration was assayed for six different p-nitrophenyl esters (C8–C18) by spectrophotometric monitoring of the p-nitrophenol released. Each kinetic run was carried out at the pH value at which the enzyme showed maximal activity (Fig. 4). The parameters derived from these kinetic runs are summarized in Table 3. As for the P. solitum 194A lipase (Fig. 4(A)), V_max values increased with increasing lengths of the fatty acid carbon chains. Conversely, K_m values decreased with increasing lengths of the carbon chains between C8 and C14, but increased when the fatty acid chains were longer than C14. Hence, although the highest activity was towards pNPS (C18; k_cat, 39.2 s⁻¹), the optimal substrate for the P. solitum 194A lipase was pNPM (C14; k_cat/K_m, 959.2 s⁻¹ mM⁻¹). As for the C. cladosporioides 194B lipase (Fig. 4(B)), both activity and affinity (Table 3) decreased with increasing lengths of carbon chain, and pNPO was the optimal substrate (C8; k_cat/K_m, 260.0 s⁻¹ mM⁻¹). As observed above (Fig. 2(B)), no activity was detected with pNPP (C16) or pNPS (C18).

Fig. 4. Steady-state kinetics of the lipases.

Note: Steady-state kinetics of the P. solitum 194A (panel (A)) and C. cladosporioides 194B (panel (B)) lipases as a function of various concentrations of p-nitrophenyl esters as substrates. Enzyme activity was assayed at pH 9.5, 37 °C (see “Materials and methods”). ● pNPO; ▼ pNPD; ■ pNPDD; ♦ pNPM ▲ pNPP;

pNPS.

Table 3. Kinetic parameters of the P. solitum 194A and C. cladosporioides 194B lipases.

Substrate	P. solitum 194A lipase			C. cladosporioides 194B lipase
	kcat (s^−1)	Km (mM)	kcat/Km (s^−1 mM^−1)	kcat (s^−1)	Km (mM)	kcat/Km (s^−1 mM^−1)
pNPO	15.4 ± 0.9	0.066 ± 0.009	233.3	6.5 ± 0.4	0.025 ± 0.003	260.0
pNPD	16.4 ± 3.4	0.041 ± 0.005	400.0	3.5 ± 0.4	0.024 ± 0.005	145.8
pNPDD	21.6 ± 2.5	0.036 ± 0.009	600.0	1.4 ± 0.1	0.046 ± 0.009	30.4
pNPM	25.9 ± 1.3	0.027 ± 0.002	959.2	0.5 ± 0.05	0.138 ± 0.023	3.6
pNPP	31.0 ± 2.5	0.063 ± 0.008	492.0	–	–	–
pNPS	39.2 ± 12.0	0.330 ± 0.120	118.8	–	–	–

Note: Results are means (SE) for three determinations from at least two different protein preparations.

Effects of metal ions on lipase activity

To evaluate the effects of metal ions on the activity of P. solitum 194A and C. cladosporioides 194B lipases, the enzymes were assayed in the presence of various cations (K⁺, Li⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺, Fe²⁺, and Pb²⁺) at a final concentration of 2 mM (Fig. 5). Cu²⁺, Fe²⁺, and Pb²⁺ severely affected P. solitum 194A lipase activity (inhibition nearly 60%). Li⁺ and Ni²⁺ were also inhibitory, although their negative effects were less marked (inhibition about 20%). Compared to the enzyme from P. solitum 194A, C. cladosporioides 194B lipase activity was less affected by the metal ions examined. Cu²⁺ and Fe²⁺ caused moderate inactivation (nearly 20%), and Pb²⁺ was even less effective (inhibition less than 10%). All the other ions left the enzyme activity unchanged, with the exception of Mg²⁺, which raised lipolytic activity by approximately 30%. In all cases, both inhibition and activation increased with increasing metal ion concentrations (data not shown).

Fig. 5002E Effects of metal ions on lipase activity.

Note: Effects of various metal ions on P. solitum 194A (dark bars) and C. cladosporioides 194B (light bars) lipase activity were determined at a final ion concentration of 2 mM. Enzyme activity was assayed as reported in “Materials and methods” and was expressed as residual activity with respect to the activity of the untreated enzyme.

Discussion

Extracellular lipases are among the most promising enzymes that have biotechnological and environmental potential, owing to their peculiar catalytic properties. The ongoing search for new lipases with improved features increasingly involves filamentous fungi, due to their capability to secrete enzymes.⁴⁾

Penicillium is one of the most investigated genera,²⁹⁾ being a good producer of various extracellular enzymes as well as a very important decomposer–recycler of organic matter of all types.^18,30) Cladosporium has been found to be able to produce cellololytic and xylanolytic enzymes, but little is known about its lipolytic enzymes.^25,31)

The present study found that two fungal strains, P. solitum 194A and C. cladosporioides 194B, isolated from domestic compost and dairy wastewater and characterized for their different lipolytic activities on media containing fatty substrates, are good lipase producers. A preliminary evaluation of their activities on solid media, measured by the lipolytic activity index, confirms the importance of this tool in detecting various lipolytic activities in micro-organisms. C. cladosporioides, previously considered to be a suspect lipolytic species,²⁵⁾ was found to be a lipase producer.

Although most biotechnological applications do not require homogeneous enzyme preparations, extensive purification sometimes enables efficient and successful usage in certain industrial processes. Moreover, pure enzymes are needed for a proper assessment of their molecular and kinetic properties and for a better understanding of kinetic mechanisms. Unfortunately, the purification of lipases often requires laborious procedures, making it difficult to obtain highly purified preparations.^8,32) The lipases from P. solitum 194A and C. cladosporioides 194B were purified to homogeneity by simple and time-saving procedures (two and three chromatographic steps, respectively), making possible accurate characterizations of their molecular and functional properties.

Most extracellular lipases from micro-organisms appear to be monomeric proteins with molecular masses of about 25–60 kDa. Moreover, some micro-organisms appear to produce at least two types of lipases, characterized by different biochemical and molecular properties.³³⁾ The P. solitum 194A and C. cladosporioides 194B lipases showed molecular masses of 42 and 30 kDa, respectively. The value obtained for P. solitum 194A was in line with the most well-known lipases from the genus Penicillium (a molecular mass in the range of 25–43 kDa),^29,34) similar to the value obtained for the type II lipases from P. camembertii (38 kDa)^5,35,36) and P. cyclopium (40–43 kDa).^5,35) Conversely, the value differed from the molecular mass of the type I P. solitum lipase previously reported (27 kDa).^5,35,37) This difference is likely to be due to an isoform of the enzyme secreted by this micro-organism. Evidence of an additional lipase secreted by P. solitum is noteworthy, because it supports the suggestion that penicillia are capable of producing more than one extracellular lipase.^37,38)

Microbial lipases can act in a wide range of pH, even though the pH optimum for bacterial enzymes is usually neutral^39–41) or alkaline,^8,42–46) and the pH optimum of fungal enzymes is in a range 4.0–8.0.³⁾ Maximal hydrolytic activity also depends on the type of ester used as substrate.⁸⁾ The pH optima for the P. solitum 194A and C. cladosporioides 194B lipases fell in an alkaline range (pH 8.5–9.7). The value obtained for P. solitum 194A differed from that obtained for the type I lipase previously reported for the same species (pH optimum, 7.0).³⁷⁾ This indicates that the enzyme we purified was a new lipase. The P. solitum 194A and C. cladosporioides 194B lipases were found to be highly heat-resistant enzymes (full activity preserved up to 40 and 48 °C, respectively, and especially T₅₀ for C. cladosporioides 194B, which equates to 60 °C) (Fig. 3), indicating that they were endowed with good protein stability. Usually, fungal lipases are less thermostable than bacterial ones (<40 °C vs. >50 °C),^47,48) and thus the new heat-resistant lipases from P. solitum 194A and C. cladosporioides 194B are particularly suitable for industrial applications.

Lipases are by definition enzymes that can hydrolyze long-chain acylglycerols (C ≥ 10), but most known lipases are also active on shorter acyl chain esters as well as on p-nitrophenyl esters, with different efficiencies.^29,49) The lipases from P. solitum 194A and C. cladosporioides 194B differed from each other in terms of maximal activity towards a range of p-nitrophenyl esters (C8–C18). Overall, the P. solitum 194A enzyme showed the characteristics of a true lipase by preferentially hydrolyzing ester substrates with a wide range of carboxylic acids, mainly long-chain fatty acid esters. The lipase from C. cladosporioides 194B stood out from the others because of its preference for medium-chain fatty acid esters, appearing to have traits of common carboxylic ester hydrolases.⁵⁰⁾ Overall, the affinities of both enzymes for their preferred substrates were similar, and significantly higher than the affinities of the most closely corresponding microbial enzymes (K_m values, μM vs. mM).^33,51) The K_m of the P. solitum 194A enzyme towards long-chain fatty acid esters was even lower (up to 14-fold vs. pNPM) than the K_m of the P. chrysogenum SNP5 lipase, which was recently reported to be a novel lipase in view of its uncommonly high affinity toward esters.⁵²⁾ Obviously, great emphasis should be placed on comparing the kinetic parameters of lipases due to the different experimental conditions adopted, the type of substrate, or the assay method.¹⁰⁾

The P. solitum 194A and C. cladosporioides 194B lipases are distinguished from other fungal extracellular lipases by their sensitivity to metal ions.^8,29,33,51) Surprisingly, the enzyme from C. cladosporioides 194B was stimulated by Mg²⁺ (nearly 30% activation), whereas the lipase activities of most microfungi usually remain almost unchanged (or even slightly inhibited) under exposure to Mg²⁺.^53,54) On the other hand, the P. solitum 194A enzyme was highly affected by Pb²⁺, Cu²⁺, and Fe²⁺ (about 60% inhibition). Ca²⁺, which has been found to affect fungal lipase activity and stability,²⁹⁾ had no effect on the activity of these novel lipases.

In conclusion, two fungal strains, P. solitum 194A and C. cladosporioides 194B, were found to be good lipase producers as they displayed different lipolytic activities on media containing fatty substrates. This study confirms lipase production by C. cladosporioides, which was previously treated as a suspect lipolytic species.²⁵⁾ Its enzyme shows preference for medium-chain fatty acid esters and a noticeably higher thermostability than the corresponding enzymes from mesophilic filamentous fungi. P. solitum 194A secretes an additional lipase, which reinforces the suggestion that filamentous fungi can produce various lipolytic enzymes. Similarly to the enzyme from C. cladosporioides 194B, the P. solitum 194A lipase displayed optimal activities at alkaline pH values, but it was more active and showed a preference for long-chain fatty acid esters.

Further research into activity and stability in organic solvents, regio- and stereo-specificity, and surfactants and inhibitors, should assess whether these new lipases can find biotechnological applications, e.g. in the manufacture of fine chemicals and detergents. Moreover, the different features of these novel lipases secreted by P. solitum 194A and C. cladosporioides 194B make the two strains suitable candidates for the treatment of lipid-rich waste, in which they can complement each other in hydrolyzing a broad spectrum of substrates.

Acknowledgments

We thank the BCCM/MUCL (Agro) Industrial Fungi and Yeasts Collection (Louvain-la-Neuve, Belgium) for molecular analysis of the strains studied here. This work was supported by the Lombardy Region ECO-BRICK Project and by Grants from the University of Pavia under the program “Fondi per Ricerca di Ateneo” (FAR).