Abstract

Penicillium restrictum lipases : A comparative study and characterization of enzymes with different degrees of purity

M.F.C.P. JESUS¹, R.N. BRANCO², G.L. SANT'ANNA JR.³, D.M.G. FREIRE² and J.G. SILVA JR1

¹Instituto de Química, Universidade Federal do Rio de Janeiro, ²Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, ³PEQ/COPPE, Universidade Federal do Rio de Janeiro, Caixa Postal 68502, CEP 21945-970, Rio de Janeiro - RJ, Brazil; Fax: (55 21) 290-6626, Phone: (021) 590-2297,

E-mail: fatima@peq.coppe.ufrj.br

(Received: January 19, 1999; Accepted: March 11, 1999)

Abstract - Penicillium restrictum was identified as a promising strain for lipase production due to enzyme production yield and thermal stability of the enzyme. This work presents results of lipase purification and enzyme stability versus pH. Ultrafiltration and precipitation with ammonium sulphate were used as initial purification steps. The partially purified enzyme preparation showed an increase in stability as pH increased. The crude enzymatic preparation was assayed with different oils and tributirin and showed a major catalytic activity for triglycerides of medium/long-chain fatty acids. Further purification steps were conducted on an analytical scale. The initial attempt to use gel filtration was abandoned as lipase lost its stability after this chromatographic procedure. The fast ion-exchange chromatography was performed on a Mono Q column, and two peaks with lipolytic activity were isolated and analysed by electrophoresis.

Keywords: lipase, Penicillium restrictum, purification, characterization, stability.

INTRODUCTION

Recently there has been an increase in interest in microbial lipases (triacylglycerol ester hydrolases E.C.3.1.1.3). There are big advantages to using microbial enzymes in comparison with lipases from other sources, and the search for new lipases showing diverse characteristics is a matter of industrial interest (Björkling et al., 1991).

From the industrial point of view, fungi are the preferred source of lipases, as they produce extracellular enzymes that facilitate downstream processing.

In spite of growing academic interest in these enzymes of great biotechnological versatility, the industrial applications of lipases are still restricted to the detergent and food industries, corresponding to 4% of the worldwide market for enzymes.

This work reports on the purification of lipases from a new and promising strain of Penicillium restrictum recently studied as a lipase producer by Freire et al. (1997^(a),(b)). In addition, this work presents experimental results on the stability of crude and partially purified lipase.

MATERIALS AND METHODS

Production and Determination of Enzymatic Activity

The production and the determination of lipase activity was conducted in accordance with the methodology described by Freire et al., 1997 ^{(a), (b)}.

Partial Purification of Lipase

Ultrafiltration: The culture broth was filtered through Whatman 44 paper and clarified using a HAWP 0.45µm membrane from Millipore. Then the crude broth was concentrated using a Desal G-80 membrane (cut off 10 kDa).

Precipitation: The proteins were precipitated at 4^ºC by the addition of (NH₄)₂SO₄ at 80% saturation. The resultant precipitate was collected by centrifugation and dissolved in 25 mM phosphate buffer pH 7.0.

Purification on an Analytical Scale

Ion exchange chromatography: The dissolved material was dialysed with a 25 mM phosphate buffer pH 7.0 and was passed (5 mL) through a MonoQ column (1 x 10 cm). Proteins were eluted in the same buffer using a NaCl stepwise gradient. The elution was studied by measuring absorbance at 280nm in a PDA detector (Waters Co.). Enzymatic activity was assayed in all eluted fractions. The active fractions were collected and pooled. Lipolytic activity and protein concentration (measured according to Peterson, 1983) in the pool were determined.

Electrophoresis

The enzymatic preparations were analysed by 8-25% SDS-PAGE gradient and by 5% IEF-PAGE in the pH range of 3 to 9. In both cases, the electrophoresis assays were carried out in PhastSystem (Pharmacia) and the proteins were stained with R-350 Coomassie Brilliant Blue according to Pharmacia's User's Manual.

Enzymatic Stability

pH stability of the crude and the partially purified enzyme preparations was determined at 37°C. Enzymatic activity was measured at the beginning and at the end of the incubation period, and activity decay results were fitted by the linear inverted model (Cardoso and Emery, 1978).

Enzymatic Hydrolysis and Determination of Kinetic Parameters

The enzymatic hydrolysis was performed with 5% (w/v) oil or triglyceride, as described by Freire et al. (1997 ^(b)). The reaction was stopped at reaction times ranging from 4 to 360 min.

The Michaelis-Menten kinetic parameters (Km and Vm) for crude enzymatic preparation were determined using different concentrations of olive oil, babassu oil and tributirin (in the range of 0.1 to 10 % (w/v)).

RESULTS AND DISCUSSION

The results from the initial stages of lipase concentration and purification are presented in Table ¹ Table 1: Partial purification of lipase from Penicillium restrictum. .

Precipitation with (NH₄)₂SO₄ largely increased the specific activity of the enzyme; a purification factor higher than those usually found in the literature was obtained. After precipitation, the enzyme could be stored at 4°C in the presence of salt, in 25 mM phosphate buffer pH 7.0 for more than a year without significant loss of enzymatic activity.

Further purification steps were carried out on an analytical scale. The dissolved material, obtained by precipitation with ammonium sulphate, was submitted to gel filtration chromatography using various matrixes (Bio Gel P-6, Sephadex G-50 and G-100, Superdex 75 and Superose 12). The initial attempt to use gel filtration as the first purification step was abandoned, as lipase lost its stability after elution. Furthermore, it seemed that the enzyme had some hydrophobic interaction with the gel matrix (Stöcklein et al., 1993), causing the elution of the enzyme to spread out, which occurred in a wide range of molecular weights (from 12,000 to 90,000 Da).

The material obtained from precipitation with ammonium sulphate was dissolved in 25 mM phosphate buffer pH 7.0 and dialysed overnight with the same buffer. A sample of 5 mL was passed through a MonoQ column, previously equilibrated with the same buffer. From the MonoQ column, two different peaks (A and B) with lipolytic activity were eluted. Peak A was eluted with 0.25 M NaCl, whereas peak B was eluted with 0.30 M NaCl (Figure ¹ ). In this step, enzyme recovery was 45% and the purification factors were 3.24 and 1.94 for peaks A and B, respectively.

Figure 1: Elution of peaks A and B with lipolytic activity from the MonoQ column. Conditions: 25mM phosphate buffer pH 7.0; flow rate: 1.5 mL/min; fractions volume: 1,5mL. Detection of protein at 280nm ( --- ). Lipolytic activity, U/mL (- - · - -). Salt gradient (- - -).

Both peaks with lipolytic activity were analysed by SDS-PAGE. A band with a molecular weight of 21,900 Da appeared in peaks A and B. Peaks were also analysed by IEF-PAGE, showing some bands of low intensity in the pH range of 4.0 to 5.5. These results indicate that there are at least 2 lipases with different characteristics produced by the fungus Penicillium restrictum.

The stability of the enzymatic preparation at 37°C and at different pH values, expressed as half-life time and decay constant, is presented in Table ² Table 2: Half-life time t½, constant of inactivation and correlation coefficient of the linear inverted model for crude and partially purified enzymatic preparations at different values of pH at 37°C*. . A decrease of 10 to 16 times the half-life time of the crude enzymatic preparation was observed when pH was increased from 7.0 to 8.0 and 7.0 to 9.0, respectively. However, for the partially purified enzyme, a decrease of about 2.5 times was observed when pH was raised from 7.0 to 8.0. That improvement in enzyme stability may be attributed to the removal, in the initial purification steps, of substances that contribute to enzyme instability, or it may be caused by the presence of a residual amount of (NH₄)₂SO₄ from the second purification step. These data are in accordance with the literature, since the majority of fungal lipases present optimal pH stability in the range of 6.0 to 8.0 and is unstable at pH values above 8.0.

Figure ² presents the hydrolysis progress curves (hydrolysis percentage) for different triglycerides (5 % w/v), using the crude enzyme preparation.

Figure 2: Percentage of hydrolysis by Penicillium restrictum lipase in olive oil (a), babassu oil (b) and tributirin (c).

Olive oil (Figure ² a) has a high content of oleic acid C18:1 (71.1%) and is usually taken as a standard substrate for the determination of lipolytic activity. The catalytic activity of lipase was 20 U/mL and the percentage of hydrolysis was below 35%. These results may indicate some degree of lipase inhibition by long-chain fatty acids.

The crude Penicillium restrictum lipase presented a high level of catalytic activity with babassu oil (30 U/mL). That was an expected result as the fungus was isolated from a rich source of that oil. Babassu oil has a high content (45 %) of lauric acid (C12:0), a medium-chain fatty acid. However, as hydrolysis occurs, the free fatty acids may cause severe lipase inhibition, as the maximum percentage of hydrolysis (after 6 hours) was only 28% (Figure ² b).

The hydrolysis of tributirin (C4:0) led to a low level of activity (4.0 U/mL). A low and constant hydrolysis rate was observed for up to 2 hours of reaction (Figure 2c). These results indicate that the enzyme has a low level of hydrolytic activity towards short-chain triglycerides.

Michaelis-Menten kinetics constants were determined from a set of assays like those shown in Figure ² by varying substrate concentrations. The kinetic parameters thus obtained are presented in Table ³ Table 3: Michaelis-Menten kinetics constants of the crude Penicillium restrictum lipase. .

In relation to tributirin, this enzyme presented a V_max value about 2.6 times smaller than those obtained with the two oils tested. Although the Km value of tributirin was lower than those obtained for the two oils, this result does not indicate a higher affinity of the lipase for the triglyceride. The hydrolysis of lipids occurs in a heterogeneous medium and lipase acts at the lipid-water interface; thus, the concentration of molecules at the interface is a key parameter that determines the rate of hydrolysis (Jaeger et al., 1994). Triglycerides that are as highly soluble in water as tributirin can mask the kinetic results.

CONCLUSIONS

The initial steps of lipase purification (ultrafiltration and/or precipitation) led to an enhancement of specific activity. In addition, these steps contributed to improving enzyme stability at 4°C (by more than a year) and at pH 8.0.

After the ion-exchange chromatography, two peaks with lipolytic activity were obtained. These peaks presented a coincident band with a molecular weight of 21,900 Da. IEF-PAGE showed some bands in the pI range from 4.0 to 5.5.

The Penicillium restrictum lipase showed a high level of catalytic activity towards medium and long-chain triglycerides.

REFERENCES

Björkling, F.; Godtfedsen, S.E. and Kirk, O., The Future Impact of Industrial Lipases, TIBTECH, 9, 360-363 (1991).

Cardoso, P. and Emery, A.N., A New Model to Describe Enzyme Inactivation, Biotechnology and Bioengeneering, 20, 1471-1477 (1978).

Freire, D.M.; Gomes, P.M.; Bon, E.P.S. and Sant`Anna Jr., G.L., Lipase Production by a New Promising Strain of Penicillium restrictum, Revista de Microbiologia, 28 (Suppl. 1), 6-12 (1997a).

Freire, D.M.; Teles, E.M.F.; Bon, E.P.S. and Sant`Anna Jr., G.L., Lipase Production by Penicillium restrictum in a Bench Scale Fermenter: Effect of Carbon and Nitrogen Nutrition, Agitation and Aeration, Appl. Biochem. Biotechnol., vol. 63-65, 409-421 (1997b).

Jaeger, K-E.; Ransac, S.; Dijkstra, B.W.; Colson, C.; Heuvel, M. and Misset, O., Bacterial Lipases, FEMS Microbiology Reviews, 15, 29-63 (1994).

Peterson, G.L., Determination of Total Protein, Methods in Enzymology, 91, 95-105 (1983).

Stöcklein, W.; Sztajer, H.; Menge, U. and Schmid, R.D., Purification and Properties of a Lipase from Penicillium expansum, BBA, 1168, 181-189 (1993).

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