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Production of lipase from Geotrichum sp and adsorption studies on affinity resin

Abstract

There is a growing interest in microbial lipase production due to its great potential for industrial applications such as food additives, industrial reagents and stain removers, as well as for medical applications. Specially for medical applications a high degree of purity is required, which is accomplished with high resolution chromatographic techniques. Affinity chromatography is considered a very high resolution chromatographic technique. In this work the adsorption isotherms and kinetics of the adsorption of lipase from Geotrichum sp on biospecific resin were determined. The resin was prepared using EAH sepharose 4B gel (Pharmacia), made to react with oleic acid as the specific ligand.The lipase was produced in a five-liter fermenter, with both complex and synthetic media. Fermentation conditions were a temperature of 30°C, an aeration of 1VVM and an agitation of 400 rpm. Maximum lipase activity was around 28 U/ml after 10 hours of fermentation for the complex medium. The kinetic model and parameters were determined by dynamic fitting to experimental results using the fourth-order Runge-Kutta method.

lipase; adsorption; affinity resin; kinetic constant


Production of lipase from Geotrichum sp and adsorption studies on affinity resin

E. S. KAMIMURA, O. MENDIETA, H. H. SATO, G. PASTORE and F. MAUGERI¹ Department of Food Engineering, School of Food Engineering, State University of Campinas (UNICAMP), C. P. 6121, CEP: 13081-970, Campinas-SP, Brazil

¹e-mail: maugeri@ceres.fea.unicamp.br

(Received: January 19, 1999: Accepted: March 4, 1999)

Abstract - There is a growing interest in microbial lipase production due to its great potential for industrial applications such as food additives, industrial reagents and stain removers, as well as for medical applications. Specially for medical applications a high degree of purity is required, which is accomplished with high resolution chromatographic techniques. Affinity chromatography is considered a very high resolution chromatographic technique. In this work the adsorption isotherms and kinetics of the adsorption of lipase from Geotrichum sp on biospecific resin were determined. The resin was prepared using EAH sepharose 4B gel (Pharmacia), made to react with oleic acid as the specific ligand.The lipase was produced in a five-liter fermenter, with both complex and synthetic media. Fermentation conditions were a temperature of 30°C, an aeration of 1VVM and an agitation of 400 rpm. Maximum lipase activity was around 28 U/ml after 10 hours of fermentation for the complex medium. The kinetic model and parameters were determined by dynamic fitting to experimental results using the fourth-order Runge–Kutta method.

Keywords: lipase, adsorption, affinity resin, kinetic constant.

INTRODUCTION

Lipases (acylglycerol acylhydrolases, E C 3.1.1.3) catalyze the hydrolysis of ester bonds at a lipid-water interface. They may exhibit specificity for the position of acid in a triglyceride in their natural substrate. Lipases also exhibit stereochemical specificity when reacting with a wide variety of substrates in organic solvents (Baillargeon, 1990).

About 20% of all biotransformations reported today are performed with lipases (Gitlesen et al., 1997). The use of specific microbial lipases to catalyze interesterification reactions has aroused considerable interest because of certain advantages over chemical catalysis. Biotransformations can compete with optimized chemical production if improved techniques, which involve the choice of convenient methods of downstream processing, are employed.

Isolation and purification of lipases from different sources (mainly microorganisms and mammals) have recently been reported. Purification procedures were usually based on multistep series of nonspecific techniques such as ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. During the past decades, affinity chromatography has been increasingly used, facilitating the purification of lipases (Taipa et al., 1992).

Affinity chromatography is an efficient technique for the isolation and purification of biological macromolecules. It is distinguished from other methods because it is mainly based on biological properties. The main advantage of this technique is a high degree of purification of the targeted enzyme that can be obtained in a single step in the overall process of separation.

Most of the commercial lipase preparations available today are crude preparations in which only a small fraction is protein and only a part of the protein is lipase (Gitlesen et al., 1997). Because of their structural and conformational complexity, proteins represent a specially challenging case for development of reliable adsorption model (Finette et al., 1997).

The aim of this work was to prepare an oleic acid affinity resin and investigate the adsorption of crude lipase produced from Geotrichum sp in biospecific resin as a purification method.

MATERIALS AND METHODS

Microrganism

The strain of Geotrichum sp. (Macêdo, 1995) was isolated and the stock culture was maintained by the Food Biochemistry Laboratory of the Food Science Department at UNICAMP.

Extracellular Lipase Production

Lipase production was started with a 10 % (vol/vol) inoculum of Geotrichum sp. in a Bioflow III (New Brunswick Scientific) fermenter. The working volume was 5 liters at 400 rpm and 1 VVM at a temperature of 30oC. T

he complex culture medium was composed of 5% corn steep liquor, 0.5% ammonium nitrate and 1% olive oil (Mendieta et al., 1998). The synthetic medium was composed of 1.5 g/l KH2PO4, 1.0 g/l NH4Cl, 1.2 g/l MgSO4.7 H2O, 20 g/l yeast extract (Difco Beta Lab.), 17 mg/l ZnSO4, 17 mg/l Mn SO4, 17 mg/l FeSO4 and 1% (v/v) olive oil (Sidebottom et al., 1991).

Activity Determination

Lipase activity was measured by a titrimetric assay with NaOH 0.05 N using emulsified olive oil as the substrate. One ml of enzyme was added to 5 ml emulsion containing 25% (vol./vol.) olive oil and 75% (vol./vol.) gum arabic and 2 ml 10mM phosphate buffer at pH 7. The assay was carried out at 37°C during a 30-minute incubation. After this time interval, the reaction was stopped by adding 15 ml acetone-ethanol 1:1 (vol./vol.) and the amount of fatty acids was then titrated. One unit of lipase activity was defined as the amount of enzyme that released 1 µmole of fatty acid per minute under these conditions (Macêdo, 1995).

Biomass Determination

Dry weight of cells was determined directly by centrifuging a known volume at 10000xg during 10 minutes in a Sorvall RC 26 Plus centrifuge. The pellet was washed once in distilled water, then resuspended in a small volume of distilled water and dried overnight at 100°C in a preweighed dry petri dish to constant weight (Henriette et al., 1993).

Protein Determination

Protein concentrations of crude fermentation broth were determined according to Lowry et al., 1951. Bovine serum albumin was used as the standard protein. One ml of sample was mixed with 5 ml 0.01% CuSO4, 0.02% Na-tartrate and 2% CaCO3 in 0.1M NaOH. The mixture was left for 10 minutes and then mixed with Folin Ciocalteau's phenol reagent diluted 1:2 in distilled water. After incubation during 30 minutes at room temperature, absorbance at 750 nm was measured with a spectrophotometer. Distilled water (1ml) treated the same way was used as a reference.

Affinity Chromatography Resin

Oleic acid affinity chromatography resin was obtained from EAH Sepharose 4B gel (Pharmacia) as described by Haas et al. (1992). Oleic acid (Sigma) used as a ligand reacted with EAH Sepharose® 4B gel (20 ml from Pharmacia) and 1-ethyl-3-(3-dimetyl-aminopropyl) carbodiimide in 70% aqueous dimethylformamide. This reaction was gently mixed at 25°C for about 24 hours. The pH was held at about pH 4.2 by addition of 1N HCl, mainly during the first 3 hours. The resin was washed in a column with 700 ml 80% ethanol and then with 400 ml 0.1N NH4OH and 400 ml 80% ethanol twice. These were followed by 400 ml methanol 80%. Finally, the column was washed with 1 liter of 10 mM phosphate buffer at pH 7 containing 0.03% sodium azide.

Kinetic Isotherms

The filtrate of crude broth with 60 ml of lipase solution in 10 mM phosphate buffer at pH 7 was mixed with 10 g of wet resin in a stirred reactor at 25°C. All the experiments lasted 120 minutes. Samples were quantitated by enzymatic activity in order to construct kinetic adsorption profiles.

Adsorption Isotherms

Experimental data were obtained from batch adsorption with 1g wet resin in 6 ml of different concentrations of crude lipase. The filtered crude broth had a lipase activity of around 23 U/ml. Other enzymatic activities were obtained by adding 10 mM phosphate buffer at pH 7. After a two hour incubation in the shaker (25°C and 130 rpm), the amount of adsorbed lipase was determined from the remaining activity in solution.

Calculation of kd, qm, k1 and k2

The kinetic equation of lipase adsorption can be expressed by Equation (1), which is transformed in to Equation (2) at equilibrium:

Equations (1) and (2) were used to determine the kinetic parameters by means of dynamic and linear fitting to experimental data. In Equations (1) and (2) q is the amount of lipase activity adsorbed per unit weight, qm is the maximum amount of lipase activity adsorbed per unit weight, C is the amount of lipase activity in liquid phase, k1 and k2 are kinetics constants and Kd (equal to k2/k1) is the equilibrium constant. The symbol * in Equation (2) represents concentrations at the equilibrium.

RESULTS AND DISCUSSION

Extracellular Lipase Production

Most microbial lipases are extracellular. They are excreted through the external membrane into the culture medium. Optimisation of fermentation conditions for microbial lipases is of great importance, since culture conditions influence the properties of the enzyme produced as well as the ratio of extracellular to intracellular lipases. The amount of lipase produced is dependent on several environmental factors, such as cultivation temperature, pH, nitrogen composition, carbon and lipids sources, concentration of inorganic salts and the availability of oxygen. Lipase production is stimulated by lipids such as butter, lard, olive oil and fatty acids (Aires-Barros et al., 1994).

Lipase was produced from Geotrichum sp using complex and synthetic media with 1% olive oil as the carbon source (Mendieta et al., 1998). The complex medium, composed of 5% corn steep liquor, 0.5% ammonium nitrate and 1% olive oil, produced a more stable lipase with enzymatic activity of about 23 U/ml after 12 hours of fermentation. Protein content was about 4.60 mg/ml (Figure 1 ). Maximum dry biomass (9.4 g/l) was reached after 12 h, and the pH decreased slowly during the early stage of fermentation but increased again up to pH 4.7 in the stationary phase. Maximum lipase activity was 28 U/ml after 10 hours of fermentation.

Figure 1: Typical time for lipase activity, dry biomass and pH in fermenter with complex medium.The experiment was carried out at 1 VVM, 400 rpm, 30°C, without pH control.

In Figure 2 it can be seen that lipase produced in the synthetic medium wasn't as stable as the lipase produced in the complex medium at the end of fermentation. Maximum lipase activity was about 20 U/ml after 14 hours, and an almost 50% loss of activity was observed 2 hours after the maximum activity level was reached. The pH of the fermentation was maintained at 4.5 during lipase production with the addition of ammonia solution. The dry biomass was about 12 g/l after 16 hours of fermentation.

Figure 2: Typical time for lipase activity, dry biomass and pH in fermenter with synthetic media.The experiment was carried out at 1 VVM, 400 rpm, 30°C, with pH control.

Typical fermentation for lipase production by Geotrichum sp are shown in Figures 1 and 2 .

Using the same microorganism in a medium containing 1.5% deffated soybean flour, 1% wheat flour, 3% yeast extract, 0.2% NH4NO3 at 30°C for 2 days in shaker flasks, Macêdo (1995) described an enzymatic activity of 1.7 U/ml. Enzyme production in this present work was thus almost 13 times higher using the complex medium in a stirred fermenter.

Tsujisaka et al. (1973) produced a lipase from Geotrichum candidum with a medium composed of 5% corn steep liquor, 0.5% ammonium nitrate and 1% soy bean oil as the carbon source. Final lipase activity was about 66 U/ml (enzyme assay conditions were 30°C, 500 rpm and 60 minutes).

According to Shimada et al. (1992), in the production by induction of lipase from Geotrichum candidum by long-chain fatty acids in a medium containing 5% corn steep liquor, 0.5% NH4OH with 1% (v/v) soy bean or olive oils, lipase activity was about 80 U/ml. Large amounts of lipase were produced in this medium with a high nitrogen concentration and a low carbon concentration.

Adsorption Isotherm

The affinity of adsorption is reflected in the initial slope of the adsorption isotherm, or better still, in the Kd value (affinity constant). According to Gelluk et al. (1992), the initial slope of isotherms for purified lipase is steeper than that for crude lipase, indicating that the purified lipase, and therewith most probably the lipase in the crude preparation, has a relatively high affinity for sorbent. The difference in slope can be explained by the presence of other protein molecules in the crude broth.

The enzymatic activity of the adsorbed lipase is measured in relation to the equilibrium concentration in the liquid phase. The results of free enzymatic activity versus the adsorbed amount at equilibrium are shown in Figure 3 (a). A Langmuir-type curve (Equation 2), fitted to these experimental data, is represented by the solid line. Figure 3 (b) shows the linear fitting of the Langmuir equation to obtain values for qm and Kd, which are 98.90 U/g and 2.41 U/ml, respectively. It can be seen that the isotherm for lipase adsorption can be described by the Langmuir equation with good agreement.

Figure 3: Adsorption isotherm for lipase from Geotrichum sp on acid oleic affinity resin in 10 mM phosphate buffer at pH 7 and 25°C. (a) - The solid line represents the fitted Langmuir equation (Equation 2). (b) - Linear fitting for the determination of qm and Kd.

Adsorption Kinetics

According to Gitlesen et al. (1997), lipase adsorption to solid surfaces includes several steps. The lipase molecule in the bulk phase must be transported to the surface, either by convection or by diffusion. Even in well-stirred systems there is a stagnant layer close to the surface that must be penetrated by diffusion. The lipase is then adsorbed at the solid surface at a definite rate. After adsorption macroscopic or microscopic rearrangements of the protein structure can occur. Desorption of the adsorbate is not common and the process is apparently irreversible, although there are examples of reversibility in adsorption on hydrophobic supports.

In order to assess the adsorption behaviour of lipase on the affinity resin, efficiency of adsorption is defined as:

Table 1 Table 1: Experimental results for efficiency adsorption (%) and the fitted values for qm, Kd, k1 and k2. shows initial lipase activity, the efficiency value in percentage, qm, Kd and the fitted kinetics constant (k1 and k2) for all kinetics adsorption assays.

It can be seen in Figure 4 and Table 1 Table 1: Experimental results for efficiency adsorption (%) and the fitted values for qm, Kd, k1 and k2. that efficiency and qm, are lower for recovered resins (runs a and b with 44.03% and 54.88%, respectively) than for fresh prepared resins experiments c and d about 63%. It is also interesting to observe in Figure 4 that adsorption of lipase on the affinity resin is very fast. Practically more than 90% of the total adsorbed enzyme is adsorbed during the first 20 minutes of reaction. This means that there is high affinity between enzyme and resin, and this behaviour is useful for purification process design.

Figure 4: Time for lipase activity in the liquid for different initial enzyme concentrations and for fresh and reused affinity resins: (a) Co = 7.29 U/ml and recovered resin; (b) Co = 8.76 U/ml and recovered resin; (c) Co = 9.74 U/ml and fresh resin and (d) Co= 12.03 U/ml and fresh resin.

It can also be observed in Table 1 Table 1: Experimental results for efficiency adsorption (%) and the fitted values for qm, Kd, k1 and k2. that dissociation constant was the same for all experiments (a, b, c and d) and maximum adsorption capacity increased with the increase in initial concentration of the enzyme. However, the decrease in efficiency for recovered resins suggests that the regeneration step should be studied carefully in order to be improved.

Figure 5 shows the dynamic fitting of Equation (1) to the experimental data in order to obtain k1, k2 and qm for each kinetic assay. It was observed that k1 and k2 are essentially the same for all runs, in contrast to qm values, which changed according to the efficiency of the resin. Table 1 Table 1: Experimental results for efficiency adsorption (%) and the fitted values for qm, Kd, k1 and k2. shows that k1 and k2 are 6x10-2 ml.U-1. min-1 and 0.14 min-1, respectively and qm 23.77, 40.86, 61.28 and 70.73 U/g for experiments (a), (b), (c) and (d), respectively. The fitting procedure was performed using the fourth-order Runge-Kutta algorithm to integrate Equation (1) and the least squares procedure to optimise fitting. Results are assembled in Table 1 Table 1: Experimental results for efficiency adsorption (%) and the fitted values for qm, Kd, k1 and k2. .

Figure 5: Dynamic fitting of lipase adsorption on biospecific resin. Solid lines represent the predicted curve from Equation (1) and square symbols, the experimental data. For all runs k1 and k2 were similar, 6.0x10-2 ml.U-1.min-1 and 0.14 min-1 respectively, with qm changing according to the resin: (a) qm = 23.77 U/g ( Co=7.29 U/ml and with recovered resin); (b) qm = 40.86 U/g ( Co= 8.76 U/ml and with recovered resin); (c) qm = 61.28 U/g ( Co = 9.74 U/ml and with fresh prepared resin) and (d) qm = 70.72 U/g (Co=12.03 U/ml and with fresh prepared resin).

CONCLUSIONS

In this work it has been shown that a complex medium composition allows to maximization of lipase production. The culture medium composed of 5% corn steep liquor, 0.5% ammonium nitrate and 1% olive oil was shown to produce a more stable lipase from Geotrichum sp with enzymatic activity of about 23 U/ml after 12 hours of fermentation. This represented 13 times the production found in previous work with the same microganism under different culture conditions.

The adsorption efficiency of freshly prepared resin was shown to be about 60%. The same resin showed lower efficiency after being recovered, which represents a drawback for this affinity resin.

Experimental results showed that adsorption of enzymatic activity using crude lipase can be described well by Langmuir isotherms. Lipase adsorption is very fast and it has been shown that equilibrium reached after 2 hours of reaction. This biospecific resin is showed a qm of as high as 98.90 U/g and Kd of 2.41 U/ml. The kinetic constants that were determined by dynamic fitting using the fourth-order Runge-Kutta algorithm were 6x10-2 ml.U-1.min-1 and 0.14 min-1 for k1 and k2, respectively.

NOMENCLATURE

C* = amount of enzymatic activity in the liquid phase at equilibrium (U/ml)

k1 = adsorption constant (U.ml-1.min-1)

k2 = desorption constant (min-1)

Kd = (k2/k1) Langmuir or dissociation constant (U/ml)

qm = maximum amount of enzymatic activity adsorbed per unit weight (U/g)

q* = amount of enzymatic activity adsorbed per unit weight at equilibrium, (U/g)

U = 1µmol of fatty acid per minute, under conditions established in Materials and Methods section

ACKNOWLEDGEMENTS

This work received financial support from Fapesp and CNPq-Padct.

REFERENCES

Aires-Barros, M.R.; Taipa, M.Â. and Cabral, M.S.J., Isolation and Purification of Lipases, Lipases their Structure, Biochemistry and Application, chapter 12, pp. 243-270, Cambridge University Press, 1994.

Baillargeon, M.W., Purification and Specifity of Lipases from Geotrichum candidum, Lipids, vol. 25(12), pp. 841-848, 1990.

Finette, G.M.S.; Baharin, B.S.; Qi-Ming Mao and Hearn, M.T.W., Adsorption Behavior of Multicomponent Protein Mixtures Containing a1 -Proteinase Inhibitor with the Anion Exchanger, 2-(Diethylamino)ethyl-Sepherodex, Biotechnol. Prog., vol. 13, pp. 265-275, 1997.

Gelluk, M.A.; Norde, W.; Van Kalsbeek, H.K.A.I. and Van't Riet, K. Adsorption of Lipase from Candida rugosa on Cellulose and Its Influence on Lipolytic Activity, Enzyme Microb. Technol., vol. 14, pp. 748-754, 1992.

Gitlesen, T.; Bauer, M. and Adlercreutz, P., Adsorption of Lipase on Polypropylene Powder, Biochimica et Biophysica Acta, 1345, pp. 188-196, 1997.

Haas, M.J.; Chichowicz, D.L. and Bailey, D.G., Purification and Characterization of an Extracellular Lipase from Fungus Rhizopus delemar, Lipids, vol. 27 (8), pp. 571-586, 1992.

Henriette, C.; Zenebi, S.; Aumaitre, M.F.; Petitdemange, E. and Petitdemange, H. Protease and Lipase by a Strain of Serratia marcescens (532S), J. of Industrial Microbiology, vol. 12, pp. 129-135, 1993.

Lowry, O.H.; Rosebrough, N.J.; Farr, A.L. and Randall, R.J., Protein Measurement with Folin-Phenol reagent, Journal of Biological Chemistry, 193, pp. 265-275, 1951.

Macêdo, G.A., Produção, Purificação, Caracterização Bioquímica e Aplicações de Lipase de Geotrichum sp., Master's thesis, DCA-FEA-UNICAMP, 1995.

Mendieta, O.; Kamimura, E.S.; Costa, F.A.A.;, Sato, H.H.; Pastore, G. and Maugeri, F., Producción y Purificación Parcial de Lipasa de Geotrichum sp., II Congresso Iberoamericano de Ingenieria de Alimentos, Bahia Blanca, Argentina, march 24-27, 1998.

Shimada, Y.; Sugihara, A.; Nagao, T. and Tominaga, Y., Induction of Geotrichum candidum Lipase by Long-Chain Fatty Acids. J. of Fermentation and Bioengineering, vol. 74(2), pp. 77-80, 1992.

Sidebottom, C.M.; Charton, E.; Dunn, P.P.J.; Mycock, G.; Davies, C.; Sutton, J.L.; Macrae, A.R. and Slabas, A.R., Geotrichum candidum Produces Several Lipases with Markedly Different Substrate Specificities, Eur. J. Biochem., 202, pp. 485-491, 1991.

Taipa, M.Â.; Aires-Barros, M.R. and Cabral, J.M.S., Purification of Lipases, Journal of Biotechnology, 26, pp. 111-142, 1992.

Tsujisaka, Y.; Iwai, M. and Tominaga, Y., Purification, Crystallization and Some Properties of Lipase Geotrichum candidum Link, Agr. Biol. Chem., vol. 37 (6), pp. 1457-1464, 1973.

  • Aires-Barros, M.R.; Taipa, M.Â. and Cabral, M.S.J., Isolation and Purification of Lipases, Lipases their Structure, Biochemistry and Application, chapter 12, pp. 243-270, Cambridge University Press, 1994.
  • Baillargeon, M.W., Purification and Specifity of Lipases from Geotrichum candidum, Lipids, vol. 25(12), pp. 841-848, 1990.
  • Finette, G.M.S.; Baharin, B.S.; Qi-Ming Mao and Hearn, M.T.W., Adsorption Behavior of Multicomponent Protein Mixtures Containing a1 -Proteinase Inhibitor with the Anion Exchanger, 2-(Diethylamino)ethyl-Sepherodex, Biotechnol. Prog., vol. 13, pp. 265-275, 1997.
  • Gelluk, M.A.; Norde, W.; Van Kalsbeek, H.K.A.I. and Van't Riet, K. Adsorption of Lipase from Candida rugosa on Cellulose and Its Influence on Lipolytic Activity, Enzyme Microb. Technol., vol. 14, pp. 748-754, 1992.
  • Gitlesen, T.; Bauer, M. and Adlercreutz, P., Adsorption of Lipase on Polypropylene Powder, Biochimica et Biophysica Acta, 1345, pp. 188-196, 1997.
  • Haas, M.J.; Chichowicz, D.L. and Bailey, D.G., Purification and Characterization of an Extracellular Lipase from Fungus Rhizopus delemar, Lipids, vol. 27 (8), pp. 571-586, 1992.
  • Henriette, C.; Zenebi, S.; Aumaitre, M.F.; Petitdemange, E. and Petitdemange, H. Protease and Lipase by a Strain of Serratia marcescens (532S), J. of Industrial Microbiology, vol. 12, pp. 129-135, 1993.
  • Lowry, O.H.; Rosebrough, N.J.; Farr, A.L. and Randall, R.J., Protein Measurement with Folin-Phenol reagent, Journal of Biological Chemistry, 193, pp. 265-275, 1951.
  • Macędo, G.A., Produçăo, Purificaçăo, Caracterizaçăo Bioquímica e Aplicaçőes de Lipase de Geotrichum sp, Master's thesis, DCA-FEA-UNICAMP, 1995.
  • Mendieta, O.; Kamimura, E.S.; Costa, F.A.A.;, Sato, H.H.; Pastore, G. and Maugeri, F., Producción y Purificación Parcial de Lipasa de Geotrichum sp, II Congresso Iberoamericano de Ingenieria de Alimentos, Bahia Blanca, Argentina, march 24-27, 1998.
  • Shimada, Y.; Sugihara, A.; Nagao, T. and Tominaga, Y., Induction of Geotrichum candidum Lipase by Long-Chain Fatty Acids. J. of Fermentation and Bioengineering, vol. 74(2), pp. 77-80, 1992.
  • Sidebottom, C.M.; Charton, E.; Dunn, P.P.J.; Mycock, G.; Davies, C.; Sutton, J.L.; Macrae, A.R. and Slabas, A.R., Geotrichum candidum Produces Several Lipases with Markedly Different Substrate Specificities, Eur. J. Biochem., 202, pp. 485-491, 1991.
  • Taipa, M.Â.; Aires-Barros, M.R. and Cabral, J.M.S., Purification of Lipases, Journal of Biotechnology, 26, pp. 111-142, 1992.
  • Table 1: Experimental results for efficiency adsorption (%) and the fitted values for qm, Kd, k1 and k2.
  • Publication Dates

    • Publication in this collection
      15 Sept 1999
    • Date of issue
      June 1999

    History

    • Accepted
      04 Mar 1999
    • Received
      19 Jan 1999
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