Biotechnology Letters 22: 1511–1514, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
1511
An esterification method for determination of lipase activity K.R. Kiran, S. Hari Krishna, C.V. Suresh Babu, N.G. Karanth & S. Divakar∗ Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute, Mysore 570 013, India ∗ Author for correspondence (Fax: +91-821-517 233; E-mail:
[email protected]) Received 9 June 2000; Revisions requested 28 June 2000/17 July 2000; Revisions received 14 July 2000/25 July 2000; Accepted 25 July 2000
Key words: butyl butyrate, butyric acid, esterification, lipase
Abstract The choice of a lipase for an esterification reaction can be determined from the esterification reaction between butyric acid (0.16 M) and butanol (0.33 M) at 50 ◦ C and agitated with 10 mg lipase. The decrease in butyric acid is measured by titration and 1 unit of lipase activity is defined as 1 µmol butyric acid consumed per min per mg lipase.
Introduction Various methods are currently available for the determination of lipase activity – usually involving hydrolysis (Jensen 1983). Titration of fatty acids (Tietz & Eiereck 1966, Jensen 1971, Erdmann et al. 1991) released from tributyrin or triacetin or a long-chain unsaturated oil such as sunflower seed or olive oil is one of the most common methods reported. There are however, large differences in activites measured by tributyrin hydrolysis and by hydrolysis of longchain substrates (Erdmann et al. 1991). Colorimetric methods are quick and simple (Hirayama & Matsuda 1972, Theimer & Rosnitschik 1978) as are fluorimetric methods (Theimer & Rosnitschik 1978). Unfortunately, different methods often give different results (Erdmann et al. 1991). Recently, lipase-catalyzed esterification reactions have received extensive importance due to numerous products that can be obtained viz., monoacylglycerols etc., flavour esters and surfactant esters (Kloosterman et al. 1988, Mukherjee 1990, Hari Krishna et al. 1999a,b, Kiran et al. 1999, 2000). In this context, a knowledge of the esterification activities of lipases from different sources as well as lipase preparations obtained through different procedures like immobilization are essential to explore a large number of hitherto unreported esterification reactions. Currently
the enzyme activities for these reactions are often reported in terms of the hydrolytic activity. However, it is well known that esterification and hydrolytic activities do not agree with each other as the requirement and the reaction rates are completely different for these two reactions. Therefore, a researcher in this area is often ignorant of the choice of lipase for a particular esterification reaction. The choice made on the basis of the hydrolytic activity of the lipase is likely to be misleading. A few methods based on interesterification activities are available (Graille et al. 1987, Furutani et al. 1995). However, they are not suitable for comparing the ability of lipases in esterification reactions, as the reaction conditions are quite different. This has prompted us to develope a procedure based on an esterification reaction for determining activity of lipases for comparison and selection purposes, which is reported in this paper.
Materials and methods Lipases Four different kinds of lipases (triacylglycerol hydrolases, EC 3.1.1.3) – immobilized lipase from Rhizomucor meihei, Lipozyme IM20 (BM) from Boehringer Mannheim (Germany), Lipozyme IM20 (Novo) a gift
1512 Table 1. Statistical significance of esterification and hydrolytic activitiesa . Enzymeb (mg)
Esterification Meanc Standard deviation
t-stat
P-value
Chirazyme (2.5) Chirazyme (5) Chirazyme
0.28 0.42 0.42
0.07 0.09 0.09
−5.46 −3.64 −5.30
1.72E-05 0.003 2.43E-05
7.34
2.76
3.05
0.008
Lipozyme IM20(BM)d Lipozyme IM20(Novo)e PPLf
0.47
0.09
−5.25
2.69E-05
0.68
0.11
−3.28
0.006
0.49
0.11
−3.54
0.004
0.89
0.18
−2.97
0.009
0.04
0.01
−6.89
8.47E-08
2.85
1.15
−0.18
0.43
0.03
0.01
−4.21
0.001
0.57
0.26
−3.39
0.005
0.01
0.003
−4.23
0.001
1.09
0.75
−1.47
0.09
Lipozyme IM20(Novo)g PPLg
Hydrolysis Meanc Standard deviation
t-stat
P-value
a Both hydrolytic and esterification activities were determined at 50 ◦ C and results are from an average of 5
experiments Error in activity measurements will be ± 5–10%.
b Amount of the enzyme employed: For the hydrolytic method, uniformly 1 mg Chirazyme and 5 mg PPL,
Lipozyme IM20(BM) and Lipozyme IM20 (Novo). For the esterification method, 10 mg Lipozyme IM20(BM), Lipozyme IM20(Novo), PPL and Chirazyme. In case of Chirazyme 2.5 mg and 5 mg employed are shown in parentheses. Unless otherwise stated 10 mg lipase was employed. c Mean values of esterification/hydrolytic activity are in µmol min−1 mg−1 lipase. d Lipozyme IM20(BM) – Lipozyme IM-20 from Boehringer Mannheim (Germany). e Lipozyme IM20(Novo) – Lipozyme IM-20 from Novo Nordisk (Denmark). f PPL from Sigma (USA). g Once used lipase from previous esterification reaction.
from Novo Nordisk (Denmark), lipase from Rhizomucor miehei (Chirazyme L-9 lyo, hereafter designated as Chirazyme) from Boehringer Mannheim (Germany) and porcine pancreatic lipase [PPL, a type II, crude enzyme preparation (steapsin)] from Sigma (USA) – were employed. Chemicals Butyric acid, butanol, methanol, NaOH, n-heptane, tributyrin and other solvents and chemicals used were all of analytical reagent grade and were procured from Sd. Fine Chemicals Ltd. (Mumbai, India). Butyric acid, butanol and n-heptane were distilled once before use. Esterification activity measurements A stock solution containing 1.35 ml butyric acid (0.16 M) and 2.7 ml butanol (0.33 M) in 85.95 ml nheptane was prepared for the analyses. The experiment with each lipase was set up with six 50 ml stoppered conical flasks containing 3 ml stock solution with the appropriate quantity of the lipase (see Table 1). The
flasks were incubated from 30 to 60 ◦ C in a shaking water bath for up to 120 min after which period the contents of the flask were directly titrated, after adding 1 ml methanol, with 0.02 M NaOH, using phenolphthalein as indicator. The blank with 3 ml of the stock solution in a stoppered conical flask without the enzyme was titrated as mentioned above to determine the total acid content of the reaction mixture. Butanol did not react with butyric acid in the absence of the lipase within 2 h employed for measuring the activity. The esterification activity was determined by the following relation: Esterification activity =
V × M × 100 units, E×T
(1)
where V is the difference in volume in ml of NaOH between the blank and samples after time T (period of incubation in min), which is a measure of the butyric acid consumed due to esterification, M is the molarity of NaOH and E is the amount of the enzyme employed in mg. One unit of esterification activity is defined as 1 µmol butyric acid consumed in the esterification reaction per min per mg lipase.
1513 Table 2. Comparison between the methods for determining the hydrolytic and esterification activities of lipasesa .
Method of estimation Procedure Activity units (µmol min−1 mg−1 lipase) Lipozyme IM20(BM) Lipozyme IM20(Novo) PPL Chirazyme
Hydrolysis
Esterification
Butyric acid released pH adjusted to 9.5 using NaOH
Butyric acid reacted Titrating unreacted acid with NaOH
0.68 ± 0.11 0.89 ± 0.18 2.85 ± 1.15 7.35 ± 2.76
0.47 ± 0.09 0.49 ± 0.11 0.04 ± 0.01 0.42 ± 0.09
a Error in measurements will be ± 5–10%. Lipozyme IM20(BM) – Rhizomucor miehei lipase from M/s. Boehringer Mannheim, Germany. Lipozyme IM20(Novo) – Rhizomucor miehei lipase from M/s. Novo Nordisk, Denmark. Chirazyme – Rhizomucor miehei lipase from M/s. Boehringer Mannheim, Germany. PPL – Porcine pancreatic lipase from M/s. Sigma, USA.
Hydrolytic activity measurements Tributyrin emulsion was prepared in phosphate buffer (10 mM, pH 7.0) at a concentration of 0.33 M. Sodium benzoate (0.2% w/v) was used as a preservative and gum acacia (0.5% w/v) used as an emulsifying agent. Sodium dodecyl sulfate (50 µl 10% w/v solution in water) was also added. Tributyrin (4 ml) and phosphate buffer (8 ml) mixture was incubated with the respective lipase at 50 ◦ C and agitated at 175 rpm. A blank was also performed in the absence of lipase. Samples were withdrawn at intervals of time and assayed by the pH-stat procedure, in which the pH of the sample was adjusted to 9.5 using 0.02 M NaOH (Lavayre & Baratti 1982). The hydrolytic activity was also determined from Equation (1) above, where V is now a measure of the butyric acid released due to hydrolysis. One unit of hydrolytic activity is defined as 1 µmol butyric acid released per min per mg lipase.
Results and discussion Esterification is slower than hydrolysis. Depending on the source, method of preparation and immobilization, esterification activities of lipases vary. In order to compare the esterification activities of a wide variety of lipases, a set of reaction conditions has to be standardized whereby accurate measurement of lipase activities can be made with reproducibility and reliability. Several reaction systems were tried for these measurements like lipase catalyzed esterification of isoamyl alcohol, butanol, α-terpineol, isopropanol,
benzyl alcohol and phenols. Of these, esterification of butanol with butyric acid gave measurable results with the greatest accuracy in a short span of time and with minimum amount of the lipase. The other esterification reactions gave very low conversions or poor linearity in the esterification profiles studied. The esterification activity units are based on measurement of initial rates, which may also vary with temperature and method of immobilization of lipase and the lipase source. The esterification of butanol with butyric acid was studied at 30, 40, 50 and 60 ◦ C. At 30 and 40 ◦ C, the extent of conversion was not satisfactory for accurate measurement of titre volumes (especially PPL, because of its lower activity). At 60 ◦ C, the reactions did not exhibit any regularity in esterification behaviour. However, 50 ◦ C was the best, showing a regular linear behaviour. Hence, only data obtained at this temperature are shown (Table 1). The results were highly reproducible with high confidence levels (>99.6%). Although the water activity, aw , of the reaction system was not determined, these reactions were conducted with substrates and solvents which were rendered almost anhydrous by pretreatment procedures prior to such esterification experiments. Table 2 lists the differences between the esterification and hydrolytic methods for determining lipase activities. The hydrolytic procedure registered a very small difference in titre volume of the order of 1–2 ml against 2–22 ml in case of the esterification procedure. These corresponded to 5% esterification within a duration of 2 h in case of PPL and >90% esterification in case of Lipozyme IM20(BM)/Lipozyme
1514 IM20(Novo)/Chirazyme with respect to the substrate concentrations employed. In spite of the relatively low titre value observed for PPL, the linearity was found to be good. Although Lipozyme IM20 (BM), Lipozyme IM20 (Novo) and Chirazyme showed identical and higher esterification values than PPL, these four lipases exhibited wide variation in hydrolytic activity values. A comparison between once-used lipase and fresh lipase [Table 1 – Lipozyme IM20 (Novo) and PPL] does not show much variation in hydrolytic activity values, but exhibits drastic difference in esterification activity values. This information is relevant and important for probable repeated use in esterification reactions as well as from the technology viewpoint. Although esterification activities of lipases depend on the nature of the substrate, this method gives a good indication within a reasonable duration of 90 to 150 min which is better than what is detected by hydrolytic activity measurements for different lipases. Further, this esterification procedure can also be employed for native enzymes, although this manuscript describes the work on immobilized systems only.
Acknowledgements One of us (SD) acknowledges Department of Biotechnology, India for the financial assistance rendered. KRK and SHK acknowledge the award of Senior Research Fellowships from Council of Scientific and Industrial Research, India.
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