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Enhancing reaction rate for transesterification reaction catalyzed by Chromobacterium lipase
Enzyme and Microbial Technology 36 (2005) 896–899
Enhancing reaction rate for transesterification reaction catalyzed by Chromobacterium lipase Ipsita Roy, Munishwar N. Gupta ∗ Department of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India Received 10 September 2003; accepted 8 January 2005
Abstract Precipitation of Chromobacterium viscosum lipase as an interfacial layer by addition of ammonium sulphate and t-butanol [in a process called three phase partitioning (TPP)] led to an increase in the initial rate of tranesterification in iso-octane by 4.9 times. It is shown that the TPP-treated lipase also showed salt activation and further increase in activity in the presence of sorbitol. Thus, in synergy with these strategies, the TPP treatment resulted in a more efficient design of catalytic transesterification; the overall increase in transesterification as a result of the optimization was about 15 times. © 2005 Elsevier Inc. All rights reserved. Keywords: Enzymes in organic media; Lipase; Three phase partitioning; Transesterification reactions
1. Introduction Enzymes in organic solvents are finding increasing applications in organic synthesis [1–3]. Simultaneously, there have been some concerns about the relatively low catalytic power observed under such non-aqueous conditions as compared to aqueous buffers. Some approaches which have been attempted for enhancing enzyme activity in organic media have been pH tuning [4], immobilization [5], use of microwaves [6] and addition of potassium chloride during lyophilization [7]. Recently, we have observed that three phase partitioning (TPP) of Proteinase K resulted in enhancement of its catalytic power in aqueous buffers [8]. The process, originally described as a bioseparation strategy [8], consists of adding less than ‘salting out’ amount of ammonium sulphate and tbutanol to the aqueous solution of the enzyme. The enzyme floats as an interfacial layer between the top t-butanol phase and the lower aqueous phase. The X-ray diffraction pattern of Proteinase K, after being subjected to TPP, showed unusually high ‘B-factor’, signifying overall increase in the flexibility of the protein molecule [8]. ∗
Corresponding author. Fax: +91 11 2658 1073. E-mail address: mn [email protected] (M.N. Gupta).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.01.022
The decreased molecular flexibility of the enzyme powder suspended in low water media has been partly shown to account for the low turnover number of enzymes under low water conditions [9]. Thus, TPP appeared to be a suitable strategy for enhancing the overall flexibility of the enzyme in organic solvents. It was hoped that such a TPP-treated enzyme, with a more flexible structure, would have higher catalytic efficiency. As lipase-catalyzed transesterifications constitute the most frequently used class of enzymes for organic synthesis [2,3,10], a lipase from Chromobacterium viscosum was chosen for this study.
2. Materials and methods 2.1. Materials Lipase (from C. viscosum) was a product of Asahi Corporation, Japan. All other chemicals were of analytical grade. The organic solvents used in this work were of low water grade [<0.0075% water ,v v−1 ] and were further dried by gen˚ molecular sieves (Central Drug House, tle shaking with 3 A India) overnight before use.
I. Roy, M.N. Gupta / Enzyme and Microbial Technology 36 (2005) 896–899
897
2.2. Method
Table 1 Three phase partitioning of Chromobacterium lipase at 25 ◦ C
2.2.1. Three phase partitioning of enzyme Lipase (3 ml, 3 mg ml−1 , 0.02 M Tris–HCl, pH 7.8; containing 0.83 mg protein per mg solid powder) was saturated with 50% (w v−1 ) ammonium sulphate. This was followed by the addition of 6 ml of t-butanol, the solution was vortexed and allowed to stand at 25 ◦ C for 1 h. The solution was then centrifuged (2000 × g, 10 min). The lower aqueous and upper organic layers were separated using a pasteur pipette. The interfacial precipitate was dissolved in 1 ml of 0.02 M Tris–HCl, pH 7.8, and dialyzed against the same buffer for 16 h with frequent changes; the dissolved precipitate was then lyophilized. The native, untreated enzyme (200 mg) was dissolved in 10 ml of 0.02 M Tris–HCl, pH 7.8 and lyophilized as before. This is referred to as the pH-tuned enzyme [4] and has been used throughout this work as the control.
Sample
Total activity (U)
Starting After three phase partitioning Aqueous layer Precipitate
2.2.2. Effect of additives For salt activation, 10 mg of the pH-tuned enzyme was added to 10 mg K2 HPO4 and 0.98 g KCl and the solid was dissolved in 40 ml of distilled water and the pH adjusted to 7.8. This solution was then lyophilized and used further as described above. For working with sorbitol, pH-tuned enzyme (10 mg) was dissolved in 2 ml of 0.02 M sodium phosphate buffer, pH 7.8, containing 20 mg of sorbitol and lyophilized. For removal of sorbitol, the lyophilized enzymes (control and TPP-treated) were washed with 2 ml each of acetonitrile. 2.2.3. Assay of lipase activity The activity of lipase in aqueous media was monitored by following the rate of hydrolysis of p-nitrophenyl palmitate [11]. In low water media, the lyophilized samples of untreated and TPP-treated [8] lipases were suspended in isooctane (dried over molecular sieves), followed by the addition of the substrates, tributyrin and 1-hexanol [10]. Both the enzymes and the substrates had been previously equilibrated to the same level of water activity, aw , against saturated salt solutions [10]. The progress of the transesterification reaction was monitored by gas chromatography [10]. Each experiment was carried out six times and the standard deviation among the values in each set was within 5%.
Total protein (mg)
Specific activity (U mg−1 )
Increase in activity (%)
14,28,645
7.5
1,90,486
–
19,048 45,71,664
0.1 7.4
1,90,480 6,17,792
– 320
The activity of lipase was measured using p-nitrophenyl palmitate as the substrate.
activity of the precipitated enzyme resulted not due to purification of the enzyme, but because of the increase in inherent catalytic power of the enzyme (as reflected in the increase in total activity units). While lipases have been employed in aqueous media as well for the production of free fatty acids, their (transesterification) reactions in low water containing organic solvents have found wide applications in synthesis and resolutions of esters, acids and alcohols [2,3]. Hence, the focus of the work was to evaluate the TPP-treated lipase for transesterification in organic solvents containing low amounts of water. As enzymes are known to show higher rates in non-polar solvents, iso-octane, a fairly non-polar solvent, was chosen for this study. Fig. 1 shows the effect of various hydration levels on the activity of untreated and TPP-treated lipase. (Water activity, aw , is widely accepted as an indicator of hydration level of the enzyme in non-aqueous enzymology [3,6].) It is seen that at all hydration levels, TPP-treated enzyme showed higher reaction rates. As observed earlier for the untreated lipase [10], the water activity at the level of 0.84 in iso-octane
3. Results and discussion The two main parameters which are known to affect three phase partitioning are the amounts of ammonium sulphate and t-butanol added to the enzyme solution [8]. It was found that 50% (w v−1 ) ammonium sulphate saturation and double volume of t-butanol (added to 9.0 mg lipase in 3 ml of 0.02 M Tris–HCl buffer, pH 7.8) gave 3.2 times increase in the lipase activity in aqueous buffer (Table 1). The data show that TPP did not separate significant amount of protein, as only 0.1 mg was left behind in the aqueous layer. The increase in specific
Fig. 1. Initial tranesterification rates of Chromobacterium lipase, untreated () and TPP-treated (䊉), at various water activities.The enzyme (10 mg) was suspended in 1.8 ml of dried iso-octane containing tributyrin (250 mM) and 1-hexanol (250 mM) after equilibrating the enzyme and the solvent separately at varying water activities and shaken in an orbital shaker (200 rpm) at 40 ◦ C.
898
I. Roy, M.N. Gupta / Enzyme and Microbial Technology 36 (2005) 896–899
Fig. 2. Effect of different additives on the initial rate of transesterification of (a) untreated and (b) TPP-treated. The enzyme was suspended in 1.8 ml of dried iso-octane (with 0.05% water) containing 0.1% dimethyl sulphoxide (×); 0.1% ethylene glycol (); 0.2% dimethyl formamide () or 0.2% diethanolamine ().
gave the maximum initial rate even in the case of TPP-treated enzyme. The latter showed 4.9 times more activity than the untreated enzyme at this hydration level. Salt activation has been suggested to be another approach for enhancing the activity of enzymes in organic solvents. While some dramatic enhancements in reaction rates due to salt activation have been reported in few cases [12], the effects observed have been marginal in other cases [6,13]. It was found that both untreated and TPP-treated enzymes showed 3.9 and 2.6 times increase, respectively, with salt activation approach. This represents an increase of about 10 times in the initial rate of transesterification for the TPP-treated enzyme from the untreated enzyme (without added salt). Thus, salt activation can be used in synergy with TPP-treatment for enhancing transesterification rate of the lipase. Triantafyllou et al. [13] have examined the effect of adding other polar solvents in the reaction medium (containing, 0.05% water, v v−1 ) of lipase from Candida rugosa. Fig. 2 shows the behaviour of C. viscosum lipase under similar conditions. In general, various polar solvents behaved in a similar way in this case as well. The low concentrations of polar solvents did not affect the enzyme activity; concentrations 0.2% were found to lower the initial rates of transesterification. More important, TPP-treated enzyme showed a similar trend with the addition of these polar solvents. Thus, in this respect as well, the TPP-treated enzyme behaved similar to the behaviour of enzymes in organic media. It is noteworthy that even at 0.4% (v v−1 ) of polar solvents, the level of activity shown by TPP-treated enzyme was greater than the activity of the untreated enzyme even when no polar solvent was present. Thus, one can deal with substrates with wider range of stabilities and still obtain reasonable transesterification rates by using TPP-treated enzymes. Polyols have been found to help enzymes in retaining essential water layer when placed in organic solvents. Tri-
antafyllou et al. [10] have examined the effect of sorbitol addition on the activity of C. viscosum. Fig. 3 shows the effect of the presence of sorbitol in the lyophilized preparation of C. viscosum lipase and the corresponding TPP-treated enzyme. As far as the behaviour of untreated enzyme was concerned, it was, of course, similar to the earlier report [10]. Again, TPPtreated enzyme showed similar behaviour. The optimum aw (0.84) was the same for both untreated and TPP-treated enzymes. The TPP-treated enzyme with sorbitol showed 14.6 times increase in activity over the untreated enzyme (without sorbitol) and 5.2 times enhancement in activity over even the untreated enzyme with sorbitol.
Fig. 3. Initial tranesterification rates of Chromobacterium lipase, untreated () and TPP-treated (䊉), at various water activities. Broken lines represent initial rates after the addition of sorbitol, while solid lines represent activities obtained after the removal of sorbitol. Controls were run to check for the residual activity when untreated () and TPP-treated () lipases were washed with acetonitrile.
I. Roy, M.N. Gupta / Enzyme and Microbial Technology 36 (2005) 896–899
It has been shown earlier that Chromobacterium lipase is resistant to solvent inactivation and thus retains its activity after sorbitol is washed away with pyridine [10]. Fig. 3 shows the effect of sorbitol removal by acetonitrile in the case of both untreated and TPP-treated lipase from C. viscosum. In agreement with the earlier work, sorbitol removal did lead to a decrease in enzyme activity in all the cases. One reason for high stability (and low activity) of enzymes under low water conditions is that the side chains of amino acids (which otherwise would have formed weak bonds with water in aqueous media) interact with each other and result in rigid enzyme structure. Additives like sorbitol, in anhydrous media, interact with these side chains, making the rigid structure flexible, enhancing the activity [10]. Removal of sorbitol, presumably restores the status quo, and results in rigid structure [10]. This leads to decrease in enzyme activity. It was observed (Fig. 3) that (i) in the case of the untreated and TPP-treated enzymes, the activity decreased by 85 and 67%, respectively, as a result of acetonitrile wash; (ii) in the case of untreated enzyme containing sorbitol, the activity dropped to a level identical to that of the untreated enzyme (without sorbitol), which had been washed with acetonitrile; (iii) in the case of TPP-treated enzyme containing sorbitol, washing with acetonitrile resulted in the loss in activity till a level which was marginally higher than the activity of the TPP-treated lipase (without sorbitol). It did not reach the level of activity of TPPtreated enzyme (without sorbitol), washed with acetonitrile. Thus, the presence of sorbitol seems to have helped the TPPtreated enzyme more than the untreated enzyme in retaining an active structure. TPP has been reasonably successful as a bioseparation strategy in quite a few cases [14,15]. Its application in enhancing catalytic rates has, so far, been limited and confined exclusively to working in aqueous media [8]. The data given above indicate that TPP shows sufficient promise for obtaining enzymes with better catalytic power in both aqueous and non-aqueous media. Unfortunately, the molecular mechanism of TPP is not well established and is supposed to involve multiple phenomena like conventional salting out, Morton’s n-butanol extraction method, isoionic precipitation, cold cosolvent precipitation and osmolyte and kosmotropic precipitation of proteins [14]. To conclude, the strategy of three phase partitioning can be used in conjunction with addition of KCl or sorbitol and optimization of the right hydration level. The higher initial rates obtained with TPP-treated lipase along with other strategies obviously constitute a more efficient design for catalysis. So far, while individual strategies have been attempted for obtaining more active enzyme preparations for functioning in organic media, this work shows that combina-
899
tion of strategies may give a preparation with better catalytic properties.
Acknowledgements The financial support provided by Department of Science and Technology, Government of India, is gratefully acknowledged. Partial financial support was also provided by Council for Scientific and Industrial Research (technology mission on oilseeds, pulses and maize and Extramural division).
References [1] Okahata T, Mori T. Lipid-coated enzymes as efficient catalysts in organic media. Trends Biotechnol 1997;15:50–4. [2] Gupta MN. In: Gupta MN, editor. Methods in non-aqueous enzymology. Basel: Birkhauser Verlag; 2000. [3] Halling P. Enzymatic conversions in organic and other low-water media. In: Drauz K, Waldmann H, editors. Enzyme catalysis in organic solvents. Weinheim: Wiley-VCH; 2002. p. 259–85. [4] Gupta MN. Enzyme function in organic solvents. Eur J Biochem 1992;203:25–32. [5] Bosley J, Peilow A. Immobilization of lipases for use in non-aqueous reaction systems. In: Gupta MN, editor. Methods in nonaqueous enzymology. Basel: Birkhauser Verlag; 2000. p. 51–69. [6] Roy I, Gupta MN. Non-thermal effects of microwaves on protease-catalyzed esterification and transesterification. Tetrahedron 2003;59:5431–6. [7] Khmelnitsky YL, Rich JO. Biocatalysis in nonaqueous solvents. Curr Opin Chem Biol 1999;3:47–53. [8] Singh RK, Gourinath S, Sharma S, Roy I, Gupta MN, Betzel C, et al. Enhancement of enzyme activity through three phase partitioning: ˚ resolution. crystal structure of a modified serine protease at 1.5 A Protein Eng 2001;14:307–13. [9] Broos J, Visser AJ, Engbersen JF, Verboom W, van Hoek A, Reinhoudt DN. Flexibility of enzymes suspended in organic solvents probed by time-resolved fluorescence anisotropy. Evidence that enzyme activity and enantioselectivity are directly related to enzyme flexibility. J Am Chem Soc 1995;117:12657–63. [10] Triantafyllou AO, Wehtje E, Adlercreutz P, Mattiasson B. Effect of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media. Biotechnol Bioeng 1995;45:406–14. [11] Winkler UK, Stuckmann M. Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol 1979;138:663–70. [12] Dordick JS. Designing enzymes for use in organic solvents. Biotechnol Prog 1992;8:259–67. [13] Triantafyllou AO, Adlercreutz P, Mattiasson B. Influence of the reaction media on enzyme activity in bioorganic synthesis: behaviour of lipase from Candida rugosa in the presence of polar additives. Biotechnol Appl Biochem 1993;17:167–73. [14] Dennison C, Lovrein R. Three-phase partitioning: purification and concentration of proteins. Protein Expr Purif 1997;11:149–61. [15] Roy I, Gupta MN. Three phase affinity partitioning of proteins. Anal Biochem 2002;300:11–4.
Enhancing reaction rate for transesterification reaction catalyzed by Chromobacterium lipase Ipsita Roy, Munishwar N. Gupta ∗ Department of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India Received 10 September 2003; accepted 8 January 2005
Abstract Precipitation of Chromobacterium viscosum lipase as an interfacial layer by addition of ammonium sulphate and t-butanol [in a process called three phase partitioning (TPP)] led to an increase in the initial rate of tranesterification in iso-octane by 4.9 times. It is shown that the TPP-treated lipase also showed salt activation and further increase in activity in the presence of sorbitol. Thus, in synergy with these strategies, the TPP treatment resulted in a more efficient design of catalytic transesterification; the overall increase in transesterification as a result of the optimization was about 15 times. © 2005 Elsevier Inc. All rights reserved. Keywords: Enzymes in organic media; Lipase; Three phase partitioning; Transesterification reactions
1. Introduction Enzymes in organic solvents are finding increasing applications in organic synthesis [1–3]. Simultaneously, there have been some concerns about the relatively low catalytic power observed under such non-aqueous conditions as compared to aqueous buffers. Some approaches which have been attempted for enhancing enzyme activity in organic media have been pH tuning [4], immobilization [5], use of microwaves [6] and addition of potassium chloride during lyophilization [7]. Recently, we have observed that three phase partitioning (TPP) of Proteinase K resulted in enhancement of its catalytic power in aqueous buffers [8]. The process, originally described as a bioseparation strategy [8], consists of adding less than ‘salting out’ amount of ammonium sulphate and tbutanol to the aqueous solution of the enzyme. The enzyme floats as an interfacial layer between the top t-butanol phase and the lower aqueous phase. The X-ray diffraction pattern of Proteinase K, after being subjected to TPP, showed unusually high ‘B-factor’, signifying overall increase in the flexibility of the protein molecule [8]. ∗
Corresponding author. Fax: +91 11 2658 1073. E-mail address: mn [email protected] (M.N. Gupta).
0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.01.022
The decreased molecular flexibility of the enzyme powder suspended in low water media has been partly shown to account for the low turnover number of enzymes under low water conditions [9]. Thus, TPP appeared to be a suitable strategy for enhancing the overall flexibility of the enzyme in organic solvents. It was hoped that such a TPP-treated enzyme, with a more flexible structure, would have higher catalytic efficiency. As lipase-catalyzed transesterifications constitute the most frequently used class of enzymes for organic synthesis [2,3,10], a lipase from Chromobacterium viscosum was chosen for this study.
2. Materials and methods 2.1. Materials Lipase (from C. viscosum) was a product of Asahi Corporation, Japan. All other chemicals were of analytical grade. The organic solvents used in this work were of low water grade [<0.0075% water ,v v−1 ] and were further dried by gen˚ molecular sieves (Central Drug House, tle shaking with 3 A India) overnight before use.
I. Roy, M.N. Gupta / Enzyme and Microbial Technology 36 (2005) 896–899
897
2.2. Method
Table 1 Three phase partitioning of Chromobacterium lipase at 25 ◦ C
2.2.1. Three phase partitioning of enzyme Lipase (3 ml, 3 mg ml−1 , 0.02 M Tris–HCl, pH 7.8; containing 0.83 mg protein per mg solid powder) was saturated with 50% (w v−1 ) ammonium sulphate. This was followed by the addition of 6 ml of t-butanol, the solution was vortexed and allowed to stand at 25 ◦ C for 1 h. The solution was then centrifuged (2000 × g, 10 min). The lower aqueous and upper organic layers were separated using a pasteur pipette. The interfacial precipitate was dissolved in 1 ml of 0.02 M Tris–HCl, pH 7.8, and dialyzed against the same buffer for 16 h with frequent changes; the dissolved precipitate was then lyophilized. The native, untreated enzyme (200 mg) was dissolved in 10 ml of 0.02 M Tris–HCl, pH 7.8 and lyophilized as before. This is referred to as the pH-tuned enzyme [4] and has been used throughout this work as the control.
Sample
Total activity (U)
Starting After three phase partitioning Aqueous layer Precipitate
2.2.2. Effect of additives For salt activation, 10 mg of the pH-tuned enzyme was added to 10 mg K2 HPO4 and 0.98 g KCl and the solid was dissolved in 40 ml of distilled water and the pH adjusted to 7.8. This solution was then lyophilized and used further as described above. For working with sorbitol, pH-tuned enzyme (10 mg) was dissolved in 2 ml of 0.02 M sodium phosphate buffer, pH 7.8, containing 20 mg of sorbitol and lyophilized. For removal of sorbitol, the lyophilized enzymes (control and TPP-treated) were washed with 2 ml each of acetonitrile. 2.2.3. Assay of lipase activity The activity of lipase in aqueous media was monitored by following the rate of hydrolysis of p-nitrophenyl palmitate [11]. In low water media, the lyophilized samples of untreated and TPP-treated [8] lipases were suspended in isooctane (dried over molecular sieves), followed by the addition of the substrates, tributyrin and 1-hexanol [10]. Both the enzymes and the substrates had been previously equilibrated to the same level of water activity, aw , against saturated salt solutions [10]. The progress of the transesterification reaction was monitored by gas chromatography [10]. Each experiment was carried out six times and the standard deviation among the values in each set was within 5%.
Total protein (mg)
Specific activity (U mg−1 )
Increase in activity (%)
14,28,645
7.5
1,90,486
–
19,048 45,71,664
0.1 7.4
1,90,480 6,17,792
– 320
The activity of lipase was measured using p-nitrophenyl palmitate as the substrate.
activity of the precipitated enzyme resulted not due to purification of the enzyme, but because of the increase in inherent catalytic power of the enzyme (as reflected in the increase in total activity units). While lipases have been employed in aqueous media as well for the production of free fatty acids, their (transesterification) reactions in low water containing organic solvents have found wide applications in synthesis and resolutions of esters, acids and alcohols [2,3]. Hence, the focus of the work was to evaluate the TPP-treated lipase for transesterification in organic solvents containing low amounts of water. As enzymes are known to show higher rates in non-polar solvents, iso-octane, a fairly non-polar solvent, was chosen for this study. Fig. 1 shows the effect of various hydration levels on the activity of untreated and TPP-treated lipase. (Water activity, aw , is widely accepted as an indicator of hydration level of the enzyme in non-aqueous enzymology [3,6].) It is seen that at all hydration levels, TPP-treated enzyme showed higher reaction rates. As observed earlier for the untreated lipase [10], the water activity at the level of 0.84 in iso-octane
3. Results and discussion The two main parameters which are known to affect three phase partitioning are the amounts of ammonium sulphate and t-butanol added to the enzyme solution [8]. It was found that 50% (w v−1 ) ammonium sulphate saturation and double volume of t-butanol (added to 9.0 mg lipase in 3 ml of 0.02 M Tris–HCl buffer, pH 7.8) gave 3.2 times increase in the lipase activity in aqueous buffer (Table 1). The data show that TPP did not separate significant amount of protein, as only 0.1 mg was left behind in the aqueous layer. The increase in specific
Fig. 1. Initial tranesterification rates of Chromobacterium lipase, untreated () and TPP-treated (䊉), at various water activities.The enzyme (10 mg) was suspended in 1.8 ml of dried iso-octane containing tributyrin (250 mM) and 1-hexanol (250 mM) after equilibrating the enzyme and the solvent separately at varying water activities and shaken in an orbital shaker (200 rpm) at 40 ◦ C.
898
I. Roy, M.N. Gupta / Enzyme and Microbial Technology 36 (2005) 896–899
Fig. 2. Effect of different additives on the initial rate of transesterification of (a) untreated and (b) TPP-treated. The enzyme was suspended in 1.8 ml of dried iso-octane (with 0.05% water) containing 0.1% dimethyl sulphoxide (×); 0.1% ethylene glycol (); 0.2% dimethyl formamide () or 0.2% diethanolamine ().
gave the maximum initial rate even in the case of TPP-treated enzyme. The latter showed 4.9 times more activity than the untreated enzyme at this hydration level. Salt activation has been suggested to be another approach for enhancing the activity of enzymes in organic solvents. While some dramatic enhancements in reaction rates due to salt activation have been reported in few cases [12], the effects observed have been marginal in other cases [6,13]. It was found that both untreated and TPP-treated enzymes showed 3.9 and 2.6 times increase, respectively, with salt activation approach. This represents an increase of about 10 times in the initial rate of transesterification for the TPP-treated enzyme from the untreated enzyme (without added salt). Thus, salt activation can be used in synergy with TPP-treatment for enhancing transesterification rate of the lipase. Triantafyllou et al. [13] have examined the effect of adding other polar solvents in the reaction medium (containing, 0.05% water, v v−1 ) of lipase from Candida rugosa. Fig. 2 shows the behaviour of C. viscosum lipase under similar conditions. In general, various polar solvents behaved in a similar way in this case as well. The low concentrations of polar solvents did not affect the enzyme activity; concentrations 0.2% were found to lower the initial rates of transesterification. More important, TPP-treated enzyme showed a similar trend with the addition of these polar solvents. Thus, in this respect as well, the TPP-treated enzyme behaved similar to the behaviour of enzymes in organic media. It is noteworthy that even at 0.4% (v v−1 ) of polar solvents, the level of activity shown by TPP-treated enzyme was greater than the activity of the untreated enzyme even when no polar solvent was present. Thus, one can deal with substrates with wider range of stabilities and still obtain reasonable transesterification rates by using TPP-treated enzymes. Polyols have been found to help enzymes in retaining essential water layer when placed in organic solvents. Tri-
antafyllou et al. [10] have examined the effect of sorbitol addition on the activity of C. viscosum. Fig. 3 shows the effect of the presence of sorbitol in the lyophilized preparation of C. viscosum lipase and the corresponding TPP-treated enzyme. As far as the behaviour of untreated enzyme was concerned, it was, of course, similar to the earlier report [10]. Again, TPPtreated enzyme showed similar behaviour. The optimum aw (0.84) was the same for both untreated and TPP-treated enzymes. The TPP-treated enzyme with sorbitol showed 14.6 times increase in activity over the untreated enzyme (without sorbitol) and 5.2 times enhancement in activity over even the untreated enzyme with sorbitol.
Fig. 3. Initial tranesterification rates of Chromobacterium lipase, untreated () and TPP-treated (䊉), at various water activities. Broken lines represent initial rates after the addition of sorbitol, while solid lines represent activities obtained after the removal of sorbitol. Controls were run to check for the residual activity when untreated () and TPP-treated () lipases were washed with acetonitrile.
I. Roy, M.N. Gupta / Enzyme and Microbial Technology 36 (2005) 896–899
It has been shown earlier that Chromobacterium lipase is resistant to solvent inactivation and thus retains its activity after sorbitol is washed away with pyridine [10]. Fig. 3 shows the effect of sorbitol removal by acetonitrile in the case of both untreated and TPP-treated lipase from C. viscosum. In agreement with the earlier work, sorbitol removal did lead to a decrease in enzyme activity in all the cases. One reason for high stability (and low activity) of enzymes under low water conditions is that the side chains of amino acids (which otherwise would have formed weak bonds with water in aqueous media) interact with each other and result in rigid enzyme structure. Additives like sorbitol, in anhydrous media, interact with these side chains, making the rigid structure flexible, enhancing the activity [10]. Removal of sorbitol, presumably restores the status quo, and results in rigid structure [10]. This leads to decrease in enzyme activity. It was observed (Fig. 3) that (i) in the case of the untreated and TPP-treated enzymes, the activity decreased by 85 and 67%, respectively, as a result of acetonitrile wash; (ii) in the case of untreated enzyme containing sorbitol, the activity dropped to a level identical to that of the untreated enzyme (without sorbitol), which had been washed with acetonitrile; (iii) in the case of TPP-treated enzyme containing sorbitol, washing with acetonitrile resulted in the loss in activity till a level which was marginally higher than the activity of the TPP-treated lipase (without sorbitol). It did not reach the level of activity of TPPtreated enzyme (without sorbitol), washed with acetonitrile. Thus, the presence of sorbitol seems to have helped the TPPtreated enzyme more than the untreated enzyme in retaining an active structure. TPP has been reasonably successful as a bioseparation strategy in quite a few cases [14,15]. Its application in enhancing catalytic rates has, so far, been limited and confined exclusively to working in aqueous media [8]. The data given above indicate that TPP shows sufficient promise for obtaining enzymes with better catalytic power in both aqueous and non-aqueous media. Unfortunately, the molecular mechanism of TPP is not well established and is supposed to involve multiple phenomena like conventional salting out, Morton’s n-butanol extraction method, isoionic precipitation, cold cosolvent precipitation and osmolyte and kosmotropic precipitation of proteins [14]. To conclude, the strategy of three phase partitioning can be used in conjunction with addition of KCl or sorbitol and optimization of the right hydration level. The higher initial rates obtained with TPP-treated lipase along with other strategies obviously constitute a more efficient design for catalysis. So far, while individual strategies have been attempted for obtaining more active enzyme preparations for functioning in organic media, this work shows that combina-
899
tion of strategies may give a preparation with better catalytic properties.
Acknowledgements The financial support provided by Department of Science and Technology, Government of India, is gratefully acknowledged. Partial financial support was also provided by Council for Scientific and Industrial Research (technology mission on oilseeds, pulses and maize and Extramural division).
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