Optimising biocatalyst design for obtaining high transesterification activity by α-chymotrypsin in non-aqueous media
© Solanki and Gupta; 2008
Received: 27 October 2007
Accepted: 12 February 2008
Published: 12 February 2008
Enzymes are often used in organic solvents for catalyzing organic synthesis. Two enzyme preparations, EPRP (enzyme precipitated and rinsed with n-propanol) and PCMC (protein coated microcrystals) show much higher activities than lyophilized powders in such systems. Both preparations involve precipitation by an organic solvent. The clear understanding of why these preparations show higher catalytic activity than lyophilized powders in organic solvents is not available.
It was found that EPRPs of α-chymotrypsin prepared by precipitation with n-propanol in the presence of trehalose contained substantial amount of trehalose (even though trehalose alone at these lower concentrations was not precipitated by n-propanol). The presence of trehalose in these EPRPs resulted in much higher transesterification rates (45.2 nmoles mg-1min-1) as compared with EPRPs prepared in the absence of trehalose (16.6 nmoles mg-1min-1) in octane. Both kinds of EPRPs gave similar initial transesterification rates in acetonitrile. Use of higher concentrations of trehalose (when trehalose alone also precipitates out), resulted in the formation of PCMCs, which showed higher transesterification rates in both octane and acetonitrile. SEM analysis showed the relative sizes of various preparations. Presence of trehalose resulted in EPRPs of smaller sizes.
The two different forms of enzymes (EPRP and PCMC) known to show higher activity in organic solvents were found to be different only in the way the low molecular weight additive was present along with the protein. Therefore, the enhancement in the transesterification activity in EPRPs prepared in the presence of trehalose was due to: (a) better retention of essential water layer for catalysis due to the presence of the sugar. This effect disappeared where the reaction media was polar as the polar solvent (acetonitrile) is more effective in stripping off the water from the enzyme; (b) reduction in particle size as revealed by SEM. In the case of PCMC, the enhancement in the initial rates was due to an increase in the surface area of the biocatalyst since protein is coated over the core material (trehalose or salt).
It is hoped that the insight gained in this work would help in a better understanding for designing high activity biocatalyst preparation of non-aqueous media.
The possibility of using enzymes in low water containing organic solvents has expanded their applications in organic synthesis [1–4]. In many cases, improved regioselectivity and enantioselectivity have been observed [5, 6]. The considerable success obtained in such applications in numerous cases has obscured the fact that reaction rates in such media tend to be extremely slow. For an organic chemist, reaction times of 24–72 h are not a novelty as many chemical transformations, catalysed by chemical catalysts, often take that kind of time. Hence, the early successes with use of enzymes in organic synthesis in non-aqueous media had created considerable excitement [7, 8]. In the last decade or so, it has been realized that reaction rates in non-aqueous media are considerably lower than those in the conventional aqueous media. For example, the catalytic efficiency of subtilisin for hydrolytic reactions (esterase activity in water) is 5.94 × 103 M-1s-1 whereas its catalytic efficiency for transesterification using the same ester (in hexane) is merely 1.04 × 10-1 M-1s-1. In fact, formation of alkyl esters of long chain fatty acids (biodiesel) takes only an hour or so when alkali is used as the catalyst; lipase catalyzed formation takes more than 24 h . Hence the common perception (based upon all our experience in conventional enzymology) that enzymes are more efficient catalysts than chemical catalysts is often not valid in non-aqueous enzymology. Yet, there are many advantages of using enzymes as catalysts. Hence, the reasons for slowness of enzyme catalyzed reactions in organic solvents have been investigated . Among the many reasons, an important one to emerge is that in lyophilized powders (the frequently used forms of enzymes employed in organic solvents), enzymes, in fact, have conformations which are very different from native structures. While these conformational changes are reversed when enzymes are dissolved in water, these remain permanant when enzymes are used as powders in organic solvents. Again, numerous strategies have been described in the literature to obtain enzyme activation in non-aqueous media . Two simple and elegant strategies are based upon avoiding lyophilization. In propanol rinsed enzyme preparations (PREP) , the bulk water from immobilized enzyme is removed by rinsing with n-propanol. In protein coated microcrystals (PCMC) , protein is coprecipitated along with a salt (or another low molecular weight organic compound) by an organic solvent. Both PREP and PCMCs show much higher rates as compared to lyophilized powders in organic solvents [13, 14]
Few years back, we described EPRPs . The idea was to merely adapt PREP concept to free enzymes. It was found that more active EPRPs were formed if an optimum amount of buffer salts were present . The role of the buffer salts in that preliminary work was not investigated. One possibility was that this optimum amount of salt prevented any structural damage to the protein structure. Many small molecular weight additives are known to protect enzymes during exposure to high temperature , during lyophilization  and during catalysis in organic solvents [18, 19]. Disaccharides have been found to be an effective additive as a protector of enzymes activity during both thermal stress  as well as lyophilization [9, 21]. The present work was initiated to find out whether presence of disaccharides offers any protection to the enzyme during its exposure to organic solvent during precipitation by an organic solvent for formation of EPRPs.
Results and discussion
The effects of 1 percent (wv-1) disaccharides and PEG-6000 on the activity (initial rates) of α-chymotrypsin precipitated by n-propanol.
Initial rate (nmoles mg-1min-1.)
Trehalose + PEG-6000(1 percent wv-1)
PEG-6000 (1 percent wv-1)
The effects of trehalose concentration on the activity (initial rates) of the α-chymotrypsin precipitated by n-propanol in octane. The experiments were carried out in triplicate. The percentage error in a set was within 7 percent.
Starting trehalose (percent wv-1)
percent trehalose precipitated
Enzyme loadinga (weight percentage)
Initial rateb (nmoles mg-1min-1)
Visual morphology of enzyme precipitate
Precipitation of trehalose in propanol (control)
Saturated trehalose solution
In all cases, 0.83 mg of enzyme was precipitated. Initially, until the addition of 10 percent (w/v) trehalose, the percentage amount of trehalose precipitated increased with the increase in the starting concentration of trehalose. As trehalose alone did not precipitate until this concentration, it can be assumed that the presence of the protein caused the trehalose to co-precipitate. Beyond this percentage (w/v) trehalose, trehalose precipitates as micro crystalline material even when present alone. This precipitation is much faster than protein precipitation which leads to a protein being coated on the microcrystals . It is interesting to note that over this concentration range, percentage trehalose getting precipitated decreased with the increase in the percentage (w/v) of starting trehalose. Enzyme loading continued to decrease progressively throughout, as the actual amount of trehalose being precipitated continued to progressively increase.
The effects of trehalose concentration on the activity (initial rates) of α-chymotrypsin precipitated by n-propanol in acetonitrile.
Trehalose concentration (wv-1)
Initial rate (nmoles mg-1min-1)
The two different forms of enzymes (EPRP and PCMC) known to show higher activity in organic solvents were found to be different only in the way the low molecular weight additive was present along with the protein. In PCMC, a more defined microcrystals compound is formed. This, of course, is well described in a number of earlier papers. [12, 15].
Based upon FT-IR data, it appears that the presence of trehalose did not affect the structure of the enzyme after precipitation. So, the enhancement in the transesterification activity in EPRPs prepared in the presence of trehalose was due to the actual presence of trehalose during the reaction. This was largely due to (a) better retention of essential water layer for catalysis due to the presence of the sugar . This effect disappeared where reaction media was polar as the polar solvent (acetonitrile) is more effective in stripping off the water from the enzyme [1–4], (b) reduction in particle size revealed by SEM.
As trehalose concentration became high enough for trehalose to precipitate out even when present alone, trehalose precipitated faster than the protein. This led to the well known preparation called PCMCs. As both EPRPs and PCMCs described here contained trehalose, the observed higher initial rates with PCMCs can be attributed to the fact that in these biocatalyst designs, the protein was coated over the crystalline material. This created a large biocatalyst surface. It also agrees with the observation that both trehalose and potassium sulphate worked more or less the same as the core material in PCMCs.
It is hoped that the insight gained in this work would help in a better understanding for designing high activity biocatalyst preparation for use in non-aqueous media. It may be added that with the recent work with ionic liquids [24, 25] the range of low water containing non-aqueous media continues to expand.
α-chymotrypsin (from Bovine pancreas, Cat. No. C-4129), N-acetyl-L-phenylalanine ethyl ester and N-benzoyl-L-tyrosine ethyl ester (BTEE) were purchased from Sigma Chemical (St. Louis, MO, USA). Octane, n-propanol and acetonitrile (anhydrous grade with water content less than 0.001) were obtained from Sigma Chemical (St. Louis, MO, USA) and J.T. Baker (Phillipsburg, NJ, USA) respectively. Trehalose, sucrose, lactose, PEG-6000, n-propanol and tris(hydroxymethyl)aminomethane buffer were obtained from Merck (Mumbai, India). PMDA was obtained from Eastman Kodak Company (Rochester, NY, USA). All solvents were dried over molecular sieves prior to use. All other chemicals employed were of analytical grade.
Protein concentrations were determined according to procedure described by Bradford using bovine serum albumin as the standard .
Precipitation of α-chymotrypsin by n-propanol in the presence of trehalose
α-chymotrypsin was precipitated by n-propanol according to previous methods [12, 15, 27]. The α-chymotrypsin (10 μl of 100 mgml-1 in 10 mM potassium phosphate buffer, pH 7.8) was added to saturated aqueous solutions of trehalose (of different volumes), the total volume was then made up to 100 μl, before being cooled at 4°C. This solution was then added dropwise to dry, chilled (4°C) n-propanol (3 ml) with constant shaking (150 rpm). After allowing the suspension to be shaken for 30 min at 4°C, it was then centrifuged (12,000 g) for 5 min at 4°C. The precipitate was then rinsed three times with dry n-propanol (chilled), before replacing the n-propanol with the desired anhydrous organic solvent. After rinsing the precipitate with organic solvent, the reaction was carried out.
The catalytic activities of α-chymotrypsin precipitates (1 mg of solid enzyme) in organic solvents were determined with reference to the transesterification reaction between N-acetyl-L-phenylalanine ethyl ester (10 mM) and n-propanol (1 M) in organic solvent (2 ml). The reaction mixture was incubated at 25°C with on an orbital shaker (continuous at 150 rpm). The progress of the reaction was monitored by withdrawing aliquots at different time intervals that were then analysed by HPLC .
The samples were analysed by HPLC for the presence of the transesterification product using a ZORBAX SB-C18 column (Agilent Technologies, USA). The eluent consisted of 5 percent glacial acetic acid, 55 percent water and 40 percent acetonitrile, and had a flow rate of 1 ml min-1. Detection of the product was carried out with a UV detector at 258 nm.
The amount of trehalose precipitated in the presence of α-chymotrypsin was determined by HPLC using a TSKgel™ column (Supelco, USA). The eluent consisted of 0.1 M of Na2SO4 in 0.1 M of phosphate buffer (pH 6.7) with a flow rate of 1 ml min-1, with detection carried out using an RI detector.
FT-IR analysis of enzyme preparations
The spectra were recorded on a Nicolet™ FTIR 6700 optical spectrophotometer. Different formulations of α-chymotrypsin were suspended in n-propanol. The samples were placed in a demountable cell fitted with zinc selenide windows and 500 μm thick Teflon spacers. All spectra were corrected for solvent background. The data collection was carried out using OMNIC 2.1 software. A total of 250 scans were signal averaged at a resolution of 2 cm-1 [29–31].
KS thanks the University Grant Commission (UGC) for the Junior Research Fellowship. This work was supported by funds obtained from the Government of India's Department of Science and Technology (DST).
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