Lipases (also called triacylglycerol hydrolases,EC 3.1.1.3) catalyze a wide range of reactions,e.g.,hydrolysis,esterification,transesterification,alcoholysis,and aminolysis [1]. Most lipases have a catalytic triad composed of Ser-His-Asp residues. Lipase molecules generally consist of hydrophilic and hydrophobic parts with interfacial activation,and the catalytic site is concealed under a short amphipathic helix,which acts as a “lid” in hydrophobic interactions [2,3]. Lipase-catalyzed reactions in non-aqueous media are increasingly finding applications in the fields of food additives,pharmaceuticals,flavors,chemical materials,and bioenergy [4, 5, 6, 7, 8].
Lipozyme TL IM is a versatile enzyme and has been widely used in industrial processes and laboratory tests,particularly in the interesterification of bulk fats and the production of frying fats,because it is inexpensive and commercially available [9, 10, 11]. The development of a fast and reliable analytical method for characterizing the catalytic properties of Lipozyme TL IM is therefore imperative. At present,most methods for lipase activity determination are based on hydrolytic assays such as the routinely used titrimetric protocol for olive oil emulsions,ultraviolet (UV) spectrophotometry using p-nitrophenol (pNP) esters as substrates,and chromatographic methods [12, 13, 14]. However,the synthetic activities of the lipases in organic solvents do not correspond to the hydrolytic activities in aqueous solutions [15,16]. Enzymes identified using hydrolytic approaches are not necessarily suitable for the desired synthetic reactions. It is therefore important to establish an assay for the synthetic activities of lipases based on esterification or transesterification. Most of the reported approaches are based on gas chromatography (GC),high- performance liquid chromatography,or fluorometric detection [17, 18, 19],which are extremely time consuming,expensive,and require expensive equipment. Teng et al. [20] reported transesterification between p-nitrophenyl palmitate (pNPP) and ethanol,in which the liberated pNP was extracted into an aqueous alkaline phase and then the extract was detected at 410 nm to estimate the lipase synthetic activity. The detection accuracy was significantly affected by the concentration of the alkaline liquor. Goujard et al. [21] studied transesterifications between vinyl esters and alkyl alcohols and evaluated the lipase activity by measuring the decre ase in the UV absorbance at 200 nm. However,the solvents,alkyl alcohols,and acetaldehyde have specific UV absorbances at 200 nm,and these interfere with the spectrophotometric measurements.
In this study,we attempted to develop a novel lipase activity assay method for determining the properties of Lipozyme TL IM-catalyzed transesterification. pNPP,a well-known chromogenic substrate for lipase hydrolysis activity determination,was selected as the substrate in the present work. The lipase-catalyzed transesterification between pNPP and n-butanol in n-hexane was used as a model reaction (Scheme 1). The amount of released product was estimated spectrophotometrically at 310 nm after dilution of sample aliquots with ethanol instead of NaOH solution; this improved the detection accuracy. The method,which can be used for microorganism high- throughput screening and for identifying large libraries of mutants with lipase synthetic activity,uses a commercially available substrate,has a simple detection procedure,and gives accurate test results. The properties of Lipozyme TL IM-catalyzed transesterification,namely solvent effect,acyl acceptor specificity,alcohol tolerance,optimum reaction temperature,and thermostability,were also investigated using the UV spectrophotometric method. Characterization of the transesterification properties of Lipozyme TL IM will be helpful in industrial processes and laboratory tests.
CAL-B (Lipozyme 435,lipase B from Candida antarctica immobilized on macroporous polyacrylate resin,10 000 U/g),RML (Lipozyme RM IM,Mucor miehei immobilized on ionic resin,20 000 U/g),and TLL (Lipozyme TL IM,Thermomyces lanuginosus immobilized on silica,50 000 U/g) were supplied by Novozymes A/S (Bagsvaerd,Denmark). PFL (Lipase AK,Pseudomonas fluorescens lipase,20 000 U/g),ANL (Lipase A,Aspergillus niger lipase,120 000 U/g),RNL (Rhizopus niveus lipase,1500 U/g),and CRL (Lipase AY,Candida rugosa lipase,700 000 U/g) were obtained from the Sigma-Aldrich (Shanghai) Trading Co.,Ltd. (Shanghai,China).
pNPP was purchased from Sigma-Aldrich (Shanghai,China),and pNP was provided by the Aladdin Chemistry Co.,Ltd. (Shanghai,China). Other chemicals used were of analytical grade. To avoid spontaneous hydrolysis caused by the presence of water,all solvents and reactants were pretreated using 4 & A ring molecular sieves.
A typical reaction mixture for the lipase synthetic activity assay consisted of 10 mmol/L pNPP and 1 mol/L n-butanol in n-hexane (1 mL). The experiments were carried out in 10 mL centrifuge tubes. To start the reaction,lipase (1 mg) was added to the reaction mixture. The mixture was incubated at 30 °C with a shaking speed of 200 r/min for 10 min. After reaction,clear supernatant (30 μL) was removed and immediately mixed with ethanol (3 mL). A control experiment was conducted using the same conditions and procedures but without addition of an enzyme. The diluent was detected at 310 nm against a blank control using a UV-visible spectrophotometer (GE Ultrospec 1100 pro,Amersham,USA). The conversion was calculated based on a calibration curve. All experiments were conducted in triplicate.
To validate the proposed assay,the transesterifications in non-aqueous solution were also monitored using a gas chromatograph (Agilent 6890N,Agilent Technologies,USA) equipped with a flame ionization detector (FID) and an HP-5 column (30 m x 0.32 mm x 0.25 μm,Agilent Technologies). The carrier gas was nitrogen with an inlet flow rate of 1 mL/min and a split ratio of 1:20. The oven temperature was maintained at 150 °C. Supernatant (100 μL) was removed and diluted with ethyl acetate (1:10). GC analysis of the prepared samples and pNP calibration were carried out to quantify the collected conversion data.
The conversion of pNPP to pNP was monitored using UV absorbance changes at 310 nm based on the different molar extinction coefficients of the substrate and product. The conversion was calculated using the equation c = 104(A1 − A0)/(ε2 − ε1) x 100%,where A0 and A1 are the absorbances of the blank control and sample,respectively,at 310 nm,and ε1 and ε2 are the molar extinction coefficients of pNPP and pNP,respectively,at 310 nm.
To measure the maximum absorption wavelength of pNP in ethanol and investigate UV interference from the substrate (pNPP) and solvent (ethanol),scans at 200−400 nm of pNP (0.1 mmol/L in ethanol),pNPP (0.1 mmol/L in ethanol),and ethanol were carried out. The results (Fig. 1) showed that pNP had a maximum absorption peak at 310 nm (1.341); relatively low absorbances for pNPP (0.193) and ethanol (0.067) were detected at the same wavelength. Palmitate,the other product of transesterification between pNPP and an alcohol,made no significant contribution to the absorbance at 310 nm (data not shown). Based on the above results,310 nm was considered to be the optimum wavelength.
The standard calibration curves for pNP and pNPP are shown in Fig. 2. The absorbance at 310 nm versus concentration from 0.01 to 0.1 mmol/L for pNP (R2 = 0.9977) and pNPP (R2 = 0.9988) both showed good linear relationships. On the basis of the calibration curves,the molar extinction coefficient of pNP in ethanol at 310 nm was found to be 11.63 x 103 mol−1 L cm−1,which is 11 times higher than that of pNPP (1.06 x 103 mol−1 L cm−1).
It was therefore possible to estimate the transesterification activity for pNPP and a short-chain alcohol (n-butanol) by assessing the absorbance at 310 nm because of the good linear relationships shown by the standard curves and the difference between the molar extinction coefficients of pNP and pNPP.
To validate the effectiveness of the methodology,seven commercially available lipases with a wide range of activities were investigated using the UV spectrophotometric assay and GC-FID assay. Most of the data shown in Fig. 3 correlated well with the conversions determined using conventional GC analysis. Lipozyme TL IM gave the highest conversion (45%) of pNPP in the UV spectrophotometric assay. Lipozyme TL IM was therefore selected for the kinetic study.
Figure 4 shows the kinetic curves for the Lipozyme TL IM-catalyzed transesterification reaction between pNPP and n-butanol at different biocatalyst concentrations. The biocatalyst loading was tested in the range 2-20 mg/mL; all other parameters were constant. The reaction was also carried out without Lipozyme TL IM,and the results showed that the absorbance at 310 nm was constant during the whole process (data not shown). As expected,with increasing enzyme concentration,the reaction rate increased,and a steady conversion was obtained when the reaction time was extended. Moreover,the initial rate increased proportionally to the lipase concentration (data not shown). These results confirmed the possibility of monitoring the synthetic activity through transesterification using the proposed UV spectrophotometric method.
A similar model reaction to that used by Teng et al. [20] was adopted in this study. However,the major breakthrough associated with this method is that the lipase transesterification activity is detected in ethanol instead of NaOH solution. The extinction coefficient of pNP is strongly pH dependent,and chemical hydrolysis of the remaining pNPP occurs in alkaline solution [22]. The measurement accuracy is therefore significantly improved using the proposed method.
Organic solvents have a direct effect on enzymatic activity and stability in lipase-catalyzed transesterification. We investigated the influences of eight commonly used solvents on Lipozyme TL IM-catalyzed transesterification between pNPP and n-butanol. It has been reported that a minimum quantity of water surrounding the immobilized lipase is essential for maintaining enzymatic activity. Hydrophobic solvents are therefore preferable to hydrophilic solvents because the latter cause stripping of the essential water layer around the enzyme [1]. Among the tested solvents,n-hexane (logP = 3.76) was found to be the optimum solvent for the Lipozyme TL IM-catalyzed transesterification reaction,giving the maximum conversion,11.16% (Table 1). n-Hexane is a common hydrophobic reagent and is widely used as a solvent in biocatalysis,giving favorable enzymatic activities [23, 24, 25]. Soumanou et al. [26] reported Lipozyme TL IM-catalyzed methanolysis of vegetable oil in n-hexane,achieving a high conversion,97% in 24 h.
Lipozyme TL IM-catalyzed transesterifications of pNPP with various alcohols,i.e.,methanol,ethanol,n-propanol,isopropanol,and n-butanol were studied. As shown in Fig. 5,the highest conversion,approximately 9.36%,was achieved for transesterification between pNPP and n-butanol. The variations in the conversion as a result of using different alcohols can be attributed to the influences of various factors such as the molecular size of the alcohol,solubility in the reaction medium,and the affinity of the lipase for a particular alcohol [27]. Similar effects of the alcohol on conversion have been reported in the literature for lipase-catalyzed esterification and transesterification reactions [28]. n-Butanol was also used as the optimum acyl acceptor in the lipase-catalyzed transesterification of ethyl 3-phenylpropanoate [29]. Based on the above results,n-butanol was the optimum acyl receptor for lipase-catalyzed transesterification.
The effect of n-butanol concentration on conversion was studied in the range 0.01-2 mol/L to determine the alcohol tolerance of Lipozyme TL IM. As shown in Fig. 6,the maximum conversion,12.02%,was obtained using 1 mol/L n-butanol. The conversion increased rapidly as the n-butanol concentration increased from 0.01 to 0.2 mol/L. From 0.2 to 1 mol/L,a slight increase in the conversion was also obtained. When the concentration of n-butanol was further increased to 2 mol/L,the conversion decreased. This could be attributed to an inhibitory effect of the high n-butanol concentration on Lipozyme TL IM. The enzyme is hydrophobic and n-butanol contains a hydrophobic tail and a polar head. There may be hydrophobic- hydrophobic interactions between the enzyme and n-butanol,which would increase the n-butanol residence time. As a result of close contact with neighboring hydrophobic residues,the enzyme-n-butanol complex would be partially dehydrated,which may destabilize the native conformation of the enzyme. A large number of studies of the alcohol tolerance of Lipozyme 435 have been reported,but there have been few reports on Lipozyme TL IM. Yadav et al. [30] reported Lipozyme 435-catalyzed transesterification of methyl acetoacetate with n-butanol; the optimum n-butanol concentration was 2.5 mol/L. Yadav et al. [31] and Talukder et al. [32] reported that the optimum alcohol concentrations in lipase-catalyzed transesterification were 0.75 and 0.1 mol/L,respectively. The effect of alcohol concentration on enzymatic activity varied depending on the reaction type.
In general,the reaction temperature influences the conversions and stabilities of biocatalysts. The optimum reaction temperature for Lipozyme TL IM-catalyzed transesterification between pNPP and n-butanol was evaluated by performing the reaction at different temperatures. As shown in Fig. 7,the highest enzymatic transesterification activity was obtained at 65 °C in n-hexane. The Lipozyme TL IM activity improved with increasing temperature from 30 to 65 °C in n-hexane (boiling point: 68.7 °C). With further increases in temperature in the range 65-80 °C in isooctane (boiling point: 99.2 °C),maximum conversion was achieved at 75 °C,but the enzymatic activity was lower than at 65 °C in n-hexane. Hence,65 °C was the optimum temperature for Lipozyme TL IM-catalyzed transesterification of pNPP in n-hexane. Zhang et al. [33] reported that the optimum reaction temperature for Lipozyme TL IM-catalyzed interesterification for the modification of margarine fats was 70 °C. Kohr et al. [34] reported the enzymatic production of biodiesel by Lipozyme TL IM-catalyzed transesterification between palm oil and methanol at 40 °C,with a conversion of 98%. The differences among the optimum reaction temperatures for Lipozyme TL IM-catalyzed transesterification have been reported to be associated with the lengths of the acyl donors [35].
The thermostability is the length of time during which an enzyme remains active before undergoing conformational changes [36]. In other words,the useful lifetime of a biocatalyst is dictated by its thermostability. Thermostability is therefore a significant parameter in characterizing enzyme catalytic properties. However,a specific study of Lipozyme TL IM thermostability in transesterifications between pNPP and short-chain alcohols has not previously been reported. In this study,the thermostability of Lipozyme TL IM was investigated at various incubation temperatures. The residual activity at 65 °C (Fig. 8) demonstrated that Lipozyme TL IM is resistant to this temperature,but the residual activity at 75 °C decreased with increasing incubation time. This was probably the result of partial inactivation of the enzyme because elevated temperature is one of the most common causes of protein denaturation and deactivation [37].
A novel modified UV spectrophotometric method for the assay in organic solvents of lipases with transesterification activity was described. The major advantages of this assay were a simple detection method,few steps,accurate test results,and low instrument requirements. The method could be applied to microorganism screening and the identification of mutants with lipase synthetic activity. The kinetics of Lipozyme TL IM-catalyzed transesterification between pNPP and n-butanol in n-hexane was also investigated. The properties of Lipozyme TL IM-catalyzed transesterification were determined using this method. It was found that Lipozyme TL IM showed the highest catalytic activity in n-hexane at 65 °C using n-butanol as the acyl acceptor with favorable alcohol tolerance (1 mol/L) and excellent thermostability (65 °C in n-hexane).
2014, Vol. 35 Issue (4): 553-559
PDF (493KB)
2014, Vol. 35 Issue (4): 553-559
PDF (493KB)