Improving activity and enantioselectivity of lipase via immobilization on macroporous resin for resolution of racemic 1- phenylethanol in non-aqueous medium
© Li et al.; licensee BioMed Central Ltd. 2013
Received: 14 June 2013
Accepted: 23 October 2013
Published: 29 October 2013
Burkholderia cepacia lipase (BCL) has been proved to be capable of resolution reactions. However, its free form usually exhibits low stability, bad resistance and no reusability, which restrict its further industrial applications. Therefore, it is of great importance to improve the catalytic performance of free lipase in non-aqueous medium.
In this work, macroporous resin NKA (MPR-NKA) was utilized as support for lipase immobilization. Racemic transesterification of 1-phenylethanol with vinyl acetate was chosen as model reaction. Compared with its free form, the enzyme activity and enantioselectivity (ees) of the immobilized lipase have been significantly enhanced. The immobilized BCL exhibited a satisfactory thermostability over a wide range of temperature (from 10 to 65°C) and an excellent catalytic efficiency. After being used for more than 30 successive batches, the immobilized lipase still kept most of its activity. In comparison with other immobilized lipases, the immobilized BCL also exhibits better catalytic efficiency, which indicates a significant potential in industrial applications.
The results of this study have proved that MPR-NKA was an excellent support for immobilization of lipase via the methods of N2 adsorption–desorption, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and Fourier transform-infrared spectroscopy (FT-IR). The improvement of enzyme activity and ees for the immobilized lipase was closely correlated with the alteration of its secondary structure. This information may contribute to a better understanding of the mechanism of immobilization and enzymatic biotransformation in non-aqueous medium.
In recent years, lipase (EC 184.108.40.206) has been widely applied in biotransformation reactions in aqueous and non-aqueous medium, because it can be used to catalyze hydrolysis and transesterification reactions, as well as synthesis of esters . Especially, the ability of lipases to perform enantioselective biotransformation in preparation of pharmaceutical intermediates and chiral building blocks has made them increasingly attractive and promising . Particular attention has been paid to the Burkholderia cepacia strain, which can produce versatile enzyme and be widely used for biodegradation, biological control and hydrolyzing biotransformation in various reactions . The lipase from Burkholderia cepacia lipase (BCL) has high stability, alcohol tolerance and activity suitable for a broad spectrum of reactions substrates and media . However, its free form usually exhibits low stability, bad resistance and no reusability, which restrict its further application in industry . In most cases, these disadvantages of directly using free form lipase are common phenomena in other enzyme-catalyzed reactions [6, 7]. Thus, the issue focusing on how to improve the catalytic properties of free lipase (such as activity, thermal stability and reusability) in non-aqueous medium is an important topic.
Immobilization has been proved to be one of the most useful strategies to improve catalytic properties of free enzyme . There are several conventional immobilization approaches, such as adsorption, entrapment, encapsulation, and covalent binding [9, 10]. Among them, adsorption is advantageous because of its procedural simplicity, low cost, high efficiency and ease of industrial application. Immobilized lipases via adsorption methods have been used in many reactions, such as ester synthesis, biodiesel production and enrichment of polyunsaturated fatty acids [5, 11, 12]. So far, various materials have been employed as supports for enzyme immobilization . However, usage of MPRs in the resolution reaction has rarely been explored. Although many studies showed that immobilization could greatly enhance the catalytic performance of enzyme [14, 15], till now, to the best of our knowledge, it is still unclear that why immobilization can enhance the activity and tolerance of lipases. Thus, it is important to elucidate the possible mechanism of this enhancement.
For this purpose, in this study, several methods (N2 adsorption–desorption, SEM, EDS and FT-IR) were employed to characterize the immobilized lipase in order to investigate probable mechanism for the enhancements of enzyme activity and enantioselectivity after immobilization. The enantioselective transesterification of racemic 1-phenylethanol with vinyl acetate was chosen as the model reaction so as to evaluate the enzyme activity/enantioselectivity (ees) and to compare the catalytic efficiency between the free and immobilized lipases in non-aqueous medium , because secondary alcohols are often used as target substrates in lipase-catalyzed resolution reactions . In addition, 1-phenylethanol is an essential building block and synthetic intermediate in many fields, such as fragrance in cosmetic industry, solvatochromic dye in chemical industries, ophthalmic preservative and inhibitor of cholesterol intestinal adsorption in pharmaceutical industries . Moreover, numerous reports on transesterification of racemic 1-phenylethanol with vinyl acetate are available in the literature, we can easily compare the catalytic activity of immobilized BCL with other enzyme catalysts under similar reaction conditions.
Therefore, based on the above analysis, the main objectives of this work are: (1) to compare the properties of the free lipase and the immobilized lipase on MPRs based on the reaction parameters, such as temperature, water content, substrate molar ratio, and reaction time; (2) to investigate probable mechanism for the significant improvement of enzyme activity and enantioselectivity through various characterizations of the immobilized lipase; and (3) further to compare the catalytic efficiency between the immobilized BCL and other immobilized lipases.
Results and discussion
Properties of MPRs used in the present study
Particle size (μm)
Specific surface area (m2/g)
Average pore diameter (nm)
As shown in Table 1, MPRs were synthesized from inexpensive styrene. Actually, they had relatively low price ($ 5-12/kg). The result in Figure 1 showed that the enzyme activity and immobilization efficiency both were highest as compare with the other MPRs, when BCL was immobilized on MPR-NKA. The reason was mainly attributed to different specific surface area and pore diameter. Among these five types of MPRs, NKA had relatively higher specific surface area and average pore diameter (> 20 nm). Gao et al.  has also made similar conclusion that pore diameter of resin influences immobilization degree where immobilization degree increased with the increment of pore diameter. Therefore, MPR-NKA was chosen as the immobilization matrix in the following experiments.
Effect of substrate molar ratio on enzyme activity/eesof the free and immobilized BCL
Effect of water content on enzyme activity/eesof the free and immobilized BCL
Effect of temperature on enzyme activity/eesof the free and immobilized BCL
Effect of reaction time on conversion/eesof the free and immobilized BCL
Operational stability and reusability of the immobilized BCL
The BJH pore size distributions of MPR-NKA
SEM and EDS analysis
Moreover, some researchers have reported that the inner surface may not be fully utilized for lipase adsorption even if the pore size is big enough during the immobilization process [11, 28]. As shown in the Figure 8 (a,b), the outer and internal surface of pure MPR-NKA were full of various pores before adsorption. After immobilization, surface of MPR-NKA was covered by lipase, and pores on the surface of MPR-NKA could not be found (in Figure 8c), which indicates that the lipase has almost been adsorbed by the MPR-NKA. It meant that the internal surface of MPR-NKA had been fully utilized, which was the possible reason for the high thermostability, organic solvent tolerance and operational stability of BCL immobilized on MPR-NKA.
Secondary structure analysis of the free and immobilized BCL by FT-IR spectroscopy
Quantitative estimation of the secondary structure elements of free and immobilized BCL
α- Helix (%)
β- Sheet (%)
β- Turn (%)
Random coil (%)
28.3 ± 1.09
21.4 ± 2.21
25.8 ± 0.91
24.5 ± 0.21
11.8 ± 3.21
42.6 ± 1.21
15.9 ± 0.81
29.8 ± 0.71
Comparison with other immobilized lipases
Compared with other immobilized lipases, the immobilized BCL exhibited a much higher catalytic efficiency. Chua et al. reported that immobilized lipase ChiroCLEC-PC (cross-linked enzyme crystals of Pseudomonas cepacia lipase) was used for the resolution of racemic 1-phenylethanol in organic solvents (including heptane) with different log P values, while the maximal initial rate of reaction was 473.5 ± 10 μmol/min and the reaction reached equilibrium conversion at 45% after 100 mins of reaction . Compared with the cross-linked enzyme crystals method, the immobilized BCL showed a better catalytic efficiency (based on initial reaction rate and final conversion value). Wang et al. reported that lipase from B. cepacia was encapsulated inside zirconia particles by biomimetic mineralization of K2ZrF6. After 48 h reaction under the optimal conditions, their immobilized lipase reached 49.9% with higher ees of 99.9%, however, after 6 cycles, the conversion and ees were only 43% and 85%, respectively . Compared with the approach of encapsulating lipase within zirconia induced by protamine, our immobilized BCL exhibited a better reusability in the successive batch experiments.
In order to compare the catalytic efficiency between our immobilized BCL and several commercially available immobilized lipases usually used in literature, the ee s and conversions of Novozyme 435, Lipozyme RM IM, and Lipozyme TL IM were measured respectively. Under the same conditions of substrate molar ratios (vinyl acetate to racemic 1-phenylethanol) 4:1; reaction time 0.5 h, reaction temperature 35°C, 0.1 g immobilized lipase and 5 mL solvent (heptane), their ee s was 75%, 24% and 15%, respectively. The corresponding conversions were 43.3%, 2.6% and 4.8%, respectively. It can be seen that our immobilized BCL (ee s 99%; conversion 49%) is much better than the commercially avaialable immobilized lipases in catalyzing enantioselective transesterification of 1-phenylethanol with vinyl acetate.
In this study, results were significantly enhanced in terms of enzyme activity and ees when BCL immobilized on MPR-NKA. Compared with the free BCL, the immobilized BCL had better thermostability and excellent reusability in non-aqueous medium. Combined strategies (N2 adsorption–desorption, SEM and EDS) were used to characterize the immobilized lipase, which proved that MPR-NKA was an excellent support for lipase immobilization. FT-IR analysis also indicated that improvement of enzyme activity and ees was closely correlated with the alteration of its secondary structure. Compared with the other immobilized lipases, the immobilized BCL exhibits a better catalytic efficiency, indicating a great potential for industrial applications.
Lipase from B. cepacia was purchased from Amano Enzyme Inc. (Nagoya, Japan). Racemic and optically pure 1-phenylethanol was got from Alfa Aesar Co., Ltd (P. R. China). 1-Phenylethyl acetate; (R)- and (S)- 1-phenylethanol were bought from Sigma-Aldrich Co., Ltd (St. Louis, Missouri, USA). Novozym 435 (from Candida antarctica), Lipozyme RM IM (from Rhizomucor miehei) and Lipozyme TL IM (from Thermomyces lanuginose) were purchased from Sigma-Aldrich Co., Ltd (St. Louis, Missouri, USA). MPR was product of Tianjin Nankai Hecheng S & T Co., Ltd (China). All organic solvents and other reagents were of analytical grade and were obtained commercially from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Preparation of immobilized lipase
The procedures of immobilization were described as follows: 1 g MPR and 5 mL 99% ethanol was added into a 25 mL tube, then the mixture was put in 30°C shaking incubator at 200 rpm for 2 h to wash out the residual catalyst and impurities. The ethanol was removed after MPR precipitated to the bottom of the tube. The residual MPR was washed with distilled water for three times. 5 mL 0.05 M phosphate buffer (pH 7) was mixed with the residual MPR, the mixture was kept for 12 h at 30°C. Then, the buffer was removed. After this pretreatment, MPR was kept in the tube. 0.8 g free BCL powder was dissolved in the 5 mL 0.05 M phosphate buffer (pH 7), this solution was loaded into the tube to mix with the MPR. The tube was stirred in a rotary shaker with a speed of 200 rpm at 30°C for 2 h. The suspension was separated after MPR precipitated to the bottom of the tube. The immobilized BCL (MPR adsorbing the lipase) was washed with 5 mL 0.05 M phosphate buffer (pH 7) for three times to remove the unadsorbed lipase in the surface of the MPR, and then, protein content of the lipase solution and washed water was determined by the method of Bradford . Five types of MPRs were used so as to choose the best immobilization support.
Lipase activity and protein content measurements
Before usage, the organic solvent was dried over 4 Å molecular sieves. Under the above mentioned conditions, reactions were carried out in 5 mL pure heptane, containing 1 mmol racemic 1-phenylethanol, 4 mmol vinyl acetate and 0.1 g free or immobilized BCL. The reaction mixture was put in a 50 mL stoppered flask at 35°C and 200 rpm for 1 h. These conditions were used except when the reaction parameters (molar ratio; temperature; reaction time) needed to be changed in the following text. The above experiments were all conducted in triplicate. After the reactions, the free or immobilized lipase was removed by centrifugation. Then, the samples were filtered through a 0.44 μm filter and analyzed by HPLC.
Analysis and calculation
The samples were analyzed by HPLC (Model 2300–525 SSI. Co., Ltd USA) using a Chiralcel OD-H column (4.6 mm × 250 mm, Daicel Chemical, Japan). Samples (5 μL) were eluted by a mixture of n-hexane: 2-propanol (95:5, v/v) at a rate of 1.0 mL/min, and detected at a wavelength of 254 nm (Model 525 UV Detector SSI. Co., Ltd USA). The retention time of (R)- and (S)-1-phenylethanol in the Chiralcel OD-H column was 7.28 and 8.23 min, respectively.
where, C represents the substrate conversion, ee s stands for the substrate enantiomeric excess, S0 and R0 respectively represent the concentrations of the (S)- and (R)-enantiomers of 1-phenylethanol before reaction, S and R are the concentrations of the (S)- and (R)-enantiomers of 1-phenylethanol after reaction.
Characterization of the immobilization support with N2adsorption–desorption
The specific surface area, pore volumes, and average pore diameters were measured by nitrogen adsorption–desorption equipment (ASAP 2020 V4.00, Micromeritics Instrument Ltd, Shanghai). The specific areas of the MPR-NKA were calculated by the Brunauer–Emmett–Teller (BET) method, and the distributions of pore diameters were estimated by the desorption branches of the isotherms with the Barrett–Joyner–Halenda (BJH) model.
Characterization of the immobilized BCL by SEM and EDS
The immobilized BCL was analyzed with SEM and EDS (Nova Nano SEM 450, FEI Company, Eindhoven, Netherlands). The samples were coated with gold using a sputter coating system and measured at an acceleration voltage of 5 kV.
The samples were mixed with KBr and pressed into pellets. FT-IR measurements in the region of 400-4000 cm-1 were recorded at 25°C by Vextex 70 FT-IR spectrometer (Bruker, Germany) with the nitrogen-cooled, mercury–cadmium– tellurium (MCT) detector. The spectrum acquisition (all samples were overlaid on a zinc selenide ATR accessory) is from IR spectra. The infrared spectrum of KBr has been subtracted from the infrared spectrum during each measurement. The conditions of the measurements were as follows: 20 kHz scan speed, 4 cm-1 spectral resolution, 128 scan co-additions, and triangular apodization. The secondary structure element content was estimated by software PeakFit version 4.12 according to the method described by Yang et al. .
The authors are deeply indebted to Muhammad Jawed Chanesra for his kind assistance in language editing. This work is financially supported by the National Natural Science Foundation of China (Nos. 31070089, 31170078 and J1103514), the National High Technology Research and Development Program of China (2011AA02A204), the Innovation Foundation of Shenzhen Government (JCYJ20120831111657864), and the Innovation Foundation of HUST (2011TS100). Many thanks are indebted to Analytical and Testing Center of HUST for their valuable assistances in SEM, EDS and FT-IR measurement.
- Sellami M, Aissa I, Frikha F, Gargouri Y, Miled N: Immobilized Rhizopus oryzae lipase catalyzed synthesis of palm stearin and cetyl alcohol wax esters: Optimization by response surface methodology. BMC Biotechnol. 2011, 11 (1): 68-10.1186/1472-6750-11-68.View ArticleGoogle Scholar
- Alissandratos A, Baudendistel N, Flitsch S, Hauer B, Halling P: Lipase-catalysed acylation of starch and determination of the degree of substitution by methanolysis and GC. BMC Biotechnol. 2010, 10 (1): 82-10.1186/1472-6750-10-82.View ArticleGoogle Scholar
- Wang H, Sun H, Wei D: Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction. BMC Biotechnol. 2013, 13 (1): 14-10.1186/1472-6750-13-14.View ArticleGoogle Scholar
- Liu Y, Chen D, Yan Y, Peng C, Xu L: Biodiesel synthesis and conformation of lipase from Burkholderia cepacia in room temperature ionic liquids and organic solvents. Bioresource Technol. 2011, 102 (22): 10414-10418. 10.1016/j.biortech.2011.08.056.View ArticleGoogle Scholar
- Liu T, Liu Y, Wang X, Li Q, Wang J, Yan Y: Improving catalytic performance of Burkholderia cepacia lipase immobilized on macroporous resin NKA. J Mol Catal B: Enzym. 2011, 71 (1–2): 45-50.View ArticleGoogle Scholar
- Herbst D, Peper S, Niemeyer B: Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification. J Biotechnol. 2012, 162 (4): 398-403. 10.1016/j.jbiotec.2012.03.011.View ArticleGoogle Scholar
- Klibanov AM: Why are enzymes less active in organic solvents than in water?. Trends Biotechnol. 1997, 15 (3): 97-101. 10.1016/S0167-7799(97)01013-5.View ArticleGoogle Scholar
- Garcia-Galan C, Berenguer-Murcia Á, Fernandez-Lafuente R, Rodrigues RC: Potential of different enzyme immobilization strategies to improve enzyme performance. Adv Synth Catal. 2011, 353 (16): 2885-2904. 10.1002/adsc.201100534.View ArticleGoogle Scholar
- Cao L: Immobilised enzymes: science or art?. Curr Opin Chem Biol. 2005, 9 (2): 217-226. 10.1016/j.cbpa.2005.02.014.View ArticleGoogle Scholar
- Alloue W, Destain J, Medjoub T, Ghalfi H, Kabran P, Thonart P: Comparison of Yarrowia lipolytica lipase immobilization yield of entrapment, adsorption, and covalent bond techniques. Appl Biochem Biotechnol. 2008, 150 (1): 51-63. 10.1007/s12010-008-8148-9.View ArticleGoogle Scholar
- Yan Y, Zhang X, Chen D: Enhanced catalysis of Yarrowia lipolytica lipase LIP2 immobilized on macroporous resin and its application in enrichment of polyunsaturated fatty acids. Bioresource Technol. 2013, 131: 179-187.View ArticleGoogle Scholar
- Gao Y, Tan T, Nie K, Wang F: Immobilization of lipase on macroporous resin and its application in synthesis of biodiesel in low aqueous media. Chin J Biotechnol. 2006, 22 (1): 114-118. 10.1016/S1872-2075(06)60008-3.View ArticleGoogle Scholar
- Gao S, Wang Y, Diao X, Luo G, Dai Y: Effect of pore diameter and cross-linking method on the immobilization efficiency of Candida rugosa lipase in SBA-15. Bioresource Technol. 2010, 101 (11): 3830-3837. 10.1016/j.biortech.2010.01.023.View ArticleGoogle Scholar
- Kharrat N, Ali YB, Marzouk S, Gargouri Y, Karra-Châabouni M: Immobilization of Rhizopus oryzae lipase on silica aerogels by adsorption: Comparison with the free enzyme. Process Biochem. 2011, 46 (5): 1083-1089. 10.1016/j.procbio.2011.01.029.View ArticleGoogle Scholar
- Dib I, Nidetzky B: The stabilizing effects of immobilization in D-amino acid oxidase from Trigonopsis variabilis. BMC Biotechnol. 2008, 8 (1): 72-10.1186/1472-6750-8-72.View ArticleGoogle Scholar
- Shah S, Gupta MN: Kinetic resolution of (±)-1-phenylethanol in [Bmim][PF6] using high activity preparations of lipases. Bioorg Med Chem Lett. 2007, 17 (4): 921-924. 10.1016/j.bmcl.2006.11.057.View ArticleGoogle Scholar
- Ghanem A, Aboul-Enein HY: Application of lipases in kinetic resolution of racemates. Chirality. 2005, 17 (1): 1-15. 10.1002/chir.20089.View ArticleGoogle Scholar
- Chua LS, Sarmidi MR: Immobilised lipase-catalysed resolution of (R, S)-1-phenylethanol in recirculated packed bed reactor. J Mol Catal B: Enzym. 2004, 28 (2–3): 111-119.View ArticleGoogle Scholar
- Ghamgui H, Karra-Chaâbouni M, Bezzine S, Miled N, Gargouri Y: Production of isoamyl acetate with immobilized Staphylococcus simulans lipase in a solvent-free system. Enzyme Microb Tech. 2006, 38 (6): 788-794. 10.1016/j.enzmictec.2005.08.011.View ArticleGoogle Scholar
- Persson M, Costes D, Wehtje E, Adlercreutz P: Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity. Enzyme Microb Tech. 2002, 30 (7): 916-923. 10.1016/S0141-0229(02)00033-9.View ArticleGoogle Scholar
- Xia X, Wang C, Yang B, Wang Y, Wang X: Water activity dependence of lipases in non-aqueous biocatalysis. Appl Biochem Biotech. 2009, 159 (3): 759-767. 10.1007/s12010-009-8618-8.View ArticleGoogle Scholar
- Phillips RS: Temperature modulation of the stereochemistry of enzymatic catalysis: Prospects for exploitation. Trends Biotechnol. 1996, 14 (1): 13-16. 10.1016/0167-7799(96)80908-5.View ArticleGoogle Scholar
- Blanco RM, Terreros P, Muñoz N, Serra E: Ethanol improves lipase immobilization on a hydrophobic support. J Mol Catal B: Enzym. 2007, 47 (1–2): 13-20.View ArticleGoogle Scholar
- Gao S, Wang Y, Wang W, Luo G, Dai Y: Enhancing performance of lipase immobilized on methyl-modified silica aerogels at the adsorption and catalysis processes: Effect of cosolvents. J Mol Catal B: Enzym. 2010, 62 (3–4): 218-224.View ArticleGoogle Scholar
- Ema T: Rational strategies for highly enantioselective lipase-catalyzed kinetic resolutions of very bulky chiral compounds: substrate design and high-temperature biocatalysis. Tetrahedron: Asymmetry. 2004, 15 (18): 2765-2770. 10.1016/j.tetasy.2004.06.055.View ArticleGoogle Scholar
- Schrag JD, Li Y, Cygler M, Lang D, Burgdorf T, Hecht H, Schmid R, Schomburg D, Rydel TJ, Oliver JD, et al: The open conformation of a Pseudomonas lipase. Structure. 1997, 5 (2): 187-202. 10.1016/S0969-2126(97)00178-0.View ArticleGoogle Scholar
- Wang X, Zhou G, Zhang H, Du S, Xu Y, Wang C: Immobilization and catalytic activity of lipase on mesoporous silica prepared from biocompatible gelatin organic template. J Non-Cryst Solids. 2011, 357 (15): 3027-3032. 10.1016/j.jnoncrysol.2011.04.009.View ArticleGoogle Scholar
- Chen B, Miller EM, Miller L, Maikner JJ, Gross RA: Effects of macroporous resin size on Candida antarctica lipase B adsorption, fraction of active molecules, and catalytic activity for polyester synthesis. Langmuir. 2006, 23 (3): 1381-1387.View ArticleGoogle Scholar
- Pribic R, Vanstokkum IHM, Chapman D, Haris PI, Bloemendal M: Protein secondary structure from Fourier transform infrared and/or circular dichroism spectra. Anal Biochem. 1993, 214 (2): 366-378. 10.1006/abio.1993.1511.View ArticleGoogle Scholar
- Haris PI, Severcan F: FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media. J Mol Catal B: Enzym. 1999, 7 (1–4): 207-221.View ArticleGoogle Scholar
- Foresti ML, Alimenti GA, Ferreira ML: Interfacial activation and bioimprinting of Candida rugosa lipase immobilized on polypropylene: effect on the enzymatic activity in solvent-free ethyl oleate synthesis. Enzyme Microb Tech. 2005, 36 (2–3): 338-349.View ArticleGoogle Scholar
- Wang J, Ma C, Bao Y, Xu P: Lipase entrapment in protamine-induced bio-zirconia particles: Characterization and application to the resolution of (R, S)-1-phenylethanol. Enzyme Microb Tech. 2012, 51 (1): 40-46. 10.1016/j.enzmictec.2012.03.011.View ArticleGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72 (1–2): 248-254.View ArticleGoogle Scholar
- Chen CS, Fujimoto Y, Girdaukas G, Sih CJ: Quantitative analyses of biochemical kinetic resolutions of enantiomers. J Am Chem Soc. 1982, 104: 7294-7299. 10.1021/ja00389a064.View ArticleGoogle Scholar
- Yang C, Wang F, Lan D, Whiteley C, Yang B, Wang Y: Effects of organic solvents on activity and conformation of recombinant Candida antarctica lipase A produced by Pichia pastoris. Process Biochem. 2012, 47 (3): 533-537. 10.1016/j.procbio.2011.11.017.View ArticleGoogle Scholar
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