Expression and characterization of a CALB-type lipase from <i>Sporisorium reilianum</i> SRZ2 and its potential in short-chain flavor ester synthesis

doi:10.1007/s11705-019-1889-x

Front. Chem. Sci. Eng.

2020, Vol. 14

Issue (5) : 868-879 https://doi.org/10.1007/s11705-019-1889-x

RESEARCH ARTICLE

Expression and characterization of a CALB-type lipase from Sporisorium reilianum SRZ2 and its potential in short-chain flavor ester synthesis

Jiang-Wei Shen^1,², Xue Cai^1,², Bao-Juan Dou^1,², Feng-Yu Qi^1,², Xiao-Jian Zhang^1,², Zhi-Qiang Liu^1,²(

), Yu-Guo Zheng^1,²

¹. The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, China
². Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China

Download: PDF(1138 KB) HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

Abstract

A lipase from Sporisorium reilianum SRZ2 (SRL) with 73% amino acid sequence identity to Candida antarctica lipase B (CALB) was cloned and overexpressed in Pichia pastoris. The recombinant SRL showed a preference for short-chain p-nitrophenyl esters. It achieved maximum activity at pH 8.0 and 65°C for p-nitrophenyl hexanoate (C6) with K_m and k_cat/K_m values of 0.14 mmol∙L⁻¹ and 1712 min⁻¹∙mmol∙L₋₁ at 30°C, respectively. SRL displayed excellent thermostability and pH stability, retaining more than 79% of its initial activity after incubation at 60°C for 72 h and 75% at pH 3 to 11 for 72 h. It also maintained most of its activity in the presence of inhibitors and detergents except sodium dodecyl sulfate, and it tolerated organic solvents. SRL was covalently immobilized and successfully used for ethyl hexanoate synthesis in cyclohexane or in a solvent-free system with a high conversion yield (>95%). Furthermore, high conversion yield was also achieved for the synthesis of various short-chain flavor esters when high substrate concentrations of 2 mol∙L⁻¹ were applied. This study indicated that a CALB-type lipase from S. reilianum SRZ2 showed great potential in organic ester synthesis.

Keywords lipase Sporisorium reilianum biochemical characterization short-chain flavor ester solvent-free system

Corresponding Author(s): Zhi-Qiang Liu

Just Accepted Date: 19 December 2019 Online First Date: 12 March 2020 Issue Date: 25 May 2020

Cite this article:

Jiang-Wei Shen,Xue Cai,Bao-Juan Dou, et al. Expression and characterization of a CALB-type lipase from Sporisorium reilianum SRZ2 and its potential in short-chain flavor ester synthesis[J]. Front. Chem. Sci. Eng., 2020, 14(5): 868-879.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-019-1889-x
https://academic.hep.com.cn/fcse/EN/Y2020/V14/I5/868

Fig.1 SDS-PAGE analysis of expressed and purified SRL from recombinant P. pastoris X-33-SRL. Lane M: protein molecular weight marker (15?150 kDa); lane 1: SRL production in P. pastoris culture medium supernatant; lane 2: purified SRL.

Fig.2 SRL homology model. (a) Homology-modeled SRL structure represents the secondary structure elements β-sheets in purple, α-helices in cyan, and loops in yellow (the catalytic triad Ser110-Asp192-His230 is displayed in red, the oxyanion hole in green, and the disulfide bonds in orange); (b) Molecular surfaces of predicted SRL and CALB.

Fig.3 Substrate specificity of purified SRL toward various p-NP esters. The assays were conducted at 30°C in 50 mmol?L^?¹ Tris-HCl buffer (pH 8.0) for 6 min. The activity toward p-NP hexanoate was defined as 100%.

Tab.1 Comparison of SRL and CALB kinetic parameters

Fig.4 Effects of temperature and pH on SRL enzyme activity and stability. (a) SRL activity was determined at different temperatures in 50 mmol?L⁻¹ Tris-HCl buffer (pH 8.0) using 1 mmol?L⁻¹ p-NP hexanoate as substrate; (b) SRL activity was measured at 30°C in different pH buffers using 1 mmol?L⁻¹ p-NP hexanoate as substrate; (c) SRL thermostability was determined after incubating the enzyme in 50 mmol?L⁻¹ Tris-HCl buffer (pH 8.0) at temperatures ranges of 30°C?80°C for 0?96 h, and residual activity was measured at standard activity assay conditions; (d) SRL pH stability was measured after incubating the enzyme in 50 mmol?L⁻¹ different pH buffers (3.0?11.0) at 30°C for 0?72 h, and residual activity was measured at standard activity assay conditions.

Tab.2 Effects of metal ions and inhibitors on SRL activity

Tab.3 Effects of different detergents on SRL activity

Tab.4 Effects of different organic solvents on SRL activity

Fig.5 Optimization of esterification reaction operation parameters. (a) Effect of ethanol/hexanoic acid molar ratio on the esterification reaction. The reactions were conducted in 10 mL of cyclohexane with 0.5 mol?L⁻¹ of hexanoic acid and 5 g?L⁻¹ of immobilized SRL at 40°C; (b) Effect of temperature on the esterification reaction. The reactions were conducted at different temperatures in 10 mL of cyclohexane with an ethanol/hexanoic acid molar ratio 1.0 (0.5 mol?L⁻¹) and 5 g?L⁻¹ of immobilized SRL; (c) Effect of biocatalyst content on the initial rate of the esterification reaction. The reactions were conducted in 10 mL of cyclohexane with an ethanol/hexanoic acid molar ratio 1.0 (0.5?2.0 mol?L⁻¹ ) at 45°C, and different amounts of immobilized SRL ranging from 2.5?20 g?L⁻¹ were used.

Fig.6 Effect of substrate concentration on the esterification reaction (The reactions were done in 10 mL of cyclohexane with an ethanol/hexanoic acid molar ratio of 1.0 (0.5?4.0 mol?L⁻¹) and 10 g?L⁻¹ of immobilized SRL at 40°C).

Fig.7 Esterification reaction in the solvent-free system. An ethanol/hexanoic acid molar ratio of 1.0 and 10 g?L^?¹ of immobilized SRL were used at 40°C.

Tab.5 Synthesis of various short-chain flavor esters

1	P Adlercreutz. Immobilisation and application of lipases in organic media. Chemical Society Reviews, 2013, 42(15): 6406–6436 https://doi.org/10.1039/c3cs35446f
2	P Y Stergiou, A Foukis, M Filippou, M Koukouritaki, M Parapouli, L G Theodorou, E Hatziloukas, A Afendra, A Pandey, E M Papamichael. Advances in lipase-catalyzed esterification reactions. Biotechnology Advances, 2013, 31(8): 1846–1859 https://doi.org/10.1016/j.biotechadv.2013.08.006
3	T Tan, J Lu, K Nie, L Deng, F Wang. Biodiesel production with immobilized lipase: A review. Biotechnology Advances, 2010, 28(5): 628–634 https://doi.org/10.1016/j.biotechadv.2010.05.012
4	R Fernández-Lafuente. Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst. Journal of Molecular Catalysis. B, Enzymatic, 2010, 62(3-4): 197–212 https://doi.org/10.1016/j.molcatb.2009.11.010
5	S Javed, F Azeem, S Hussain, I Rasul, M H Siddique, M Riaz, M Afzal, A Kouser, H Nadeem. Bacterial lipases: A review on purification and characterization. Progress in Biophysics and Molecular Biology, 2018, 132: 23–34 https://doi.org/10.1016/j.pbiomolbio.2017.07.014
6	E M Anderson, K M Larsson, O Kirk. One biocatalyst—many applications: The use of Candida antarctica B-lipase in organic synthesis. Biocatalysis and Biotransformation, 1998, 16(3): 181–204 https://doi.org/10.3109/10242429809003198
7	C Buerth, F Kovacic, J Stock, M Terfruchte, S Wilhelm, K E Jaeger, M Feldbrugge, K Schipper, J F Ernst, D Tielker. Uml2 is a novel CalB-type lipase of Ustilago maydis with phospholipase A activity. Applied Microbiology and Biotechnology, 2014, 98(11): 4963–4973 https://doi.org/10.1007/s00253-013-5493-6
8	M E Vaquero, L I de Eugenio, M J Martinez, J Barriuso. A novel calb-type lipase discovered by fungal genomes mining. PLoS One, 2015, 10(4): e0124882 https://doi.org/10.1371/journal.pone.0124882
9	S Park. Exploration and functional expression of homologous lipases of Candida antarctica lipase B. Korean Journal of Microbiology, 2015, 51(3): 187–193 https://doi.org/10.7845/kjm.2015.5037
10	K P Dhake, D D Thakare, B M Bhanage. Lipase: A potential biocatalyst for the synthesis of valuable flavour and fragrance ester compounds. Flavour and Fragrance Journal, 2013, 28(2): 71–83 https://doi.org/10.1002/ffj.3140
11	A G A Sá, A C Meneses, P H H Araújo, D Oliveira. A review on enzymatic synthesis of aromatic esters used as flavor ingredients for food, cosmetics and pharmaceuticals industries. Trends in Food Science & Technology, 2017, 69: 95–105 https://doi.org/10.1016/j.tifs.2017.09.004
12	S Serra, C Fuganti, E Brenna. Biocatalytic preparation of natural flavours and fragrances. Trends in Biotechnology, 2005, 23(4): 193–198 https://doi.org/10.1016/j.tibtech.2005.02.003
13	W Gao, K Wu, L Chen, H Fan, Z Zhao, B Gao, H Wang, D Wei. A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters. Microbial Cell Factories, 2016, 15(1): 41 https://doi.org/10.1186/s12934-016-0435-5
14	H D Yan, Q Zhang, Z Wang. Biocatalytic synthesis of short-chain flavor esters with high substrate loading by a whole-cell lipase from Aspergillus oryzae. Catalysis Communications, 2014, 45: 59–62 https://doi.org/10.1016/j.catcom.2013.10.018
15	P Lozano, J M Bernal, A Navarro. A clean enzymatic process for producing flavour esters by direct esterification in switchable ionic liquid/solid phases. Green Chemistry, 2012, 14(11): 3026–3033 https://doi.org/10.1039/c2gc36081k
16	A B Martins, J L Friedrich, J C Cavalheiro, C Garcia-Galan, O Barbosa, M A Ayub, R Fernández-Lafuente, R C Rodrigues. Improved production of butyl butyrate with lipase from Thermomyces lanuginosus immobilized on styrene-divinylbenzene beads. Bioresource Technology, 2013, 134: 417–422 https://doi.org/10.1016/j.biortech.2013.02.052
17	Y G Zheng, H H Yin, D F Yu, X Chen, X L Tang, X J Zhang, Y P Xue, Y J Wang, Z Q Liu. Recent advances in biotechnological applications of alcohol dehydrogenases. Applied Microbiology and Biotechnology, 2017, 101(3): 987–1001 https://doi.org/10.1007/s00253-016-8083-6
18	Z Q Liu, L Wu, L Zheng, W Z Wang, X J Zhang, L Q Jin, Y G Zheng. Biosynthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate by carbonyl reductase from Rhodosporidium toruloides in mono and biphasic media. Bioresource Technology, 2018, 249: 161–167 https://doi.org/10.1016/j.biortech.2017.09.204
19	Z Q Liu, M M Lu, X H Zhang, F Cheng, J M Xu, Y P Xue, L Q Jin, Y S Wang, Y G Zheng. Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid. International Journal of Biological Macromolecules, 2018, 116: 563–571 https://doi.org/10.1016/j.ijbiomac.2018.05.045
20	D S Dheeman, G T Henehan, J M Frias. Purification and properties of Amycolatopsis mediterranei DSM 43304 lipase and its potential in flavour ester synthesis. Bioresource Technology, 2011, 102(3): 3373–3379 https://doi.org/10.1016/j.biortech.2010.11.074
21	Y Wang, D H Zhang, N Chen, G Y Zhi. Synthesis of benzyl cinnamate by enzymatic esterification of cinnamic acid. Bioresource Technology, 2015, 198: 256–261 https://doi.org/10.1016/j.biortech.2015.09.028
22	Y H Kim, S Park. Surveying enantioselectivity of two Candida antarctica-lipase-B homologs towards chiral sec-alcohols. Bulletin of the Korean Chemical Society, 2017, 38(11): 1358–1361 https://doi.org/10.1002/bkcs.11289
23	Z Q Liu, X B Zheng, S P Zhang, Y G Zheng. Cloning, expression and characterization of a lipase gene from the Candida antarctica ZJB09193 and its application in biosynthesis of vitamin A esters. Microbiological Research, 2012, 167(8): 452–460 https://doi.org/10.1016/j.micres.2011.12.004
24	J W Shen, J M Qi, X J Zhang, Z Q Liu, Y G Zheng. Significantly increased catalytic activity of Candida antarctica lipase B for the resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate. Catalysis Science & Technology, 2018, 8(18): 4718–4725 https://doi.org/10.1039/C8CY01340C
25	U K Winkler. Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. Journal of Bacteriology, 1979, 138: 663–670
26	A B Martins, A M da Silva, M F Schein, C Garcia-Galan, M A Záchia Ayub, R Fernández-Lafuente, R C Rodrigues. Comparison of the performance of commercial immobilized lipases in the synthesis of different flavor esters. Journal of Molecular Catalysis. B, Enzymatic, 2014, 105: 18–25 https://doi.org/10.1016/j.molcatb.2014.03.021
27	V Juturu, J C Wu. Heterologous protein expression in Pichia pastoris: Latest research progress and applications. ChemBioChem, 2018, 19(1): 7–21 https://doi.org/10.1002/cbic.201700460
28	G T Eom, S H Lee, B K Song, K W Chung, Y W Kim, J K Song. High-level extracellular production and characterization of Candida antarctica lipase B in Pichia pastoris. Journal of Bioscience and Bioengineering, 2013, 116(2): 165–170 https://doi.org/10.1016/j.jbiosc.2013.02.016
29	I B B Romdhane, A Fendri, Y Gargouri, A Gargouri, H Belghith. A novel thermoactive and alkaline lipase from Talaromyces thermophilus fungus for use in laundry detergents. Biochemical Engineering Journal, 2010, 53(1): 112–120 https://doi.org/10.1016/j.bej.2010.10.002
30	Y Y Zheng, X H Guo, N N Song, D C Li. Thermophilic lipase from Thermomyces lanuginosus: Gene cloning, expression and characterization. Journal of Molecular Catalysis. B, Enzymatic, 2011, 69(3-4): 127–132 https://doi.org/10.1016/j.molcatb.2011.01.006
31	X F Zhang, G Y Yang, Y Zhang, Y Xie, S G Withers, Y Feng. A general and efficient strategy for generating the stable enzymes. Scientific Reports, 2016, 6(1): 33797 https://doi.org/10.1038/srep33797
32	X F Zhang, Y H Ai, Y Xu, X W Yu. High-level expression of Aspergillus niger lipase in Pichia pastoris: Characterization and gastric digestion in vitro. Food Chemistry, 2019, 274: 305–313 https://doi.org/10.1016/j.foodchem.2018.09.020
33	W Xie, M Huang. Immobilization of Candida rugosa lipase onto graphene oxide Fe3O4 nanocomposite: Characterization and application for biodiesel production. Energy Conversion and Management, 2018, 159: 42–53 https://doi.org/10.1016/j.enconman.2018.01.021
34	A Hiol, M D Jonzo, N Rugani, D Druet, L Sarda, L C Comeau. Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme and Microbial Technology, 2000, 26(5-6): 421–430 https://doi.org/10.1016/S0141-0229(99)00173-8
35	M Kohno, W Kugimiya, Y Hashimoto, Y Morita. Purification, characterization, and crystallization of two types of lipase from Rhizopus niveus. Bioscience, Biotechnology, and Biochemistry, 1994, 58(6): 1007–1012 https://doi.org/10.1271/bbb.58.1007
36	T Florczak, M Daroch, M C Wilkinson, A Bialkowska, A D Bates, M Turkiewicz, L A Iwanejko. Purification, characterisation and expression in Saccharomyces cerevisiae of LipG7 an enantioselective, cold-adapted lipase from the Antarctic filamentous fungus Geomyces sp. P7 with unusual thermostability characteristics. Enzyme and Microbial Technology, 2013, 53(1): 18–24 https://doi.org/10.1016/j.enzmictec.2013.03.021
37	L D Castro-Ochoa, C Rodríguez-Gómez, G Valerio-Alfaro, R Oliart Ros. Screening, purification and characterization of the thermoalkalophilic lipase produced by Bacillus thermoleovorans CCR11. Enzyme and Microbial Technology, 2005, 37(6): 648–654 https://doi.org/10.1016/j.enzmictec.2005.06.003
38	Q Sun, H Wang, H Zhang, H Luo, P Shi, Y Bai, F Lu, B Yao, H Huang. Heterologous production of an acidic thermostable lipase with broad-range pH activity from thermophilic fungus Neosartorya fischeri P1. Journal of Bioscience and Bioengineering, 2016, 122(5): 539–544 https://doi.org/10.1016/j.jbiosc.2016.05.003
39	Z B Bakir, K Metin. Purification and characterization of an alkali-thermostable lipase from thermophilic Anoxybacillus flavithermus HBB 134. Journal of Microbiology and Biotechnology, 2016, 26(6): 1087–1097 https://doi.org/10.4014/jmb.1512.12056
40	M D R Sanchez-Carbente, R A Batista-Garcia, A Sanchez-Reyes, A Escudero-Garcia, C Morales-Herrera, L I Cuervo-Soto, L French-Pacheco, A Fernández-Silva, C Amero, E Castillo, J L Folch-Mallol. The first description of a hormone-sensitive lipase from a basidiomycete: Structural insights and biochemical characterization revealed Bjerkandera adusta BaEstB as a novel esterase. MicrobiologyOpen, 2017, 6(4): e00463 https://doi.org/10.1002/mbo3.463
41	R Jallouli, G Parsiegla, F Carriere, Y Gargouri, S Bezzine. Efficient heterologous expression of Fusarium solani lipase, FSL2, in Pichia pastoris, functional characterization of the recombinant enzyme and molecular modeling. International Journal of Biological Macromolecules, 2017, 94: 61–71 https://doi.org/10.1016/j.ijbiomac.2016.09.030
42	X Zheng, X Chu, W Zhang, N Wu, Y Fan. A novel cold-adapted lipase from Acinetobacter sp. XMZ-26: Gene cloning and characterisation. Applied Microbiology and Biotechnology, 2011, 90(3): 971–980 https://doi.org/10.1007/s00253-011-3154-1
43	A Gricajeva, V Bendikiene, L Kalediene. Lipase of Bacillus stratosphericus L1: Cloning, expression and characterization. International Journal of Biological Macromolecules, 2016, 92(3): 96–104 https://doi.org/10.1016/j.ijbiomac.2016.07.015
44	W Yang, Y He, L Xu, H Zhang, Y Yan. A new extracellular thermo-solvent-stable lipase from Burkholderia ubonensis SL-4: Identification, characterization and application for biodiesel production. Journal of Molecular Catalysis. B, Enzymatic, 2016, 126: 76–89 https://doi.org/10.1016/j.molcatb.2016.02.005
45	R Bancerz, G Ginalska. Ginalska G. A novel thermostable lipase from basidiomycete Bjerkandera adusta R59: characterisation and esterification studies. Journal of Industrial Microbiology & Biotechnology, 2007, 34(8): 553–560 https://doi.org/10.1007/s10295-007-0232-6
46	S Malekabadi, A Badoei-Dalfard, Z Karami. Biochemical characterization of a novel cold-active, halophilic and organic solvent-tolerant lipase from B. licheniformis KM12 with potential application for biodiesel production. International Journal of Biological Macromolecules, 2018, 109: 389–398 https://doi.org/10.1016/j.ijbiomac.2017.11.173
47	A Glogauer, V P Martini, H Faoro, G H Couto, M Muller-Santos, R A Monteiro, D A Mitchell, E M de Souza, F O Pedrosa, N Krieger. Identification and characterization of a new true lipase isolated through metagenomic approach. Microbial Cell Factories, 2011, 10(1): 54 https://doi.org/10.1186/1475-2859-10-54
48	K Ramani, E Chockalingam, G Sekaran. Production of a novel extracellular acidic lipase from Pseudomonas gessardii using slaughterhouse waste as a substrate. Journal of Industrial Microbiology & Biotechnology, 2010, 37(5): 531–535 https://doi.org/10.1007/s10295-010-0700-2
49	A Salihu, M Z Alam. Solvent tolerant lipases: A review. Process Biochemistry, 2015, 50(1): 86–96 https://doi.org/10.1016/j.procbio.2014.10.019
50	S Dutta Banik, M Nordblad, J M Woodley, G H Peters. A correlation between the activity of Candida antarctica lipase B and differences in binding free energies of organic solvent and substrate. ACS Catalysis, 2016, 6(10): 6350–6361 https://doi.org/10.1021/acscatal.6b02073
51	Z Jin, J Ntwali, S Y Han, S P Zheng, Y Lin. Production of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in a batch reactor. Journal of Biotechnology, 2012, 159(1-2): 108–114 https://doi.org/10.1016/j.jbiotec.2012.02.013
52	S Y Han, Z Y Pan, D F Huang, M Ueda, X N Wang, Y Lin. Highly efficient synthesis of ethyl hexanoate catalyzed by CALB-displaying Saccharomyces cerevisiae whole-cells in non-aqueous phase. Journal of Molecular Catalysis. B, Enzymatic, 2009, 59(1-3): 168–172 https://doi.org/10.1016/j.molcatb.2009.02.007
53	J W Shen, J M Qi, X J Zhang, Z Q Liu, Y G Zheng. Efficient resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate by covalently immobilized mutant Candida antarctica lipase B in batch and semi-continuous modes. Organic Process Research & Development, 2019, 23(5): 1017–1025 https://doi.org/10.1021/acs.oprd.9b00066
54	X J Zhang, P X Shi, H Z Deng, X X Wang, Z Q Liu, Y G Zheng. Biosynthesis of chiral epichlorohydrin using an immobilized halohydrin dehalogenase in aqueous and non-aqueous phase. Bioresource Technology, 2018, 263: 483–490 https://doi.org/10.1016/j.biortech.2018.05.027
55	E Onoja, S Chandren, F I A Razak, R A Wahab. Enzymatic synthesis of butyl butyrate by Candida rugosa lipase supported on magnetized-nanosilica from oil palm leaves: Process optimization, kinetic and thermodynamic study. Journal of The Taiwan Institute of Chemical Engineers, 2018, 91: 105–118 https://doi.org/10.1016/j.jtice.2018.05.049
56	X Duan, Y Liu, X You, Z Jiang, S Yang, S Yang. High-level expression and characterization of a novel cutinase from Malbranchea cinnamomea suitable for butyl butyrate production. Biotechnology for Biofuels, 2017, 10(1): 223 https://doi.org/10.1186/s13068-017-0912-z
57	B Chen, H Liu, Z Guo, J Huang, M Wang, X Xu, L Zheng. Lipase-catalyzed esterification of ferulic acid with oleyl alcohol in ionic liquid/isooctane binary systems. Journal of Agricultural and Food Chemistry, 2011, 59(4): 1256–1263 https://doi.org/10.1021/jf104101z
58	G V Chowdary, S G Prapulla. Enzymatic synthesis of ethyl hexanoate by transesterification. International Journal of Food Science & Technology, 2003, 38(2): 127–133 https://doi.org/10.1046/j.1365-2621.2003.00653.x
59	N Musa, W Latip, R Abd Rahman, A Salleh, M Mohamad Ali. Immobilization of an Antarctic Pseudomonas AMS8 lipase for low temperature ethyl hexanoate synthesis. Catalysts, 2018, 8(6): 234 https://doi.org/10.3390/catal8060234

[1]

Electronic Supplementary Material

Download

[1]	BAI Shu, REN Mengyuan, WANG Lele, SUN Yan. Regioselective acylation of pyridoxine catalyzed by immobilized lipase in ionic liquid[J]. Front. Chem. Sci. Eng., 2008, 2(3): 301-307.

Viewed

Full text

Abstract

Cited

Shared

Discussed