Regulation of Ester Synthesis Activity of Rhizopus chinensis Lipase†

doi:10.7503/cjcu20180437

Abstract

Abstract:

The regaining of ester synthesis catalytic ability of extracted and expressed Rhizopus chinensis lipase was investigated in this work. After de novo folding in a mimicking membrane environment, the maximum ester synthesis specific activity of extracted lipase precipitated protein(eRCL-P) was re-obtained and even increased for over 5 times. The refolding treatment of r27RCL, a Pichia pastoris expressed lipase with a high hydrolysis activity but low ester synthesis activity, with different membrane mimicking factors also increased the organic catalysis ability of the protein. The best enhancement of ester synthesis activity was shown when r27RCL was directly mixed with detergent, which indicated the interaction between lipase and regulation factor was established during mixing stage in water solution. Moreover, seven different detergents of three diversity types were introduced to confirm the result, and the zwitterionic detergent LPC14 increased the ester synthesis activity of r27RCL for the best effect as 5.75 times. Although the circular dichroism spectrum showed that the secondary structure of r27RCL had no obviously changes, the molecular dynamics simulation demonstrated that, in organic reaction, the hydrogen bonds between the catalytic triad of r27RCL have been enhanced after mixing with detergent, which may therefore stimulate the ester synthesis activity of lipase. In addition, these results confirmed the regulation of lipase ester synthesis activity, which will surely establish the basics for further research and production.

Key words: Detergent, Lipase, Non-aqueous catalysis, Ester synthesis activity regulation, Molecular dynamics simulation

CLC Number:

O621.25

TrendMD:

ZHANG Zhang,WANG Dong,WANG Xiaolei,XU Yan. Regulation of Ester Synthesis Activity of Rhizopus chinensis Lipase^†[J]. Chem. J. Chinese Universities, 2019, 40(4): 747.

Figures/Tables 8

Fig.1 Extraction and isolation of R. chinensis lipase1. The extracted lipase from R. chinensis, eRCL; 2. the supernatant of the extract, eRCL-S; 3. the precipitate of the extract, eRCL-P; M. low molecular protein marker .

Table 1 Activity assay of R. chinensis lipase

Sample	Concentration/(mg·mL^-1)	Hydrolysis activity/(U·mg^-1)	Ester synthesis activity /(U·mg^-1)
eRCL	0.75±0.03	11.4±0.6	10.4±0.6
eRCL-S	0.35±0.02	9.8±0.08	8.1±0.04
eRCL-P	0.98±0.03	3.6±0.05	1.8±0.11
eRCL-P refolding	0.22±0.01	1.1±0.01	0.8±0.01
eRCL-P refolding with eRCL-S	0.65±0.02	8.8±0.06	9.1±0.03

Fig.2 Effect of additives on the activities of R. chinensis lipase r27RCL in refoldingr27RCL, the commercial R. chinensis lipase; MC, membrane component; TX, detergent Triton X-100; with MC, r27RCL refolding with MC; with TX, r27RCL refolding with Triton X-100.

Fig.3 Effect of adding different stages of MC and Triton X-100 on activity of lipase r27RCLMC, membrane component; TX, detergent Triton X-100; with MC in re-, adding MC in renaturation during refolding process; with TX in re-, adding Triton X-100 in renaturation during refolding process; +MC, r27RCL mixed with membrane component directly; +TX, r27RCL mixed with Triton X-100 directly.

Fig.4 Effects of different detergents on the activity of r27RCLa. r27RCL; b. CTAB; c. Façade-EM; d. C12E8; e. Trion X-100; f. DiC6PC; g. LPC14; h. CHAPS.

Fig.5 Far-UV circular dichroism spectra of lipase r27RCL(a) and r27RCL with detergent LPC14(b)

Fig.6 RMSD(A, B) and RMSF(C, D) value of r27RCL(A, C) and r27RCL with LPC14(B, D) in heptane(a) and water(b)

Fig.7 Simulated 3D structure of catalytic triad of r27RCL in different systemsCrystal structure of r27RCL was downloaded from PDB(ID 4L3W) and was showed in green. The simulated 3D structure of r27RCL in water and heptane were showed in cyan and magenta, respectively. The amino acids of catalytic triad of r27RCL and LPC14 were showed as stick.

References 34

[1]	Brzozowski A. M., Derewenda U., Derewenda Z. S., Dodson G. G., Lawson D. M., Turkenburg J. P., Bjorkling F., Huge-Jensen B., Patkar S. A., Thim L., Nature,1991, 351(6326), 491—494
[2]	Jaeger K. E., Eggert T., Curr. Opin.Biotechnol.,2002, 13(4), 390—397
[3]	Cui L. J., Xu G., Meng X., Wu J. P., Yang L. R., Chem. Ind. Eng.Prog.,2014, 33(8), 2150—2154
	(崔丽娟, 徐刚, 孟枭, 吴坚平, 杨立荣. 化工进展,2014, 33(8), 2150—2154)
[4]	Jiang L., Yu H., Chem. Res. Chinese Universities,2014, 30(3), 396—399
[5]	Stergiou P. Y., Foukis A., Filippou M., Koukouritaki M., Parapouli M., Theodorou L. G., Hatziloukas E., Afendra A., Pandey A., Papamichael E. M., Biotechnol.Adv.,2013, 31(8), 1846—1859
[6]	Wang Z., Luo W., Fu J., Li Z., Wang Z., Lü P., Chem. Res. Chinese Universities,2017, 33(6), 1—5
[7]	Larios A., García H. S., Oliart R. M., Valerio-Alfaro G., Appl. Microbiol.Biotechnol.,2004, 65(4), 373—376
[8]	Ghanem A., Aboul-Enein H. Y., Tetrahedron:Asymmetry,2004, 15(21), 3331—3351
[9]	Monteiro J. B., Nascimento M. G., Ninow J. L., Biotechnol.Lett.,2003, 25(8), 641—644
[10]	Teng Y., Xu Y., Wang D., J. Mol. Catal. B: Enzym., 2009, 57(1—4), 292—298
[11]	Shah S., Gupta M. N., Process Biochem.,2007, 42(3), 409—414
[12]	Karboune S., Archelas A., Baratti J. C., Process Biochem.,2010, 45(2), 210—216
[13]	Oda M., Kaieda M., Hama S., Yamaji H., Kondo A., Izumoto E., Fukuda H., Biochem. Eng.J.,2005, 23(1), 45—51
[14]	Aarthy M., Saravanan P., Gowthaman M., Rose C., Kamini N., Chem. Eng. Res.Des.,2014, 92(8), 1591—1601
[15]	Xu Y., Wang D., Mu X. Q., Zhao G. A., Zhang K. C., J. Mol. Catal. B: Enzym., 2002, 18(1), 29—37
[16]	Wang D., Xu Y., Teng Y., Bioprocess Biosyst.Eng.,2007, 30(3), 147—155
[17]	He Q., Xu Y., Teng Y., Wang D., Chin. J.Catal.,2008, 29(1), 41—46
[18]	Di Lorenzo M., Hidalgo A., Haas M., Bornscheuer U. T., Appl. Environ.Microbiol.,2005, 71(12), 8974—8977
[19]	Joerger R. D., Haas M. J., Lipids,1993, 28(2), 81—88
[20]	Singh A., Upadhyay V., Upadhyay A. K., Singh S. M., Panda A. K., Microb. Cell.Fact., 2015, 14, 41—51
[21]	Singh S. M., Sharma A., Upadhyay A. K., Singh A., Garg L. C., Panda A. K., Protein Expr.Purif.,2012, 81(1), 75—82
[22]	Satomura A., Kuroda K., Ueda M., PLoS One,2015, 10(5), e0124545
[23]	Akbari N., Khajeh K., Rezaie S., Mirdamadi S., Shavandi M., Ghaemi N., Protein Expr.Purif., 2010, 70(1), 75—80
[24]	Martini V. P., Glogauer A., Müller-Santos M., Iulek J., de Souza E. M., Mitchell D. A., Pedrosa F. O., Krieger N., Microb. Cell.Fact.,2014, 13, 171—185
[25]	Contreras F. X., Ernst A. M., Wieland F., Brugger B., Cold Spring Harb. Perspect. Biol.,2011, 3, 1031—1033
[26]	Laganowsky A., Reading E., Allison T. M., Ulmschneider M. B., Degiacomi M. T., Baldwin A. J., Robinson C. V., Nature,2014, 510(7503), 172—175
[27]	Takada M., Takeuchi H., Shino M., Hamano S., Ohgoh T., Biochem. Biophys. Res.Commun.,1989, 164(2), 653—663
[28]	Yamaguchi H., Miyazaki M., Biomolecules,2014, 4(1), 235—251
[29]	Kordel M., Hofmann B., Schomburg D., Schmid R. D., J.Bacteriol.,1991, 173(15), 4836—4841
[30]	Seddon A. M., Curnow P., Booth P. J., Biochim. Biophys.Acta,2004, 1666(1/2), 105—117
[31]	Otzen D. E., Biophys.J., 2002, 83(4), 2219—2230
[32]	Carrasco-LóPez C., Godoy C., De B. L. R., Fernández-Lorente G., Palomo J. M., Guisán J. M., Fernández-Lafuente R., Martínez-Ripoll M., Hermoso J. A., J. Biol.Chem., 2009, 284(7), 4365—4372
[33]	Tang Q.Y., Wang Y. H.,Mod Food Sci. & Technol., 2017, (3), 179—183
	(唐庆芸, 王永华. 现代食品科技, 2017, (3), 179—183)
[34]	Mingarro I., Abad C., Braco L., Proc. Natl. Acad. Sci. USA,1995, 92(8), 3308—3312