Biomolecular NMR Assignments

, Volume 12, Issue 1, pp 63–68 | Cite as

Backbone and Ile-δ1, Leu, Val methyl 1H, 15N, and 13C, chemical shift assignments for Rhizopus chinensis lipase

  • Meng Zhang
  • Xiao-Wei Yu
  • G. V. T. Swapna
  • Gaohua Liu
  • Rong Xiao
  • Yan Xu
  • Gaetano T. Montelione


Lipase r27RCL is a 296-residue, 33 kDa monomeric enzyme with high ester hydrolysis activity, which has significant applications in the baking, paper and leather industries. The lipase gene proRCL from Rhizopus microsporus var. chinensis (also Rhizopus chinensis) CCTCC M201021 was cloned as a fusion construct C-terminal to a maltose-binding protein (MBP) tag, and expressed as MBP-proRCL in an Escherichia coli BL21 trxB (DE3) expression system with uniform 2H,13C,15N-enrichment and Ile-δ1, Leu, and Val 13CH3 methyl labeling. The fusion protein was hydrolyzed by Kex2 protease at the recognition site Lys-Arg between residues −29 and −28 of the prosequence, producing the enzyme form called r27RCL. Here we report extensive backbone 1H, 15N, and 13C, as well as Ile-δ1, Leu, and Val side chain methyl, NMR resonance assignments for r27RCL.


NMR resonance assignments Rhizopus microsporus var. chinensis lipase 



Chemical shift index


2,2-Dimethyl-2-silapentane-5-sulfonic acid


Heteronuclear single quantum coherence


Transverse relaxation optimized spectroscopy


Nuclear overhauser effect spectroscopy


Rhizopus chinensis lipase


Isopropyl β-d-1-thiogalactopyranoside



We thank Profs. J. Hunt and T. Szyperski for helpful discussions on the RCL lipase project. Financial support from the High-end Foreign Experts Recruitment Program (GDW20123200113), Six Talent Peaks Project in Jiangsu Province (NY-010), 333 Project in Jiangsu Province (BRA2015316), NSFC (31671799), and the 111 Project (111-2-06) are greatly appreciated. This work was also supported as a Community Outreach Project of the NIH NIGMS Protein Structure Initiative, Grant U54 GM094597.


  1. Bahrami A, Assadi AH, Markley JL, Eghbalnia HR (2009) Probabilistic interaction network of evidence algorithm and its application to complete labeling of peak lists from protein NMR spectroscopy. PLoS Comput Biol 5:e1000307. doi: 10.1371/journal.pcbi.1000307 ADSCrossRefGoogle Scholar
  2. Borrelli GM, Trono D (2015) Recombinant lipases and phospholipases and their use as biocatalysts for industrial applications. Int J Mol Sci 16:20774–20840. doi: 10.3390/ijms160920774 CrossRefGoogle Scholar
  3. Czisch M, Boelens R (1998) Sensitivity enhancement in the TROSY experiment. J Magn Reson 134:158–160. doi: 10.1006/jmre.1998.1483 ADSCrossRefGoogle Scholar
  4. Davis AL, Keeler J, Laue ED, Moskau D (1992) Experiments for recording pure-absorption heteronuclear correlation spectra using pulsed field gradients. J Magn Reson 98:207–216. doi: 10.1016/0022-2364(92)90126-R ADSGoogle Scholar
  5. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax AD (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293. doi: 10.1007/BF00197809 CrossRefGoogle Scholar
  6. Diercks T, Coles M, Kessler H (1999) An efficient strategy for assignment of cross-peaks in 3D heteronuclear NOESY experiments. J Biomol NMR 15:177–180. doi: 10.1023/A:1008367912535 CrossRefGoogle Scholar
  7. Eletsky A, Kienhofer A, Pervushin K (2001) TROSY NMR with partially deuterated proteins. J Biomol NMR 20:177–180. doi: 10.1023/A:1011265430149 CrossRefGoogle Scholar
  8. Farrow NA et al (1994) Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. BioChemistry 33:5984–6003. doi: 10.1021/bi00185a040 CrossRefGoogle Scholar
  9. Florkin EM, Stotz EH (1965) In: Comprehensive biochemistry, vol 16. Elsevier Publishing Co., Amsterdam, pp 303–326Google Scholar
  10. Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (1999) A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. J Biomol Nmr 13:369–374. doi: 10.1023/a:1008393201236 CrossRefGoogle Scholar
  11. Jansson M, Li YC, Jendeberg L, Anderson S, Montelione GT, Nilsson B (1996) High-level production of uniformly 15N- and 13C-enriched fusion proteins in Escherichia coli. J Biomol NMR 7:131–141. doi: 10.1007/BF00203823 CrossRefGoogle Scholar
  12. Kay LE, Torchia DA, Bax A (1989) Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. BioChemistry 28:8972–8979. doi: 10.1021/bi00449a003 CrossRefGoogle Scholar
  13. Kay L, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665. doi: 10.1021/ja00052a088 CrossRefGoogle Scholar
  14. Kay LE, Xu GY, Singer AU, Muhandiram DR, Formankay JD (1993) A gradient-enhanced HCCH-TOCSY experiment for recording side-chain 1H and 13C correlations in H2O samples of proteins. J Magn Reson Series B 101:333–337. doi: 10.1006/jmrb.1993.1053 ADSCrossRefGoogle Scholar
  15. Lee D, Hilty C, Wider G, Wuthrich K (2006) Effective rotational correlation times of proteins from NMR relaxation interference. J Magn Reson 178:72–76. doi: 10.1016/j.jmr.2005.08.014 ADSCrossRefGoogle Scholar
  16. Martinelle M, Holmquist M, Hult K (1995) On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase. Biochim Biophys Acta 1258:272–276. doi: 10.1016/0005-2760(95)00131-U CrossRefGoogle Scholar
  17. Meissner A, Schulte-Herbrüggen T, Briand J, Sørensen OW (1998) Double spin-state-selective coherence transfer. Application for two-dimensional selection of multiplet components with long transverse relaxation times. Mol Phys 95:1137–1142. doi: 10.1080/00268979809483245 ADSGoogle Scholar
  18. Moseley HN, Monleon D, Montelione GT (2001) Automatic determination of protein backbone resonance assignments from triple resonance nuclear magnetic resonance data. Methods Enzymol 339:91–108. doi: 10.1016/S0076-6879(01)39311-4 CrossRefGoogle Scholar
  19. Moseley HN, Sahota G, Montelione GT (2004) Assignment validation software suite for the evaluation and presentation of protein resonance assignment data. J Biomol NMR 28:341–355. doi: 10.1023/b:jnmr.0000015420.44364.06 CrossRefGoogle Scholar
  20. Palmer AG, Cavanagh J, Wright PE, Rance M (1991) Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy. J Magn Reson 93:151–170. doi: 10.1016/0022-2364(91)90036-S ADSGoogle Scholar
  21. Patel RN, Szarka LJ, Partyka RA (1998) Lipase esterification processes for resolution of enantiomeric mixtures of intermediates in the preparation of taxanes. Google PatentsGoogle Scholar
  22. Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371ADSCrossRefGoogle Scholar
  23. Pervushin KV, Wider G, Wüthrich K (1998) Single transition-to-single transition polarization transfer (ST2-PT) in [15N, 1H]-TROSY. J Biomol NMR 12:345–348. doi: 10.1023/A:1008268930690 CrossRefGoogle Scholar
  24. Rance M, Loria JP, Palmer AG (1999) Sensitivity improvement of transverse relaxation-optimized spectroscopy. J Magn Reson 136:92–101. doi: 10.1006/jmre.1998.1626 ADSCrossRefGoogle Scholar
  25. Rossi P et al (2010) A microscale protein NMR sample screening pipeline. J Biomol NMR 46:11–22. doi: 10.1007/s10858-009-9386-z CrossRefGoogle Scholar
  26. Salzmann M, Pervushin K, Wider G, Senn H, Wüthrich K (1998) TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci USA 95:13585–13590. doi: 10.1073/pnas.95.23.13585 ADSCrossRefGoogle Scholar
  27. Salzmann M, Wider G, Pervushin K, Senn H, Wüthrich K (1999) TROSY-type triple-resonance experiments for sequential NMR assignments of large proteins. J Am Chem Soc 121:844–848. doi: 10.1021/ja9834226 CrossRefGoogle Scholar
  28. Schleucher J et al (1994) A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients. J Biomol NMR 4:301–306. doi: 10.1007/BF00175254 CrossRefGoogle Scholar
  29. Sha C, Yu X-W, Lin N-X, Zhang M, Xu Y (2013a) Enhancement of lipase r27RCL production in Pichia pastoris by regulating gene dosage and co-expression with chaperone protein disulfide isomerase. Enzyme Microb Technol 53:438–443. doi: 10.1016/j.enzmictec.2013.09.009 CrossRefGoogle Scholar
  30. Sha C, Yu X-W, Zhang M, Xu Y (2013b) Efficient secretion of lipase r27RCL in Pichia pastoris by enhancing the disulfide bond formation pathway in the endoplasmic reticulum. J Ind Microbiol Biotechnol 1–9. doi: 10.1007/s10295-013-1328-9
  31. Sharma S, Kanwar SS (2014) Organic solvent tolerant lipases and applications. Sci World J. doi: 10.1155/2014/625258 Google Scholar
  32. Singh AK, Mukhopadhyay M (2012) Overview of fungal lipase: a review. Appl Biochem Biotechnol 166:486–520. doi: 10.1007/s12010-011-9444-3 CrossRefGoogle Scholar
  33. Weigelt J (1998) Single scan, sensitivity-and gradient-enhanced TROSY for multidimensional NMR experiments. J Am Chem Soc 120:10778–10779. doi: 10.1021/ja982649y CrossRefGoogle Scholar
  34. Wishart DS, Sykes BD (1994) The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR 4:171–180. doi: 10.1007/BF00175245 CrossRefGoogle Scholar
  35. Xiao R et al (2010) The high-throughput protein sample production platform of the Northeast Structural Genomics Consortium. J Struct Biol 172:21–33. doi: 10.1016/j.jsb.2010.07.011 CrossRefGoogle Scholar
  36. Xu Y, Wang D, Mu XQ, Zhao GA, Zhang KC (2002) Biosynthesis of ethyl esters of short-chain fatty acids using whole-cell lipase from Rhizopus chinesis CCTCC M201021 in non-aqueous phase. J Mol Catal B 18:29–37. doi: 10.1016/S1381-1177(02)00056-5 CrossRefGoogle Scholar
  37. Yu X-W, Wang L-L, Xu Y (2009) Rhizopus chinensis lipase: gene cloning, expression in Pichia pastoris and properties. J Mol Catal B 57:304–311. doi: 10.1016/j.molcatb.2008.10.002 CrossRefGoogle Scholar
  38. Yu X, Xu Y, Wang R (2010) Enhancement of activity of Rhizopus chinensis lipase by directed evolution. J Biotechnol 150:344. doi: 10.1016/j.jbiotec.2010.09.374 Google Scholar
  39. Yu X-W, Wang R, Zhang M, Xu Y, Xiao R (2012) Enhanced thermostability of a Rhizopus chinensis lipase by in vivo recombination in Pichia pastoris. Microbial Cell Fact 11:1–11. doi: 10.1186/1475-2859-11-102 CrossRefGoogle Scholar
  40. Yu XW, Xu Y, Xiao R (2016) Lipases from the genus Rhizopus: Characteristics, expression, protein engineering and application. Prog Lipid Res 64:57–68. doi: 10.1016/j.plipres.2016.08.001 CrossRefGoogle Scholar
  41. Zhu G, Kong XM, Sze KH (1999) Gradient and sensitivity enhancement of 2D TROSY with water flip-back, 3D NOESY-TROSY and TOCSY-TROSY experiments. J Biomol NMR 13:77–81. doi: 10.1023/A:1008398227519 CrossRefGoogle Scholar
  42. Zimmerman DE et al (1997) Automated analysis of protein NMR assignments using methods from artificial intelligence. J Mol Biol 269:592–610. doi: 10.1006/jmbi.1997.1052 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina
  2. 2.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  3. 3.Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and BiochemistryRutgers, The State University of New JerseyPiscatawayUSA
  4. 4.Department of Biochemistry and Molecular BiologyRobert Wood Johnson Medical School, Rutgers, The State University of New JerseyPiscatawayUSA
  5. 5.Northeast Structural Genomics ConsortiumPiscatawayUSA

Personalised recommendations