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
Heteronuclear single quantum coherence
Transverse relaxation optimized spectroscopy
Nuclear overhauser effect spectroscopy
Rhizopus chinensis lipase
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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.
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.1000307ADSCrossRefGoogle Scholar
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/bi00185a040CrossRefGoogle Scholar
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:1008393201236CrossRefGoogle Scholar
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/BF00203823CrossRefGoogle Scholar
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/bi00449a003CrossRefGoogle Scholar
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/ja00052a088CrossRefGoogle Scholar
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.1993.1053ADSCrossRefGoogle Scholar
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
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
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.95.23.13585ADSCrossRefGoogle Scholar
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/ja9834226CrossRefGoogle Scholar
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.2013.09.009CrossRefGoogle Scholar
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
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/BF00175245CrossRefGoogle Scholar
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)00056-5CrossRefGoogle Scholar