Abstract
A microbial strain (referred to as M1) that produces an extracellular lipase was isolated from a soil sample in Vietnam, and identified as a Ralstonia species by partial sequencing of its 16S rDNA. A genomic library was constructed from Pst I fragments, and a colony showing lipase activity was selected for further analysis. Sequencing of the 4.7-kb insert in this clone (named M1-72) revealed one incomplete and three complete ORFs, predicted to encode a partial hypothetical glutaminyl tRNA synthetase (304 aa), a hypothetical transmembrane protein (500 aa), a lipase (328 aa) and a lipase chaperone (352 aa), respectively. Alignment of the insert sequence with the corresponding region of the genome of R. solanacearum GMI1000 (GenBank Accession No. AL646081) confirmed the presence in the latter of the genes for the hypothetical transmembrane protein and glutaminyl tRNA synthetase, which exhibited 89–91% identity to their counterparts in M1. However, R. solanacearum GMI1000 lacks the complete lipase-encoding gene and the major part of the chaperone-encoding gene, creating a so-called “black hole”. The deduced amino acid sequences of the products of the lipase gene lipA and chaperone gene lipB from strain M1 shared 49.3–60.3% and 23.9–32.7% identity, respectively, with those of the Burkholderia lipase/chaperone subfamily I.2. lipB is located downstream of lipA, and separated from it by only 9 bp, and each gene has a putative ribosome binding site. The mature lipase LipA, a His-tagged derivative (LipAhis), the tagged full-length chaperone LipBhis and a truncated form (ΔLipBhis) lacking the 56 N-terminal residues were expressed in Escherichia coli BL21. LipA, LipAhis and ΔLipBhis could be expressed at high levels (70, 15 and 12 mg/g wet cells, respectively) and were easily purified. However, LipBhis was expressed at a much lower level which precluded purification. The specific activity of purified LipAhis, expressed on its own, was very low (<52 U/mg). However, after co-incubation with the purified ΔLipBhis in vitro, the specific activity of the enzyme was markedly enhanced, indicating that the chaperone facilitated correct folding of the enzyme. A lipase:chaperone ratio of 1:10 was found to be optimal, yielding an enzyme preparation with a specific activity of 650 U/mg.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aoyama S, Yoshida N, Inouye S (1988) Cloning, sequencing and expression of the lipase gene from Pseudomonas fragi IFO-12049 in E. coli. FEBS Lett 242:36–40
Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177–183
Chihara-Siomi M, Yoshikawa K, Oshima-Hirayama N, Yamamoto K, Sogabe Y, Nakatani T, Nishioka T, Oda J (1992) Purification, molecular cloning, and expression of lipase from Pseudomonas aeruginosa. Arch Biochem Biophys 296:505–513
Eddine AN, Hannemann F, Schäfer W (2001) Cloning and expression analysis of NhL1, a gene encoding an extracellular lipase from the fungal pea pathogen Nectria haematococca MP VI ( Fusarium solani f. sp. pisi) that is expressed in planta. Mol Genet Genomics 265:215–224
Frenken LG, Egmond MR, Batenburg AM, Bos JW, Visser C, Verrips CT (1992) Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues. Appl Environ Microbiol 58:3787–3791
Frenken LG, Bos JW, Visser C, Muller W, Tommassen J, Verrips CT (1993a) An accessory gene, lipB , required for the production of active Pseudomonas glumae lipase. Mol Microbiol 9:579–589
Frenken LG, Groot Ad, Tommassen J, Verrips CT (1993b) Role of the lipB gene product in the folding of the secreted lipase of Pseudomonas glumae. Mol Microbiol 9:591–599
Han SJ, Back JH, Yoon MY, Shin PK, Cheong CS, Sung MH, Hong SP, Chung IY, Han YS (2003) Expression and characterization of a novel enantioselective lipase from Acinetobacter species SY-01. Biochimie 85:501–510
Hemachander C, Puvanakrishnan R (2000) Lipase from Ralstonia pickettii as an additive in laundry detergent formulations. Process Biochem 35:809–814
Hemachander C, Bose N, Puvanakrishnan R (2001) Whole cell immobilization of Ralstonia pickettii for lipase production. Process Biochem 36:629–633
Hobson AH, Buckley CM, Aamand JL, Jorgensen ST, Diderichsen B, McConnell DJ (1993) Activation of a bacterial lipase by its chaperone. Proc Natl Acad Sci USA 90:5682–5686
Ihara F, Okamoto I, Akao K, Nihara T, Yamada Y (1995) Lipase modulator protein (LimL) of Pseudomonas sp. strain 109. J Biacteriol 177:1245–1258
Iizumi T, Nakamura K, Shimada Y, Sugihara A, Tominaga Y, Fukase T (1991) Cloning, nucleotide sequencing, and expression in Escherichia coli of a lipase and its activator genes from Pseudomonas sp. KWI-56. Agric Biol Chem 55:2349–2357
Jaeger K-E, Eggert T (2002) Lipases for biotechnology. Curr Opin Biotechnol 13:390–397
Jenczmionka NJ, Maier FJ, Lösch AP, Schäfer W (2003) Mating, conidiation and pathogenicity of Fusarium graminearum, the main causal agent of the head blight disease of wheat, are regulated by the MAP kinase Gpmk1. Curr Genet 43:87–95
Jorgensen S, Skov KW, Diderichsen B (1991) Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonas genes. J Bacteriol 173:559–567
Kim EK, Jang WH, Ko JH, Kang JS, Noh MJ, Yoo OJ (2001) Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator. J Bacteriol 183:5937–5941
Kim HK, Lee JK, Kim H, Oh TK (1996) Characterization of an alkaline lipase from Proteus vulgaris K80 and the DNA sequence of the encoding gene. FEMS Microbiol Lett 135:117–121
Kok RG, Thor JJv, Nugteren-Roodzant IM, Vosman B, Hellingwerf KJ (1995) Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase. J Bacteriol 177:3295–3307
Laemmli UK (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Litthauer D, Ginster A, Skein E (2002) Pseudomonas luteola lipase: a new member of the 320-residue Pseudomonas lipase family. J Enzyme Microb Technol 30:209–215
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
Maurelli AT, Fernandez RF, Bloch CA, Rode CK, Fasano A (1998) “Black holes” and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli. Proc Natl Acad Sci USA 95:3943–3948
Ohshima-Hirayama N, Yoshikawa K, Takaaki N, Oda J (1993) Lipase from Pseudomonas aeruginosa. Production in Escherichia coli and activation in vitro with a protein from the downstream gene. Eur J Biochem 215:239–246
Quyen DT, Schmidt-Dannert C, Schmid RD (1999) High-level formation of active Pseudomonas cepacia lipase after heterologous expression of the encoding gene and its modified chaperone in Escherichia coli and rapid in vitro refolding. Appl Environ Microbiol 65:787–794
Quyen DT, Schmidt-Dannert C, Schmid RD (2003) High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase. Protein Expr Purif 28:102–110
Salanoubat M, et al (2002) Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415:497–502
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Sullivan ER, Leahy JG, Colwell RR (1999) Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family. Gene 230:277–286
Taipa MA, Liebeton K, Costa JV, Cabral JM, Jaeger KE (1995) Lipase from Chromobacterium viscosum: biochemical characterization indicating homology to the lipase from Pseudomonas glumae. Biochim Biophys Acta 1256:396–402
Wohlfarth S, Winkler UK (1988) Chromosomal mapping and cloning of the lipase gene of Pseudomonas aeruginosa. J Gen Microbiol 134:433–440
Wohlfarth S, Hoesche C, Strunk C, Winkler UK (1992) Molecular genetics of the extracellular lipase of Pseudomonas aeruginosa PAO1. J Gen Microbiol 138:1325–1335
Acknowledgements
This work was supported by the Foundation for Fundamental Sciences (Vietnamese Ministry of Science and Technology) for the fiscal years 2001–2005 and by the Korea Science and Engineering Foundation (KOSEF) 2003
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by H. Ikeda
Rights and permissions
About this article
Cite this article
Quyen, D.T., Nguyen, T.T., Le, T.T.G. et al. A novel lipase/chaperone pair from Ralstonia sp. M1: analysis of the folding interaction and evidence for gene loss in R. solanacearum. Mol Genet Genomics 272, 538–549 (2004). https://doi.org/10.1007/s00438-004-1084-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-004-1084-7