High-level Heterologous Expression And Characterization Of A Pseudomonas Fluorescens Lipase

Lipases catalyze ester synthesis and transesterification reactions with high regional and stereo selectivity in non-aqueous solvent systems. These features make lipases play an important role in the function of detergent additives, fine chemical synthesis, pharmaceutical and agrochemical productions. In non-aqueous media or organic solvent water two-phase system, organic solvent tolerant Pseudomonas lipase retained catalytic activities. A number of potential applications of lipases that are either impossible or marginal in water become quite feasible and commercially attractive in other solvents. Unfortunately, the heterogeneous expressions of Ps. lipases in E. coli led to cytoplasmic accumulation as inactive inclusion bodies, and the refolding procedure is necessary to get the active enzymes. Therefore, to increase lipase productivity in a soluble and bio-active form in a biochemically safe and economic expression system such as E. coli to an dissolvable enzymatically active form is always tremendously attracts people's attention among scientists and industrialists. Nevertheless, a high-level expression of active Ps. lipases in E. coli has not yet been reported so far. The present thesis describes the cloning, heterogeneous expression, purification and characterization of an new organic solvent tolerant lipase (PFL) of a Pseudomonas fluorescens JCM5963. In addition, special purification procedures have been developed. The characteristics of the purified enzyme were also described. The recombinant lipase possessed some various advantages existed in many other Ps. lipases, and high expression level were achieved. Great recovery was got through ammonium sulfate fractionations and the resolvation used anionics surfactant (Deoxycholic scid sodium salt) or low concentration denaturant (4 M Urea). This work may be of great significance to the increased biotechnological application in organic synthesis by supplying efficient and stable biocatalyst and have potential in industrial applications. The results are as follows: 1. Cloning and expression of the gene of PFL296A band of about 900 bp length was amplified from the chromosomal DNA of Ps. fluorescens 5963. Sequencing and gene analysis indicated an ORF of 891 bp, a full-length lipase gene motif encoding a polypeptide of 296aa residues. The nucleotide sequence data reported here were submitted to GenBank with the accession number EU310372. The translated amino acid sequence was submitted to Esther and NCBI database with the accession number ABY26520.The amplified fragments were inserted in pET28b(+) vector according to the manufacturer's instructions, and the recombinant plasmid was transformed into E. coli BLP for protein expression. The transformed strains were grown in a modified 2YT medium at 37℃until the OD600 reached 1.5. Protein expression was induced by adding IPTG to a final concentration of 0.05 mM, and then the culture was shaken for 6 h at 20℃and the cells were harvested by centrifugation. The results indicate that the recombinant protein reached to near 25% of the total protein in the cells, and the activity reaches 10 U/g wet cells using p-Nitrophenyl caprylate as substrate under 50℃.2. Purification and characterizition of recombinant PFL (rPFL)During the course of purification, an important phenomenon was observed as that rPFL was easy to form aggregates just like other members of family I lipases, therefore most of the rPFL existed in cell debris as active aggregates. This discovery is of great significance for the further expression research of Ps. lipases in an efficient and economic expression system, for this is the first time that a Ps. lipase was successfully expressed in E. coli as active form at such a high level. Considering all the results above, we conclude that these lipases exhibit an intrinsic folding capability in vitro without chaperon protein, which supports the hypothesis put forward by I. Beacham et al., that is, these subfamily I.1 lipases might be secreted via a signal peptide-independent pathway.Considering the property of forming large molecular mass aggregates, a reverse procedure of purification was taken. The supernatant and precipitate of ultrasonicated cells were treated, respectively. The ammonium sulfate fractionations was applied to the supernatant part, and cell debris were removed by washing twice with small amount of buffer, while the precipitate part were incorporated and dissolved in anionics surfactant (Deoxycholic scid sodium salt) or low concentration denaturant (4 M Urea) by mild shaking at 4℃over night. Those treatments greatly simplified the process of purification. A distinct convenient purification was achieved. Finally, Ni-NTA affinity chromatography gave a production of 8.0 mg rPFL per g of wet cells, with a purity of over 95% target protein and a specific activity of 435 U/mg. Apparent molecular mass of purified enzyme estimated by native polyacrylamide gel electrophoresis and gel filtration chromatography also indicates that there is little monomer found in the enzyme solution even at a low concentration.3.characterizition of recombinant PFL (rPFL)The results of characterization of purified enzyme indicate that rPFL was stable in broad ranges of temperatures and pH values, with the optimal temperature and pH value being 55℃and 8.8, respectively. The highest hydrolytic activity of rPFL is obtained to the substrate of pNPC8. The kinetic constants Km values was 142μM and kcat values was 26 681.2 s-1. Activity detection using olive oil and tributyrin emulsion as substrate demonstrates rPFL exhibits hydrolysis activity of 156 U/mg and 215 U/mg, respectively, which suggests that rPFL can be defined as a true lipase. Its activity was found to increase in the presence of metal ions such as Ca2+, Sn2+and some surfactants. On the contrary, Zn2+ and high concentration Ca2+ inhibits activity strongly. EDTA inhibits the activity of rPFL by 40%. Anionic surfactant SDS completely inhibits the hydrolytic activity of rPFL. The purified recombinant lipase not only exhibits high activity in wide ranges of temperatures and pH values, but also behaves quite stable under alkaline, moderate temperature conditions and even in the presence of some surfactants. The most important feature of rPFL is the high activity and extreme stability against a variety of organic solvents.The rPFL was also stable in the presence of some water-miscible organic solvents, such as water-miscible organic solvents including isopropanol and acetone ,and highly tolerant to some water-immiscible organic solvents. All these features render this novel lipase attraction for biotechnological applications in the field of organic synthesis and detergent additives.