Elsevier

Process Biochemistry

Volume 45, Issue 3, March 2010, Pages 346-354
Process Biochemistry

Chaperone-dependent gene expression of organic solvent-tolerant lipase from Pseudomonas aeruginosa strain S5

https://doi.org/10.1016/j.procbio.2009.10.008Get rights and content

Abstract

The gene coding for the intracellular organic solvent-tolerant lipase of Pseudomonas aeruginosa strain S5 was isolated from a genomic DNA library and cloned into pRSET. The cloned sequence included two open reading frames (ORF) of 1575 bp for the first ORF (ORF1), and 582 bp for the second ORF (ORF2). The ORF2, known as chaperone, plays an important role in the expression of the S5 gene. The ORF2 is located downstream of lipase gene, and functions as the act gene for ORF1. The conserved pentapeptide, Gly-X-Ser-X-Gly, is located in the ORF1. A sequence coding for a catalytic triad that resembles that of a serine protease, consisting of serine, histidine, and aspartic acid or glutamic acid residues, was present in the lipase gene. Expression of the S5 lipase gene in E. coli resulted in a 100-fold increase in enzyme activity 9 h after induction with 0.75 mM IPTG. The recombinant protein revealed a size of 60 kDa on SDS-PAGE. The Lip S5 gene was stable in the presence of 25% (v/v) n-dodecane and n-tetradecane after 2 h incubation at 37 °C.

Introduction

Many researchers have found that among lipases of various origins (animal, plant and microorganism), those from bacteria, especially from Pseudomonas species, exhibit the highest versatility, reactivity and stability in catalyzing reactions in the organic phase. For example, Ogino et al. [1] and Isken et al. [2], reported that they were able to isolate lipolytic enzymes, from the organic solvent-tolerant Pseudomonas strains LST-03 and S12.

According to Quyen et al. [3], the lipase genes can be divided into three groups, designated classes I to III and based on homology. Class III is only distantly related to the other classes. Pseudomonas lipases of classes I and II, including the broadly used lipases of P. cepacia, and P. glumae (class II), as well as those of P. aeruginosa (class I), need a chaperone; a gene that is located downstream of the lipase gene, for efficient secretion and folding of active lipase. The deduced amino acid sequences of the chaperones for these lipase genes belong to two homology groups.

A variety of lipase-encoding genes from different Pseudomonas sp. have been cloned and sequenced. However, the expression of subfamily I.1 and I.2 Pseudomonas lipases is hampered by the fact that a lipase chaperone is necessary for the correct folding of the proteins to their enzymatically active forms [4].

Previously, we reported on an organic solvent-tolerant lipase secreted by S5, a benzene, toluene, ethyl-benzene and p-xylene (BTEX) degrader strain identified as Pseudomonas aeruginosa. Studies of the effects of nutritional and physical factors on enzyme activity, as well as purification of the enzyme, were also reported [5], [6], [7]. To our knowledge, however, limited studies have reported on the expression of enzymatically active Pseudomonas lipases in E. coli systems. In those reports that are published, expression of the lipases levels are very low without the helper gene or chaperone protein [8].

The cloning and expression of an organic solvent-tolerant lipase, in the presence of the activation gene, was also reported by Ogino et al. [9], [10]; however, no stability test of the lipase in organic solvents was conducted.

In this paper, we report on the cloning of an organic solvent-tolerant lipase from a genomic library, as well as the role of ORF2 as a chaperone in the activation of the S5 lipase gene in Escherichia coli. Furthermore, a test of the organic solvent tolerance of the lipase in the present of lipase-specific foldase was performed.

Section snippets

Bacterial strains and plasmids

Pseudomonas aeruginosa strain S5 was used in this study and grown under the conditions reported by Baharum et al. [5]. The bacterial pure culture was submitted to the German collection of microorganisms and cell culture (DSMZ), and was assigned the accession number DSM 17160, as reported by Rahman et al. [7]. In order to prepare competent cells, a single colony of Escherichia coli Top 10 cells from fresh LB agar was inoculated in LB (5 ml), and incubated at 37 °C with shaking at 200 rpm until an A

Lipase gene isolation by PCR

The genomic DNA from strain S5 was amplified by PCR, using the primers described, with 60 °C as the annealing temperature in 30 cycles. A 900 bp PCR product was detected. The purified PCR product was sent for sequencing. Based on the sequencing analysis, the PCR successfully amplified the lipase gene (964 bp), which showed high homology (91%) with PAO1 Lip C. The size of the PCR product, however, was too small to code for the lipase, with a molecular weight of 60 kDa as reported by Rahman et al. [6]

Effect of IPTG concentration and induction time

The effect of the IPTG concentration in media has been studied by inducing recombinant bacterial cultures with increasing IPTG concentrations. The highest lipase activity (15.1 U/ml) was detected after 6 h induction with 0.75 mM IPTG (data not shown). The lipase activity without any induction by IPTG was 6.5 U/ml, and at 0.25 mM of IPTG the activity was 8.9 U/ml. The lipase activity was enhanced by 70% compared to no induction with 0.5 mM of IPTG. The lipase activity was slightly decreased to 13.9 U/ml

Discussion

In this study, we cloned and expressed a new organic solvent-tolerant lipase from P. aeruginosa strain S5 using a shot-gun cloning approach, after several attempts with PCR cloning failed. Previous failures to express the S5 lipase might have been due to the fact that the Pseudomonas lipase gene needs a helper or activator gene in the E. coli systems. According to Iizumi et al. [14], the Pseudomonas sp. KWI-56 lipase activity was enhanced in E. coli by the function of the activator gene that

Acknowledgements

This work was financially supported by Grant No. 09-02-04-0002 from the Malaysian Ministry of Science, Technology and Innovation (MOSTI). The authors acknowledge the contribution of Robert Coe from the University of Sheffield, United Kingdom, for his help in correcting the English language of the manuscript.

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    Present address: Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.

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