Polymer-Degrading Enzymes of Pseudomonas chloroaphis PA23 Display Broad Substrate Preferences

Abstract

Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium Pseudomonas chlororaphis PA23. We cloned these genes into Escherichia coli and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES).

Keywords: PHA depolymerase; Pseudomonas chlororaphis; biodegradation; lipase; poly(ε-caprolactone); polyethylenesuccinate; polyhydroxyalkanoate; polylactic acid.

Figure 1

Expression and purification profiles (12% SDS-PAGE) of recombinant LIP3, LIP4, and PhaZ. (A) Cell lysates of Escherichia coli BL21 (DE3) transformed with pET28a-phaZ/lip3/lip4 Lane 1–3, cell lysate of uninduced E. coli BL21 (DE3) carrying phaZ-pET28a, lip3-pET28a and lip4-pET28a; Lane 4, 5, 6, the lysate of the induced host cells containing phaZ-pET28a, lip3-pET28a and lip4-pET28a expressing PhaZ, LIP3, and LIP4, respectively.(B) SDS-PAGE-purification profile after His–Tag affinity chromatography: PhaZ (Lane 1), LIP3 (Lane 2), and LIP4 (Lane 3); Lane M, standard molecular weight marker (Thermo Fisher Scientific, Waltham, MA, USA).

Figure 2

PHA agar plate assay for testing the depolymerase/esterase activity of LIP3, LIP4, and PhaZ. The clearance zones around the wells to which 20 µg of purified LIP3, LIP4, and PhaZ enzymes were added show hydrolysis of mcl-PHA in rhodamine B-PHO agar plates after UV irradiation. As a control, a crude extract of the noninduced expression host E. coli BL21(DE3) was used.

Figure 3

Amino acid sequence alignment of LIP3 with lipases from Proteus mirabilis HI4320 (3W9U) and Pseudomonas aeruginosa (1EX9). Brown box: amino acids of the lipase box consensus sequence; Blue box: amino acid residues of the oxyanion hole; green box: amino acid residues of the closing lid domain; blue stars: show the positions of the amino acids of the catalytic triad (serine, glutamic acid, and histidine); secondary structure α-helix and β-sheet regions are shown alone at the top of the alignment. White letters with red highlights represent amino acid residues that are conserved in all the enzyme sequences and red letters in blue frame indicate conserved regions with identical amino acid residues (residues with similar properties) in one or two of the enzyme sequences.

Figure 4

Amino acid sequence alignment of LIP4 with the alpha/beta hydrolase fold-3 domain protein from Pseudomonas sp. ECU1011 (4OB8) and lipase of Thalassospira sp. GB04J01 (4V2I). Brown box: amino acids of the consensus sequence of the lipase box; blue box: amino acid residues of the oxyanion hole; green box: amino acid residues of the closing lid domain; blue asterisks: the positions of the amino acids of the catalytic triad (serine, glutamic acid, and histidine) in the sequence; the secondary structure segments of the α-helix and the β-sheet are shown alone at the top of the alignment. White letters with red highlights represent amino acid residues that are conserved in all the enzyme sequences and red letters in blue frame indicate conserved regions with identical amino acid residues (residues with similar properties) in one or two of the enzyme sequences.

Figure 5

Amino acid sequence alignment of PhaZ with the serine hydrolase CCSP0084 from Cycloclasticus sp. P1 (4I3F). Brown box: amino acids of the Lipase box consensus sequence; blue box: amino acid residues of the oxyanion hole; blue asterisks indicate the positions of the amino acids of the catalytic triad (serine, glutamic acid, and histidine); the sequence parts corresponding with α-helix and β-sheet regions are shown alone at the top of the alignment. White letters with red highlights represent amino acid residues that are conserved in all the enzyme sequences and red letters in blue frame indicate conserved regions with identical amino acid residues (residues with similar properties) in one or two of the enzyme sequences. The grey asterisks above amino acid residues 67, 88, 126, and 192 of both sequences, 75 and 126 of the PhaZ sequence, 174 of the 4I3F/A sequence, indicate positions with amino acids with similar properties.

Figure 6

Determination of the parameters of enzymatic activity of LIP3, LIP4, and PhaZ. The effects of (A) pH and (B) temperature on the activity of LIP3 (red line), LIP4 (blue line), and PhaZ (green line). Recombinant LIP3 was optimally active at pH 8.0 and 45 °C, LIP4 at pH 6.0 and 50 °C, and PhaZ showed maximum activity at pH 8.0 and 50 °C. The observed maximum activity was taken as 100%. (C) The red line shows the effect of temperature on the stability of LIP3; the blue line the effect of temperature on the stability of LIP4; the green line shows the effect of temperature on the stability of PhaZ. Thermal stability of enzymes was measured over a period of 24 h (h) at various temperatures (30–60 °C). As a control, the activity of the untreated enzyme (0.025 mg/mL) was set at 100%.

Figure 7

Determination of the activities of LIP3, LIP4, and PhaZ towards different substrates. Substrate specificity of lipases LIP3 (red bars), LIP4 (blue bars), and PhaZ (green bars) from P. chlororaphis PA23. The reaction mixture contained 1 mmol L⁻¹ of various p-nitrophenyl (pNP) alkanoates and 0.025 mg of purified enzyme under optimal conditions of the enzymes. The maximum activity observed was taken as 100%. The 100% activity for LIP3, LIP4, and PhaZ corresponded to 561.87 U mg⁻¹, 453.72 U mg⁻¹, and 860.13 U mg⁻¹ for pNP-octanoate, respectively.

Figure 8

Gel Permeation Chromatography (GPC) analyses of LIP3. The overlay of chromatograms of different polymer substrates before (control = black line) and after LIP3 treatment (red line). (A) PHBV; (B) PHHx; (C) PHO; (D) PHN; (E) PHD; (F) PLA; (G) PCL; and (H) PES. Numbers indicate the change in polymer molecular weight before and after treatment. mV, the HPLC Refractive Index Detector records the signa intensity in millivolts.

Figure 9

Gel Permeation Chromatography (GPC) analyses of LIP4. The overlay of chromatograms of different polymer substrates before (control = black line) and after LIP4 treatment (red line). (A) PHBV; (B) PHHx; (C) PHO; (D) PHN; (E) PHD; (F) PLA; (G) PCL; and (H) PES. Numbers indicate the change in polymer molecular weight before and after treatment. mV, the HPLC Refractive Index Detector records the signa intensity in millivolts.

Figure 10

Gel Permeation Chromatography (GPC) analyses of PhaZ. The overlay of chromatograms of different polymer substrates before (control = black line) and after PhaZ treatment (red line). (A) PHBV; (B) PHHx; (C) PHO; (D) PHN; (E) PHD; (F) PLA; (G) PCL; and (H) PES. Numbers indicate the change in polymer molecular weight before and after treatment. mV, the HPLC Refractive Index Detector records the signa intensity in millivolts.