Identification, Characterization, and Immobilization of an Organic Solvent-Stable Alkaline Hydrolase (PA27) from Pseudomonas aeruginosa MH38

An organic solvent-stable alkaline hydrolase (PA27) from Pseudomonas aeruginosa MH38 was expressed, characterized, and immobilized for biotechnological applications. Recombinant PA27 was expressed in Escherichia coli as a 27 kDa soluble protein and was purified by standard procedures. PA27 was found to be stable at pH 8–11 and below 50 °C. It maintained more than 80% of its activity under alkaline conditions (pH 8.0–11.0). Furthermore, PA27 exhibited remarkable stability in benzene and n-hexane at concentrations of 30% and 50%. Based on these properties, immobilization of PA27 for biotechnological applications was explored. Scanning electron microscopy revealed a very smooth spherical structure with numerous large pores. Interestingly, immobilized PA27 displayed improved thermal/chemical stabilities and high reusability. Specifically, immobilized PA27 has improved thermal stability, maintaining over 90% of initial activity after 1 h of incubation at 80 °C, whereas free PA27 had only 35% residual activity. Furthermore, immobilized PA27 showed higher residual activity than the free enzyme biocatalysts against detergents, urea, and phenol. Immobilized PA27 could be recycled 20 times with retention of ~60% of its initial activity. Furthermore, macroscopic hydrogel formation of PA27 was also investigated. These characteristics make PA27 a great candidate for an industrial biocatalyst with potential applications.


Introduction
Lipolytic enzymes from microorganisms are one of the most important biocatalysts for a wide range of industrial applications in the textile, cosmetic, paper, food, and pharmaceutical industries [1][2][3]. These enzymes are gradually replacing inorganic chemical catalysts in industrial processes due to the high yields of products, strong selectivity of products, and less environmental risks they offer. Specifically, enzymes that can retain their activity in the presence of organic solvents are highly attractive and important, as they can be used in many chemical reactions [4,5].
After an early study on an organic solvent stable lipase from Pseudomonas aeroginosa LST-03 [6], significant efforts have been done to identify novel organic solvent-stable enzymes from Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Bacillus sphaericus. To date, several organic solvent stable lipases were identified and characterized, including LipS [7], SML [8], LipYY31 [9], LC2-8 [10], PseA [11], and 205y lipase [12]. However, the number of organic solvent stable lipases is still limited, and the search for novel enzymes to satisfy the needs of practical applications is of high importance. In the present study, a novel organic solvent-stable alkaline hydrolase (PA27) from Pseudomonas aeruginosa MH38 was identified, characterized, and immobilized. Furthermore, crosslinked enzyme aggregrates and hydrogel formation of this enzyme was also discussed.
To study the catalytic properties of PA27, recombinant PA27 was purified to near homogeneity using Ni-NTA affinity chromatography. As indicated by the single band in the SDS-PAGE gel, the purified protein was homogeneous ( Figure 1A). The hydrolyzing activity of PA toward p-NP esters of different acyl chain lengths was analyzed. As shown in Figure 1B, PA27 displayed a strong preference toward p-nitrophenyl octanoate (p-NPO), although it could not hydrolyze p-nitrophenyl phosphate (p-NP) and very little activity was observed for p-nitrophenyl dodecanoate (p-NPDD).
In addition, the activity profile of PA27 at different pH values was investigated in the range of 2.0-10.0 ( Figure 1C). PA27 displayed its maximal activity at pH 9.0 and ~95% and ~70% of its maximal activity was observed at pH 10.0 and 8.0, respectively. However, only ~20% of the maximal activity was retained at pH 6.0. Activity above pH 10.0 could not be properly measured because of the autohydrolysis of the substrates.
As shown in Figure 1D, PA27 showed ~58% of its initial activity in the presence of 30% (v/v) acetone, and it retained only ~25% activity in the presence of 30% (v/v) isopropanol (i-PrOH). However, PA27 was almost inactive and retained ~3% activity in the presence of 50% acetone or 50% i-PrOH. Interestingly, as shown in Figure 1D, enzyme activity of PA27 was enhanced in benzene and hexane.
The activity of PA27 was as high as ~110% and ~125% of its original activity in the presence of 30% benzene and 50% hexane, respectively. Similar behaviors were also observed in LC2-8 or PseA [10,11,15]. For the enantioselectivity analysis of PA27, a pH shift assay was used with (R)-and (S)-methyl (R)-3-hydroxy-2-methylpropanoate ( Figure 2A). Lipolytic activity was detected based on the color change of the phenol red indicator due to acid release. After incubation with PA27, the color of the reaction mixture turned yellow in (R)-and (S)-enantiomer-containing solutions, which was also confirmed by absorbance spectra readings ( Figure 2B). The results show that PA27 prefers to hydrolyze the (R)-enantiomer when compared to its (S)-enantiomer. The catalytic properties of PA27 were investigated by activity-based native gel staining using glyceryl tributyrate and olive oil. A yellowish color was detected only in solutions containing glyceryl tributyrate, which indicated the formation of hydrolysis products. However, PA27 could not effectively hydrolyze olive oil, because no color changes were developed under these conditions ( Figure 2C,D).
An interesting feature of PA27 is its ability to form hydrogels, which was reminiscent of hen egg white lysozyme [16]. In addition, a variable degree of aggregation was also reported in an extracellular lipase from Pseudomonas aeruginosa EF2 [17].   Figure 3A,B). In the SEM images, the surface of the PA27 hydrogels resembled three-dimensional networks of globular shapes ( Figure 3C,D).

Immobilization of PA27
For immobilization, crosslinked enzyme aggregates of PA27 (CLEA-PA27) were formed by precipitating enzymes in the presence of 80% AMS with glutaraldehyde crosslinking [18]. The SEM images of the immobilized PA27 showed mainly globular structures with high packing density ( Figure 4A,B). For thermostability analysis, soluble PA27 as well as immobilized PA27 were incubated for 1 h at 70 °C and the enzyme activity was measured at various time intervals. After 1 h of incubation, free PA27 showed ~30% of its initial activity, while immobilized PA27 retained most of its initial activity ( Figure 4C). The effects of several chemicals on the activity of immobilized PA27 and soluble PA27 were compared ( Figure 4D). As expected, immobilized PA27 showed more resistance against these chemicals (i.e., Triton X-100, Tween 20, SDS, urea, and phenol) than soluble PA27. As shown in Figure 4D, immobilized PA27, compared to soluble PA27, was appreciably stable in the presence of Triton X-100 and Tween 20. SDS was found to be a strong inhibitor of PA 27. Specifically, 1.0% (v/v) SDS almost completely deactivated soluble PA27, even though immobilized PA27 retained as high as ~40% of its initial activity. Moreover, in the presence of 5 M urea, soluble PA27 showed only ~30% of its activity compared to ~105% of the immobilized PA27. Therefore, the immobilization of PA27 could effectively protect the enzymes from inactivation for a variety of industrial applications. For reusability, immobilized PA27 was reused repeatedly for 20 sequential cycles, retaining ~60% of its initial activity (Table 1).

Cloning and Purification of PA27
PA27 gene was amplified from the chromosomal DNA of P. aeruginosa MH 38 by polymerase chain reaction (PCR). Restriction enzyme sites were added (5′-NdeI and 3′-XhoI) to allow subcloning into the pET-21a expression vector. After DNA sequencing, the resulting plasmid (pET-PA27) was transformed to express PA27 in E. coli BL21 (DE3) cells. A single colony of E. coli BL21 (DE3) was inoculated into LB medium containing ampicillin (100 µg/mL) and incubated at 37 °C until OD 600 reached ~0.6. Then, isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, and bacteria were further cultured for 4 h. The bacterial cells were harvested and sonicated in cell lysis buffer (20 mM Tris-HCl (pH 8.0), 100 mM sodium chloride, and 20 mM imidazole). After centrifugation at 6000 rpm for 20 min at 4 °C, the supernatants were loaded onto a Ni-NTA column followed by extensive washing with cell lysis buffer. PA27 were then eluted with column buffer (20 mM Tris-HCl (pH 8.0), 100 mM sodium chloride, and 100 mM imidazole), and desalted with storage buffer (20 mM Tris-HCl (pH 8.0)) using a PD-10 column. The purity of recombinant PA27 was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final protein was stored at −20 °C without further modifications.

Immobilization of PA27
PA27 was immobilized by forming insoluble enzyme aggregates with ammonium sulfate (AMS) and crosslinking with glutaraldehyde [21,22]. Specifically, PA27 was precipitated with 80% (w/v) AMS at 4 °C. Then, 20 mM (final concentration 0.5 mM) glutaraldehyde was added and incubated for 12 h at 4 °C. The resulting suspension (immobilized PA27) was centrifuged at 12,000 rpm for 10 min, and washed five times with 20 mM Tris-HCl (pH 8.5) until enzyme activity was no longer detected in the supernatant. The activity of immobilized PA27 was determined by monitoring the hydrolysis of p-nitrophenyl octanoate (C 8 , p-NO). Precipitating soluble PA27 with AMS yielded ~75% residual activity compared with initial activity of PA27, which was also close to the reported data for other proteins [23,24]. Longer crosslinking time (up to 24 h) did not lead to any further changes in activity recovery. A scanning electron microscope (SUPRA 55VP, Carl Zeiss, Jena, Germany) was used to examine the surface morphology of the immobilized PA27. To determine the thermostability of the immobilized PA27 and soluble PA27, each form was incubated for 1 h at 80 °C. Aliquots of each sample were removed every 15 min and the residual activity was determined by monitoring the hydrolysis of p-nitrophenyl octanoate (C 8 , p-NO). The effects of chemicals (Triton X-100, Tween 20, SDS, urea, and phenol) on the activity of the immobilized PA27 and soluble PA27 were investigated after 1 h of incubation. The chemical stabilities were then determined by measuring the residual activity. The initial activity was defined as 100%. For reusability tests, the immobilized PA27 was recovered by centrifugation after each reaction and washed five times with 500 µL of 20 mM Tris-HCl (pH 8.5) until no significant enzyme activity was detected in the supernatant. Then, fresh enzyme substrate was added for another reaction. The immobilized PA27 was used for 20 cycles and these experiments were repeated three times.