In order to amplify the AXE gene from plasmid ER405-6, two primers were designed for PCR. According to agarose gel electrophoresis of the PCR product, one major band was apparent at approximately 800 bp and could be eluted for further processes. The axe coding sequence was cloned into the pGAPZαA vector using the XhoI/XbaI restriction sites as described earlier. This construction allowed axe coding sequence to be theoretically in-frame with the α-factor secretion signal in pGAPZαA. Following a sequence check, the construct, denoted as pGAPZ-axe, was BglII-linearized and electroporated into P. pastoris X-33. Four transformants were selected for high resistance to Zeocin (2,000 μg/mL). The genomic PCR assay revealed that all the transformants contained an integrated axe coding sequence in the genomic DNA. Among these four transformants, the transformant (pGAPZ-axe) could produce the highest AXE activity and was selected for further experiments.

The fermentation conditions for constitutive expression of the AXE were investigated in a 500 mL Hinton flask loaded with 50 mL YPD broth at 28 °C. Transformant (pGAPZ-axe) grew logarithmically from 24 to 48 hours and then entered a stationary phase (Figure 1). The biomass reached approximately 22 of the OD₆₀₀ value after 48-hours incubation. The enzyme production pattern in this transformant indicated that AXE synthesis began in the middle logarithmic phase and continued to be produced during the stationary phase where the maximum activity (526 U/mL) was reached in the culture broth. The AXE activity was about 500-times higher than that observed when expressed from an E. coli transformant [18]. The control strain, P. pastoris (pGAPZαA), could also produce esterase activity under the same culture conditions (data not shown). In the GAP promoter expression system, the cloned heterologous protein will be expressed along with cell growth if the protein is not toxic for the cell [23,24]. Such a result was observed in this study. As shown in Figure 1, the rapid production of the extracellular AXE occurred in parallel with an increase in biomass accumulation of the P. pastoris transformant.

The purification of AXE was performed as described in the Experimental Section. The results of the total purification are summarized in Table 1. The purified enzyme exhibited 0.68% of the total initial activity and there was a 26.1-fold increase in specific activity when compared with the crude culture filtrate. The amount of extracellular proteins in the culture broth of the P. pastoris transformant was fewer than that in the cell free extract of the E. coli transformant. This will facilitate the application of the enzyme in an industrial process immediately without complicated purification procedures. Most of the non-specific esterase activity from the P. pastoris host was lost in the ultrafiltration process, resulting in a decrease of the purification fold.

As shown in Figure 2, after being treated with endo-β-N-acetylglycosaminidase H for deglycosylation, the purified enzyme showed an apparent single protein band on SDS-PAGE (10% gel). The subunit size of the single protein band was estimated to be 28 kDa from its mobility relative to standard proteins by SDS-PAGE. The optimal pH and temperature of the AXE from P. pastoris transformant (pGAPZ-axe) were 8.0 and 60 °C, respectively. About 70% of the original AXE activity remained after heat treatment at 60 °C for three hours. Yang et al. [17] reported that 70% of the AXE activity from T. fusca NTU22 remained after heat treatment at 70 °C for three hours. Apparently the thermostability of the AXE from the P. pastoris transformant is lower than the enzyme from the original strain, T. fusca NTU22.

Thermophilic actinomycetes, T. fusca NTU22, which was isolated from compost soils collected in Taiwan, was used in this study [16]. P. pastoris X-33 and pGAPZαA were purchased from Invitrogen (Sandiego, CA, USA). The plasmid propagation in the expression work was accomplished with E. coli Top 10 (Invitrogen, Sandiego, CA, USA). The plasmid ER405-6 was constructed with the pGEM^®-T Easy Vector and a 800 bp inserted fragment, which possessed an AXE gene axe [18].

Yeast extract, peptone, tryptone and agar were purchased from BD (Sparks, MD, USA). Zeocin was obtained from Cayla (Toulouse, France). Restriction endonucleases and T4 DNA ligation kit were purchased from Roche (Mannheim, Germany). For polymerase chain reactions, the Vio Twin Pack Kits comprising VioTag DNA polymerase, polymerase chain reaction buffer and deoxynucleotides were obtained from Viogene (Sunnyvale, CA, USA). Ni-Sepharose™ High Performance resin was purchased from GE Healthcare (Little Chalfont, UK). PiNK Prestained Protein Ladder was purchased from GeneDirex (Las Vegas, NV, USA). The protein assay kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Endo-β-N-acetylcosaminidase H was purchased from New England Biolabs Inc. (Beverly, MA, USA). Inorganic salts and all other chemicals were purchased from Sigma (St. Louis, MO, USA).

The T. fusca NTU22 AXE gene was amplified using the primers 5′-AATTCTAGACCAAATGGACAGGTGCTGCGATACTCTTC-3′ (XhoI site is underlined) and 5′-AACTCGAGAAACGTGAGGCTGAAGCAGCGGCGAATCCGTATGAACGG-3′ (XbaI site is underlined), using ER405-6 as the template. The amplification was performed using a DNA thermal cycler (Perkin Elmer, MA, USA) with the following conditions: the first step was initiated at 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 65 °C for 30 s and 72 °C for 90 s, and the final extension was carried out at 72 °C for 7 min. The gel-purified PCR product was digested with XhoI and XbaI and cloned into XhoI and XbaI digested pGAPZαA. After being transformed into E. coli Top 10, one recombinant plasmid designated as pGAPZα-axe was selected on low salt LB agar plates (5 g/L yeast extract, 10 g/L tryptone, 5 g/L NaCl, 15 g/L agar, and adjusted pH to 7.5) containing 25 μg/mL Zeocin. The proper insert orientation was checked by restriction analysis and sequencing as described above [25].

Ten micrograms of recombinant plasmid (pGAPZα-axe) was linearized with BglII, and electroporated into P. pastoris X-33 using the following conditions: 1.5 kV, 25 μF, 200 ohm and 5 ms, using a GenePulser (Bio-Rad, CA, USA). The transformants were selected at 28 °C on YPDS agar plates (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, 20 g/L agar and 1 M sorbitol) containing 100 μg/mL Zeocin for 4–6 days. Screening for high level expression transformants was done by replicating the colonies obtained from the YPDS agar plates containing 100 μg/mL Zeocin onto YPDS agar plates with a higher Zeocin concentration (2,000 μg/mL). Transformants with higher Zeocin-resistance were obtained and checked for the integration of the construct into the P. pastoris X-33 genome by genomic PCR [26].

Biomass production of the P. pastoris transformant was evaluated by measuring the optical density at 600 nm (OD₆₀₀ value). Esterase activity was assayed as described by Kodemi et al. [27] with slight modification. We used spectrophotometric analysis with p-nitrophenyl acetate as a substrate; the release of p-nitrophenol was determined at 405 nm. One unit of enzyme activity was defined as the amount of the enzyme releasing 1 μmol of p-nitrophenol per min at 25 °C, pH 8.0.

The transformant resistant to high concentration of Zeocin was incubated in 50 mL YPD broth (10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose) in 500 mL Hinton flasks and shaken (150 rpm) at 28 °C. After the cultivation was carried out for several days, the culture broth was centrifuged at 10,000 × g for 30 min at 4 °C and finally the supernatant was tested for AXE activity.

All purification procedures were done at 4 °C in 20 mM Tris-HCl buffer (pH 7.5) unless otherwise stated. After 72-hours of cultivation of the P. pastoris transformant (pGAPZα-axe) in a 500 mL Hinton flask, the fermentation broth was centrifuged at 3,000 × g for 30 min to remove cells. The supernatant was then applied to a Ni-Sepharose™ High Performance column (1.13 cm × 8 cm) pre-equilibrated with Tris-HCl buffer. After washing with wash buffer (20 mM Tris-HCl, 500 mM sodium chloride, 40 mM imidazole; pH 7.5) to remove the unbound protein, the enzyme was eluted with elution buffer (20 mM Tris-HCl, 500 mM sodium chloride, 500 mM imidazole; pH 7.5). The eluted enzymatically active fractions were pooled and used as the purified enzyme.

The molecular mass of the purified enzyme was determined by using SDS-PAGE (10% polyacrylamide). PiNK Prestained Protein Ladder was used as molecular mass standards. The electrophoresis was carried out at 150 V for 1 h. The gel was stained with 0.27% Coomassie Brilliant Blue R-250, and destained by washing overnight with a mixture of acetic acid-methanol-water (10:20:70, V/V).

The purified enzyme from P. pastoris transformant was deglycosylated by denaturing the glycoprotein at 100 °C for 10 min, and then endo-β-N-acetylglycos-aminidase H was added to perform deglycosylation at 37 °C for 1 h. All manipulations followed the manufacturer’s instructions.