Expression of an endo-β-1,4-glucanase gene from orpinomyces PC-2 in Pichia pastoris

Abstract

The endo-β-1,4-glucanase gene celE from the anaerobic fungus Orpinomyces PC-2 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K, and integrated into the genome of a methylotrophic yeast P. pastoris GS115 by electroporation. The strain with highest endo-β-1,4-glucanase activity was selected and designed as P. pastoris egE, and cultivated in shaking flasks. The culture supernatant was assayed by SDS-polyacrylamide gel electrophoresis and showed a single band at about 52 kDa. Furthermore, the recombinant P. pastoris egE was proved to possess the ability to utilize sodium carboxymethyl cellulose as a carbon source. The recombinant endoglucanase produced by P. pastoris showed maximum activity at pH 6.0 and temperature 45 °C, indicating it was a mesophilic neutral endo-β-1,4-glucanase, suitable for denim biofinishing/washing. Further research was carried out in suitable fermentation medium in shaking flasks. The most favorable methanol addition concentration was discussed and given as 1.0%. After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1). This is the first report on expression and characterization of endo-β-1,4-glucanase from Orpinomyces in P. pastoris. The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.

Keywords: Pichia pastoris; endo-β-1,4-glucanase; heterologous expression; induction medium; neutral cellulase.

Figure 1.

(a) SDS-PAGE analysis of the culture supernatant of recombinant P. pastoris strain egE. Lane 0: the samples of P. pastoris transformed with pPIC9K at 24 h of cultivation; Lanes 1–2: the samples of P. pastoris strain egE taken at 24 h, 12 h of cultivation, respectively. Lane M: the protein molecular marker; (b) CMC utilization by recombinant P. pastoris. The P. pastoris strain harboring pPICE (1) or empty plasmid pPIC9K (2) was inoculated in YSM medium to examine the CMC utilization. The CMC-containing plate was incubated at 30 °C for 60 h; (c) PCR verification of the celE gene integrated in the chromosome of P. pastoris egE. Lane 1–4: 1, 4, 6, 10 generations of the P. pastoris egE; Lane 0: P. pastoris transformed with pPIC9K. Lane M: DNA marker.

Figure 2.

Effects of pH (a) and temperature (b) on EG activity for P. pastoris transformant egE. Each value is means of three replicates. Error bars indicate standard error.

Figure 3.

Sequence alignment between Thermoascus auranticus endoglucanase (T-Cel5) and Orpinomyces PC-2 CelE (O-CelE). The eight strictly conserved residues among GH5 endoglucanases are shown in red. The catalytic proton donor (E133 in T-Cel5) and the nucleophile (E-240 in T-Cel5) are shown in red and underlined. Other identical amino acids between the two proteins are also shown and similar amino acid residues are indicated with the + symbol.

Figure 4.

Effect of methanol addition concentration on CelE enzyme production in P. pastoris GS115 transformed with pPICE plasmid. The enzyme activity was detected in the liquid BCG medium. Methanol was supplemented every 24 h to a final concentration of 0.5% (squares), 1.0% (circles) or 1.5% (up-triangles). The BMMY medium induced by 0.5% methanol was taken for comparison (down-triangles). Data shown are the means of three independent experiments for each strain.