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| High-level Expression and Characterization of xynA Originated from Anaerobic Fungi Orpinomyces sp. PC-2 in Pichia pastoris |
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Abstract Endo-1,4-β-xylanase (xynA, EC.3.2.1.8), the most important member of xylanolytic enzymes, is widely used in feed, food, papermaking and bioenergy and other fields. The xynA from anaerobic fungi Orpinomyces sp. PC-2 has potential exploitation value, but its enzyme activity of heterologous expression is low. In the present research, according to the codon usage frequency of highly expressed genes in Pichia pastoris, a β-xylanase gene (xynA) derived from anaerobic fungi Orpinomyces sp. PC-2 was optimized, and the modified gene (xynAm) was chemical synthesized and constructed a yeast expression vector pPIC9K-xynAm. Then, the xynAm gene was expressed in Pichia pastoris GS115, and enzymatic properties of the recombinant β-xylanase were characterized. The results showed that the enzyme activity of the recombinant β-xylanase reached the maximum of 612 IU/mL in shake-flask culture at 108 h of induction. In 10 L fermenter, the enzyme activity and specific activity of the recombinant β-xylanase reached the maximum of 3 515 IU/mL and 2 411 IU/mg at 96 h of induction. The dry weight, wet weight of the yeast cells and the protein content in the culture supernatant were reached 324, 156 and 2.25 g/L, respectively, at this time. The molecular weight of the enzyme was about 42.7 kD revealed by non-denatured sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE). Enzymatic properties analysis showed that the optimum pH and reaction temperature of the purification xylanase were 55 ℃ and 6.0, and relatively more stable at 30~50 ℃ and pH 4.0~10.6, and the Km and Vmax were 27.86 mg/mL and 277.78 mg/(mL·min), respectively. Substrate specificity analysis showed that the xylanase could hydrolyze low and high viscosity arabinoxylans, oat spelt xylan, birch xylan and 4-O-methyl-D-glucurono-D-xylan but not barley (Hordeum vulgare) β-glucan and lichenan. The enzyme activity was slightly activated by 10 mmol/L Co2+ and K+, and inhibited by Mn2+, Fe2+ and SDS. This study lays a foundation for further industrial application of the β-xylanase from the anaerobic fungi Orpinomyces sp. PC-2.
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Received: 09 August 2015
Published: 01 April 2016
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