Abstract
Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0–9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s−1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.
Highlights
Plant cell walls are the main polysaccharide-containing renewable resource on earth, and are generated by photosynthesis [1]
The codon usage bias was changed by upgrading the codon adaptation index (CAI) from 0.69 to 0.83, giving a smoother GC content curve and eliminating the negative cis-acting elements
The xynA gene from P. citrinum FERM P-15944 was synthesized with codon optimization for expression in P. pastoris based on the nucleotide database
Summary
Plant cell walls are the main polysaccharide-containing renewable resource on earth, and are generated by photosynthesis [1]. Hemicellulose, a group of heteropolysaccharides, is the main polysaccharide component of plant cell walls, and in most plants, xylan (β-1,4-linked xylose residues with side branches of α-glucuronic acid and α-arabinofuranose) forms the major component and accounts for approximately one third of all renewable organic carbon on earth [2]. As xylan is a complex heteropolysaccharide, the complete conversion of the xylan requires the interaction of several main-chain- and side-chain-cleaving enzymes. Molecules 2019, 24, 3515 play a key role in xylan hydrolysis to xylooligosaccharides, which in turn can be converted to xylose by β-xylosidases [3]. Examples include serving as bio-bleaching agents in the pulp and paper industry [4], improving digestibility and enhancing the efficiency of nutrient utilization in animal feed [5,6], increasing dough volume in the bakery industry [7], clarification of juices [8], improving the filtration rate and reduction of viscosity in the brewing industry [9], and releasing xylose that can be fermented to value products, such as biofuels and xylitol [10,11]
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