Insight to Improve α-L-Arabinofuranosidase Productivity in Pichia pastoris and Its Application on Corn Stover Degradation

Abstract

α-L-arabinofuranosidase (ARA) with enhanced specific activity and in large amounts, is needed for a variety of industrial applications. To improve ARA production with engineered methylotrophic yeast Pichia pastoris, a genetically modified ara gene from Aspergillus niger ND-1 was investigated. Through codon optimization and rational replacement of α-factor signal peptide with the native propeptide (MFSRRNLVALGLAATVSA), ARA production was improved from 2.61 ± 0.13 U/mL to 14.37 ± 0.22 U/mL in shaking flask culture (a 5.5-fold increase). Results of N-terminal sequencing showed that secreted active ARA of recombinant strain p-oARA had theoretical initial five amino acids (GPCDI) comparable to the mature sequences of α-oARA (EAEAG) and αp-oARA (NLVAL). The kinetic values have been determined for ARA of recombinant strain p-oARA (Vmax = 747.55 μmol/min/mg, Km = 5.36 mmol/L), optimal activity temperature 60°C and optimal pH 4.0. Scaling up of ARA production by p-oARA in a 7.5-L fermentor resulted in remarkably high extracellular ARA specific activity (479.50 ± 12.83 U/mg) at 168 h, and maximal production rate 164.47 ± 4.40 U/mL. In studies of corn stover degradation activity, degree of synergism for ARA and xylanase was 32.4% and enzymatic hydrolysis yield for ARA + xylanase addition was 15.9% higher than that of commercial cellulase, indicating significant potential of ARA for catalytic conversion of corn stover to fermentable sugars for biofuel production.

Characterization and functional analysis of two novel thermotolerant α-l-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum

“…Under the optimal conditions, enzymes TtABF51A and EpABF62C were stable after incubation at 55 °C for 4 or 6 days, respectively. The two enzymes displayed higher thermal stabilities than many reported ABFs (Hu et al 2018 ; Maehara et al 2014 ; Sarch et al 2019 ; Tu et al 2019 ; Wang et al 2014 ; Zheng et al 2018 ).…”

“…Previous studies have indicated that debranching is a key step in the conversion of hemicellulose into monosaccharides or shorter oligosaccharides (Gao et al 2011 ; Moreira and Filho 2016 ; Zheng et al 2018 ). Endo-xylanase EpXYN1 (GH10 family) is a thermotolerant enzyme from E. parvum and can hydrolyze different xylans into xylobiose, xylotriose, and xylose (Long et al 2018b ).…”

Biochemical characterization of a novel acidophilic β-xylanase from Trichoderma asperellum ND-1 and its synergistic hydrolysis of beechwood xylan

“…The positive single colonies of α-Xyl11, α-oXyl11, and X-33 were incubated in 5 ml YPD at 30°C while shaking at 200 rpm for 12 h. The expression of transformants in buffered methanol-complex (BMMY) was analyzed as reported previously ( Zheng et al, 2018 ). Culture supernatants were examined using 12% SDS-PAGE, stained with Coomassie brilliant blue R-250 (Bio-Rad Laboratories, United States), and de-stained with 100 ml/L ethyl alcohol and 100 ml/L acetic acid.…”

Improved Production of Xylanase in Pichia pastoris and Its Application in Xylose Production From Xylan

“…The resultant recombinant plasmid was transformed into E. coli strain DH5α, as further confirmed by PCR and DNA sequencing. The expression plasmid pPICZαA encoding α-factor signal peptide and MYCTH_56237 were designed to facilitate the construction of variants as described by Zheng et al (2018).…”

“…Moreover, P. pastoris secretes less native proteins and enable the purification of recombinant protein in supernatant easier, through directing signal peptide. To intensify heterologous protein expression in P. pastoris, different procedures have been established, including the optimization of the secretory signal peptide sequence (Zheng et al, 2018). Protein expression levels have been improved with greater success prospects through regulation of the several factors such as recruitment of natural pro-peptides and gene dosage (Aw et al, 2018).…”