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.
Highlights
Second-generation ethanol production is based on plant biomass degradation
Our sequence analysis indicates that ara gene (Gene ID: MH023278) from A. niger ND-1 (GenBank Accession number MH137707) has length 1500 bp
Information available on the NCBI and CAZy database indicates that the characterized ARA belongs to GH54
Summary
Second-generation ethanol production is based on plant biomass degradation. The primary type of plant biomass is cellulose, hemicellulose and lignin (Saha, 2000). The CAZy database uses sequencebased means to classify enzyme families. The enzymes ARAs or arabinases degrade the complex polysaccharides or arabino-oligosaccharides and liberate L-arabinose (McKee et al, 2012). ARAs cleave α-(1,2), α-(1,3), or α-(1,5) linked L-arabinofuranosyl residues from polysaccharides or oligosaccharides containing arabinose (Bouraoui et al, 2016). In the CAZy database, ARAs are categorized into the different families of GH, GH2, GH3, GH43, GH51, GH54, and GH62 (Wilkens et al, 2017). GH54 contains several ARAs reported to act synergistically with various cell wall-degrading enzymes (Lagaert et al, 2014)
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