Abstract
BackgroundApplication of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs. RSDEs are generally produced by filamentous fungi, such as Penicillium oxalicum, but with very low yields, which seriously hampers industrialization of raw starch processing. Breeding assisted by random mutagenesis is an efficient way to improve fungal enzyme production.ResultsA total of 3532 P. oxalicum colonies were generated after multiple rounds of mutagenesis, by atmospheric and room-temperature plasma (ARTP) and/or ethyl methanesulfonate (EMS). Of these, one mutant A2-13 had the highest RSDE activity of 162.7 U/mL, using raw cassava flour as substrate, a yield increase of 61.1%, compared with that of the starting strain, OXPoxGA15A. RSDE activity of A2-13 further increased to 191.0 U/mL, through optimization of culture conditions. Increased expression of major amylase genes, including the raw starch-degrading glucoamylase gene, PoxGA15A, and its regulatory gene, PoxAmyR, as well as several single-nucleotide polymorphisms in the A2-13 genome, were detected by real-time reverse transcription quantitative PCR and genomic re-sequencing, respectively. In addition, crude RSDEs produced by A2-13, combined with commercial α-amylase, could efficiently digest raw corn flour and cassava flour at 40 °C.ConclusionsOverall, ARTP/EMS-combined mutagenesis effectively improved fungal RSDE yield. An RSDE-hyperproducing mutant, A2-13, was obtained, and its RSDEs could efficiently hydrolyze raw starch, in combination with commercial α-amylase at low temperature, which provides a useful RSDE resource for future starch processing.
Highlights
Application of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs
Avicel stimulated the expression of cellulase and xylanase genes, as well as induction of the pPoxEgCel5B promoter, which controls over-expression of the RSDG gene, PoxGA15A in OXPoxGA15A [15], whereas raw natural cassava flour (RNCF) induced the expression of amylase genes, including RSDE genes
In this study, we carried out ARTP/ethyl methanesulfonate (EMS)-combined mutagenesis to enhance raw starch-degrading enzyme (RSDE) production in P. oxalicum
Summary
Application of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs. A major challenge, for biorefineries, is the high cost of enzymes used for biomass degradation into fermentable sugars, which results in poor profitability [1]. Conventional enzymatic starch processing requires an initial high-temperature liquefaction step, using thermostable α-amylase, followed by saccharification with glucoamylase, after cooling below the starch gelatinization temperature, fermentation to produce bioethanol [4]. Even these enzymatic processes require a large energy input and special equipment, which increases processing costs. The liquefaction step accounts for approximately 30‒40% of the total cost of bioethanol production [5]
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