Application 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. A 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. Overall, 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.

中文翻译：

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ARTP / EMS结合多重诱变有效提高了草酸青霉中生淀粉降解酶的生产以及酶高产突变体的表征

生淀粉降解酶（RSDEs）在生物燃料生产淀粉加工中的应用可以有效降低能源消耗和加工成本。RSDE通常由丝状真菌（例如草酸青霉）产生，但收率非常低，严重阻碍了淀粉原料加工的工业化。通过随机诱变进行育种是提高真菌酶产生的有效途径。在多轮诱变后，通过大气和室温血浆（ARTP）和/或甲烷磺酸乙酯（EMS）生成了总计3532个草酸疟原虫菌落。其中，以生木薯粉为底物，一种突变体A2-13的RSDE活性最高，为162.7 U / mL，与起始菌株OXPOxGA15A相比，产率提高了61.1％。A2-13的RSDE活性进一步提高到191.0 U / mL，通过优化培养条件。通过实时逆转录定量PCR检测到主要淀粉酶基因的表达增加，包括原始的降解淀粉的葡糖淀粉酶基因PoxGA15A及其调控基因PoxAmyR以及A2-13基因组中的几个单核苷酸多态性。和基因组重测序。此外，由A2-13产生的粗制RSDE与商业α-淀粉酶结合可以在40°C的温度下有效地消化玉米粗粉和木薯粉。总体而言，ARTP / EMS结合诱变有效提高了真菌RSDE的产量。获得了RSDE高产突变体A2-13，其RSDE可以与商品α-淀粉酶在低温下有效地水解原料淀粉，为将来的淀粉加工提供了有用的RSDE资源。