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Expression of a novel α-glucosidase from Aspergillus neoniger in Pichia pastoris and its efficient recovery for synthesis of isomaltooligosaccharides
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2020-11-01 , DOI: 10.1016/j.enzmictec.2020.109653
Sandeep Kumar , Sarma Mutturi

A gene conferring α-glucosidase (AG) with high transglycosylation activity from Aspergillus neoniger (a non-niger strain belonging to section Nigri) was cloned and expressed in Pichia pastoris. As the cDNA construction retained intronic portions due to alternative splicing, the exonic portions of the gene were stitched using restriction digestion and overlap extension PCR. Pre-determined open-loop exponential feeding strategies were evaluated for methanol dosage to improve the recombinant enzyme synthesis during high-cell density cultivation in 5 L bioreactor. Specific growth rate of 0.1 h-1 resulted in the highest enzyme activity of 182.3 mU/mL in the supernatant, whereas the activity of 3.8 U/g dry cell weight was obtained in the cell pellet. There was negligible enzyme activity in the cell lysate, indicating that approximately 80 % accumulation of total enzyme is in the periplasm. Later, this unreleased fraction was extracted to 90 % yield using 25 mM cysteine. The enzyme was purified and validated using western blot analysis and MS/MS profile. The SDS PAGE analysis revealed three bands corresponding to 80, 38, and 33 kDa indicating the multimeric nature of the enzyme. Thus, obtained enzyme was utilized in synthesis of a potential prebiotic molecule, isomaltooligosaccharides (IMOs), which can be used as a sweetener and bulk filler in the food industry. This is the first report to demonstrate challenges in cloning and expression of transglycosylating α-glucosidase from Aspergillus neoniger.

中文翻译:

新型黑曲霉α-葡萄糖苷酶在毕赤酵母中的表达及其合成低聚异麦芽糖的高效回收

在毕赤酵母中克隆并表达了来自新黑曲霉(属于 Nigri 部分的非黑曲霉菌株)的具有高转糖基化活性的 α-葡萄糖苷酶 (AG) 的基因。由于 cDNA 构建由于选择性剪接而保留了内含子部分,因此使用限制性消化和重叠延伸 PCR 缝合基因的外显子部分。评估了预先确定的开环指数补料策略的甲醇剂量,以改善在 5 L 生物反应器中高细胞密度培养过程中的重组酶合成。0.1 h-1 的比生长速率导致上清液中最高的酶活性为 182.3 mU/mL,而在细胞沉淀中获得 3.8 U/g 细胞干重的活性。细胞裂解液中的酶活性可以忽略不计,表明大约 80% 的总酶积累在周质中。随后,使用 25 mM 半胱氨酸将该未释放部分提取至 90% 的产率。使用蛋白质印迹分析和 MS/MS 谱对酶进行纯化和验证。SDS PAGE 分析揭示了对应于 80、38 和 33 kDa 的三个带,表明酶的多聚体性质。因此,获得的酶被用于合成一种潜在的益生元分子,即低聚异麦芽糖 (IMO),该分子可用作食品工业中的甜味剂和散装填料。这是第一份证明从新黑曲霉克隆和表达转糖基化 α-葡萄糖苷酶面临挑战的报告。使用蛋白质印迹分析和 MS/MS 谱对酶进行纯化和验证。SDS PAGE 分析揭示了对应于 80、38 和 33 kDa 的三个带,表明酶的多聚体性质。因此,获得的酶被用于合成一种潜在的益生元分子,即低聚异麦芽糖 (IMO),该分子可用作食品工业中的甜味剂和散装填料。这是第一份证明从新黑曲霉克隆和表达转糖基化 α-葡萄糖苷酶面临挑战的报告。使用蛋白质印迹分析和 MS/MS 谱对酶进行纯化和验证。SDS PAGE 分析揭示了对应于 80、38 和 33 kDa 的三个带,表明酶的多聚体性质。因此,获得的酶被用于合成一种潜在的益生元分子,即低聚异麦芽糖 (IMO),该分子可用作食品工业中的甜味剂和散装填料。这是第一份证明从新黑曲霉克隆和表达转糖基化 α-葡萄糖苷酶面临挑战的报告。在食品工业中可用作甜味剂和散装填料。这是第一份证明从新黑曲霉克隆和表达转糖基化 α-葡萄糖苷酶面临挑战的报告。在食品工业中可用作甜味剂和散装填料。这是第一份证明从新黑曲霉克隆和表达转糖基化 α-葡萄糖苷酶面临挑战的报告。
更新日期:2020-11-01
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