RESUMEN
For applications in food industries, a fungal α-amylase from Malbranchea cinnamomea was engineered by directed evolution. Through two rounds of screening, a mutant α-amylase (mMcAmyA) was obtained with higher optimal temperature (70 °C, 5 °C increase) and better hydrolysis properties (18.6 % maltotriose yield, 2.5-fold increase) compared to the wild-type α-amylase (McAmyA). Site-directed mutations revealed that Threonine (Thr) 226 Serine (Ser) substitution was the main reason for the property evolution of mMcAmyA. Through high cell density fermentation, the highest expression level of Thr226Ser was 3951 U/mL. Thr226Ser was further used for bread baking with a dosage of 1000 U/kg flour, resulting in a 17.8 % increase in specific volume and a 35.6 % decrease in hardness compared to the control. The results were a significant improvement on those of McAmyA. Moreover, the mutant showed better anti-staling properties compared to McAmyA, as indicated by the improved sensory evaluation after 4 days of storage at 4 and 25 °C. These findings provide insights into the structure-function relationship of fungal α-amylase and introduce a potential candidate for bread-making industry.
Asunto(s)
Pan , alfa-Amilasas , alfa-Amilasas/genética , alfa-Amilasas/metabolismo , Hidrólisis , TrisacáridosRESUMEN
A novel α-amylase gene (RmAmyA) from Rhizomucor miehei was cloned and expressed in Pichia pastoris. RmAmyA showed 70% amino acid identity with the α-amylase from Rhizomucor pusillus. A high α-amylase activity of 29,794.2â¯U/mL was found through high cell density fermentation. The molecular mass of RmAmyA was determined to be 49.9â¯kDa via SDS-PAGE. RmAmyA was optimally active at 75⯰C and pHâ¯6.0, and it did not require Ca2+ to improve its activity. It exhibited broad substrate specificity towards amylose, amylopectin, soluble starch, pullulan, and cyclodextrins. High level of maltose (54%, w/w) was produced after liquefied starch was hydrolysed with RmAmyA for 16â¯h. Moreover, the addition of RmAmyA into Chinese steamed bread resulted in 7.7% increment in the specific volume, and 17.2% and 11.5% reduction in the chewiness and hardness, respectively. These results indicate that RmAmyA might be a potential candidate for applications in the food industry.
Asunto(s)
Maltosa/metabolismo , Rhizomucor/enzimología , alfa-Amilasas/metabolismo , Pan/análisis , Industria de Alimentos , Concentración de Iones de Hidrógeno , Hidrólisis , Pichia/metabolismo , Almidón/metabolismo , Especificidad por Sustrato , Temperatura , alfa-Amilasas/química , alfa-Amilasas/genéticaRESUMEN
A novel α-amylase gene (TdAmyA) with an open reading frame of 1431â¯bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314â¯U/mL was obtained with protein content of 28.7â¯mg/mL after 168â¯h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2â¯kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2â¯kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60⯰C and pHâ¯6.5, and was thermostable up to 55⯰C within pHâ¯4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8â¯h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.