来自 subsp. DSM 2910 的耐热 α-L-鼠李糖苷酶的异源表达和特性及其在芦丁生物转化中的应用。
Heterologous Expression and Characterization of a Thermostable α-L-Rhamnosidase from subsp. DSM 2910 and Its Application in the Biotransformation of Rutin.
机构信息
Department of Medical Science and Technology, Suzhou Chien-Shiung Institute of Technology, 1 Jian Xiong Road, Taicang 215411, P.R. China.
Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, P.R. China.
出版信息
J Microbiol Biotechnol. 2023 Nov 28;33(11):1521-1530. doi: 10.4014/jmb.2305.05032. Epub 2023 Jul 21.
An α-L-rhamnosidase gene from . subsp. DSM 2910 (TstRhaA) was cloned and expressed. The maximum TstRhaA activity of the protein reached 25.2 U/ml, and the molecular mass was approximately 106.6 kDa. The protein was purified 8.0-fold by Ni-TED affinity with an overall recovery of 16.6% and a specific activity of 187.9 U/mg. TstRhaA activity was the highest at 65°C and pH 6.5. In addition, it exhibited excellent thermal stability, better pH stability, good tolerance to low concentrations of organic reagents, and high catalytic activity for -nitrophenyl-α-L-rhamnopyranoside (NPR). Substrate specificity studies showed that TstRhaA exhibited a high specific activity for rutin. At 60°C, pH 6.5, and 0.3 U/ml enzyme dosage, 60 g/l rutin was converted to 45.55 g/l isoquercitrin within 150 min. The molar conversion rate of rutin and the yield of isoquercitrin were 99.8% and 12.22 g/l/h, respectively. The results suggested that TstRhaA could be used for mass production of isoquercitrin.
从. subsp. DSM 2910（TstRhaA）克隆并表达了一种 α-L-鼠李糖苷酶基因。该蛋白的最大 TstRhaA 活性达到 25.2 U/ml，分子量约为 106.6 kDa。该蛋白通过 Ni-TED 亲和纯化 8.0 倍，总回收率为 16.6%，比活为 187.9 U/mg。TstRhaA 的活性在 65°C 和 pH 6.5 时最高。此外，它表现出出色的热稳定性、更好的 pH 稳定性、对低浓度有机试剂的良好耐受性以及对 -硝基苯基-α-L-鼠李吡喃糖苷（NPR）的高催化活性。底物特异性研究表明，TstRhaA 对芦丁具有很高的比活性。在 60°C、pH 6.5 和 0.3 U/ml 酶剂量下，60 g/l 芦丁在 150 min 内转化为 45.55 g/l 异槲皮苷。芦丁的摩尔转化率和异槲皮苷的产率分别为 99.8%和 12.22 g/l/h。结果表明，TstRhaA 可用于异槲皮苷的大规模生产。
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