State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China; International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu 214122, China.
Department of Food, Nutrition and Home Sciences, National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan.
Int J Biol Macromol. 2020 Mar 1;146:965-975. doi: 10.1016/j.ijbiomac.2019.09.221. Epub 2019 Nov 25.
L-fuculose is a rare sugar that is useful for the agriculture and medicine industries. L-fucose isomerase (E.C.5.3.1.25), which is an aldose-ketose isomerase, plays a significant role in producing rare sugars. A recommended L-fucose isomerase gene was cloned from Caldanaerobius polysaccharolyticus and purified with a single band of 65 kDa using nickel-affinity chromatography, with a specific activity of 108.23 U mg. The native molecular mass existed with 214 kDa was a trimer. The purified enzyme showed a maximum activity in 1 mM Mn at 55 °C and pH 6.5 with a melting temperature (T) of 80.3 °C in the presence of one molecule per monomer. L-fucose isomerase from C. polysaccharolyticus (Capo-LfIase) exhibited the highest activity of L-fucose with K, k and K/k values of 94.2 mM, 23854 min and 253.3 min mM, respectively. Capo-LfIase showed more than 50% thermostability after 20 h of incubation at 45, 55, 65, 75 and 85 °C. The 9 putative active site residues of the L-fucose substrate were described using a homology model, and the results showed that Tyr440, Met185, Trp499 and Asn527 are the candidates of metal-binding residues, while Ser393, Glu337, Glu302, His528 and Asp361 would be involved in substrate binding. The conversion rate of L-fuculose from L-fucose was almost 28.2%, with 80 g L L-fucose, and no byproduct was found. To the best of our knowledge, Capo-LfIase produces high yield of L-fuculose from L-fucose by enzymatic methods.
L-岩藻糖是一种在农业和医药行业都很有用的稀有糖。L-岩藻糖异构酶（E.C.5.3.1.25）是一种醛糖-酮糖异构酶，在生产稀有糖方面发挥着重要作用。本文从多粘厌氧杆菌中克隆出一个推荐的 L-岩藻糖异构酶基因，并用镍亲和层析法纯化出一个 65 kDa 的单带，比活为 108.23 U mg。天然存在的 214 kDa 三聚体的分子质量。该酶的最适反应温度和 pH 分别为 55°C 和 6.5，在 1 mM Mn 存在下，其最大活性为 80.3°C，每个单体有一个熔解温度（T）。多粘厌氧杆菌来源的 L-岩藻糖异构酶（Capo-LfIase）对 L-岩藻糖表现出最高的活性，其 K、k 和 K/k 值分别为 94.2 mM、23854 min 和 253.3 min mM。Capo-LfIase 在 45、55、65、75 和 85°C 下孵育 20 小时后，仍保持超过 50%的热稳定性。使用同源模型描述了 L-岩藻糖底物的 9 个可能的活性位点残基，结果表明 Tyr440、Met185、Trp499 和 Asn527 是金属结合残基的候选者，而 Ser393、Glu337、Glu302、His528 和 Asp361 则参与底物结合。用 80 g L L-岩藻糖作为底物时，L-岩藻糖转化为 L-岩藻酮的转化率几乎达到 28.2%，且没有发现副产物。据我们所知，Capo-LfIase 通过酶法从 L-岩藻糖生产出高产量的 L-岩藻酮。
