The application of agarose oligosaccharides has garnered great attention, with their biological activities varying among different structures. However, it still meets a great bottleneck for the targeted production of odd-numbered neoagarooligosaccharides (NAOSs), such as neoagarotriose (NA3), due to the lack of one-step hydrolases. In this work, the α-agarase AgaA33 and β-galactosidase BgaD were synergistically used to prepare NA3 with agarose as a substrate. Additionally, an l-arabinose isomerase CaLAI from Clostridium acetobutylicum was characterized to valorize low-value byproducts (d-galactose) by forming d-tagatose, which exhibited good thermal stability without the need for additional metal ions. Under the optimal reaction conditions, the production of NA3 and d-galactose catalyzed by these three enzymes was 0.40 and 0.15 g/L, respectively. The artificial three-enzyme-based cascade transformation system not only achieved the highest production of NA3 until now but also allowed for the valorization of d-galactose, providing a wiser application route for agarose utilization.

中文翻译：

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人工级联转化生物系统，用于通过更明智的琼脂糖利用对奇数新琼脂低聚糖和 d-塔加糖进行一罐法生物制造


琼脂糖低聚糖的应用引起了极大的关注，其生物活性在不同结构中有所不同。然而，由于缺乏一步水解酶，它仍然遇到了奇数新琼脂低聚糖 （NAOSs） 靶向生产的巨大瓶颈，例如新琼脂三糖 （NA3）。在本工作中，α-琼脂糖酶 AgaA33 和 β-半乳糖苷酶 BgaD 协同用于制备以琼脂糖为底物的 NA3。此外，来自乙酰丁酸梭菌的 l-阿拉伯糖异构酶 CaLAI 通过形成 d-塔加糖来评估低价值副产物 （d-半乳糖），其表现出良好的热稳定性，而无需额外的金属离子。在最佳反应条件下，这三种酶催化的 NA3 和 d-半乳糖产量分别为 0.40 和 0.15 g/L。基于人工三酶的级联转化系统不仅实现了迄今为止最高的 NA3 产量，而且还允许 d-半乳糖增值，为琼脂糖利用提供了更明智的应用途径。