Qu Wu, Wang Dingquan, Wu Jie, Chan Zhuhua, Di Wenjie, Wang Jianxin, Zeng Runying
Marine Science and Technology College, Zhejiang Ocean University, Zhoushan, China.
Third Institute of Oceanography, Ministry of Natural Resources, Xiamen, China.
Front Microbiol. 2020 Sep 24;11:574771. doi: 10.3389/fmicb.2020.574771. eCollection 2020.
Bioactivities, such as freshness maintenance, whitening, and prebiotics, of marine neoagaro-oligosaccharides (NAOS) with 4-12 degrees of polymerization (DPs) have been proven. However, NAOS produced by most marine β-agarases always possess low DPs (≤6) and limited categories; thus, a strategy that can efficiently produce NAOS especially with various DPs ≥8 must be developed. In this study, 60 amino acid residues with no functional annotation result were removed from the C-terminal of agarase AgaM1, and truncated recombinant AgaM1 (trAgaM1) was found to have the ability to produce NAOS with various DPs (4-12) under certain conditions. The catalytic efficiency and stability of trAgaM1 were obviously lower than the wild type (rAgaM1), which probably endowed trAgaM1 with the ability to produce NAOS with various DPs. The optimum conditions for various NAOS production included mixing 1% agarose (w/v) with 10.26 U/ml trAgaM1 and incubating the mixture at 50°C in deionized water for 100 min. This strategy produced neoagarotetraose (NA4), neoagarohexaose (NA6), neoagarooctaose (NA8), neoagarodecaose (NA10), and neoagarododecaose (NA12) at final concentrations of 0.15, 1.53, 1.53, 3.02, and 3.02 g/L, respectively. The NAOS served as end-products of the reaction. The conditions for trAgaM1 expression in a shake flask and 5 L fermentation tank were optimized, and the yields of trAgaM1 increased by 56- and 842-fold compared with those before optimization, respectively. This study provides numerous substrate sources for production and activity tests of NAOS with high DPs and offers a foundation for large-scale production of NAOS with various DPs at a low cost.
已证实聚合度（DP）为4 - 12的海洋新琼脂寡糖（NAOS）具有多种生物活性，如保鲜、美白和益生元活性。然而，大多数海洋β-琼脂酶产生的NAOS聚合度较低（≤6）且种类有限；因此，必须开发一种能够高效生产尤其是各种DP≥8的NAOS的策略。在本研究中，从琼脂酶AgaM1的C末端去除了60个无功能注释结果的氨基酸残基，发现截短的重组AgaM1（trAgaM1）在一定条件下有能力产生各种DP（4 - 12）的NAOS。trAgaM1的催化效率和稳定性明显低于野生型（rAgaM1），这可能赋予了trAgaM1产生各种DP的NAOS的能力。生产各种NAOS的最佳条件包括将1%琼脂糖（w/v）与10.26 U/ml的trAgaM1混合，并在50°C的去离子水中孵育混合物100分钟。该策略分别产生了终浓度为0.15、1.53、1.53、3.02和3.02 g/L的新琼脂四糖（NA4）、新琼脂六糖（NA6）、新琼脂八糖（NA8）、新琼脂十糖（NA10）和新琼脂十二糖（NA12）。这些NAOS作为反应的终产物。优化了trAgaM1在摇瓶和五升发酵罐中的表达条件，与优化前相比，trAgaM1的产量分别提高了56倍和842倍。本研究为高DP的NAOS的生产和活性测试提供了大量底物来源，并为低成本大规模生产各种DP的NAOS奠定了基础。
