Abstract：
Rare sugar was defined as a sugar that occurs in very small quantities in nature. Among them, l-ribose and d-tagatose were of high added value and useful as pharmaceutical intermediate for anti-HBV drugs or low calorie sweetener in food industry. Bio-production of the two rare sugar from biomass waste has not been investigated. Hence, development of a feasible and efficient co-production method was of practical usage. However, lack of suitable biocatalyst has become a bottleneck. By sequence alignment and analysis, a C-terminal α-helix from l-arabinose isomerase (L-AI) family was selected as a tool for protein engineering. This α-helix was ligated to C-terminal of Lactobacillus fermentum L-AI (LFAI) and significantly enhanced its thermostability and robustness for both l-arabinose and galactose catalysis. The mutant LFAI-C4 enzyme was immobilized by alginate and antimicrobial peptide poly-l-lysine, and was used to convert pretreated corncob acid hydrolysate (PCAH) into l-ribulose and d-tagatose in the presence of boric acid. In addition, we identified and immobilized a novel thermostable mannose-6-phosphate isomerase from Bacillus subtilis (BsMPI-2) which was efficient in catalyzing retaining l-ribulose into l-ribose and showing no activity on d-tagatose. The dual immobilized enzymes (LFAI-C4 and BsMPI-2) system co-produced 191.9 g/L l-ribose and 80.1 g/L d-tagatose, respectively. Showing a total yield of 46.6% from l-arabinose to l-ribose, which was the highest among reported. The dual immobilized enzymes system preserved 82% activity after 40 batches reaction, showing excellent potentials for industrial use. This study presents a promising alternative for rare sugar production from low-value raw material and showed satisfied conversion rate, product concentration, and operation stability.
摘要：
稀有糖被定义为自然界中非常少量的糖。其中,l-核糖和d-塔格糖具有高附加值，可用作食品工业中抗HBV药物或低热量甜味剂的药物中间体。尚未研究从生物质废物中生物生产两种稀有糖。因此，开发一种可行、高效的联产方法具有实际应用价值。然而,缺乏合适的生物催化剂已成为瓶颈。通过序列比对和分析，选择来自1-阿拉伯糖异构酶（L-AI）家族的C末端α-螺旋作为蛋白质工程的工具。该α-螺旋连接到发酵乳杆菌L-AI（LFAI）的C末端，并显着增强了其热稳定性和对l-阿拉伯糖和半乳糖催化的鲁棒性。突变体LFAI-C4酶被藻酸盐和抗菌肽聚赖氨酸固定，并用于在硼酸存在下将预处理的玉米芯酸水解产物(PCAH)转化为l-核酮糖和d-塔格糖。此外,我们从枯草芽孢杆菌（BsMPI-2）中鉴定并固定了一种新型的热稳定甘露糖-6-磷酸异构酶，该酶可有效催化将l-核糖保留为l-核糖，并且对d-塔格糖没有活性。双固定化酶（LFAI-C4和BsMPI-2）系统共同产生191.9g/L的l-核糖和80.1g/L的d-塔格糖，分别。从l-阿拉伯糖到l-核糖的总收率为46.6%，这是报告中最高的。双固定化酶系统在40批反应后保留了82%的活性，显示出良好的工业应用潜力。这项研究提出了一个有希望的替代稀有糖生产从低价值的原料，并显示出满意的转化率，产品浓度,和运行稳定性。
