Mannooligosaccharides (MOSs) are one of the most commonly used biomass-derived feed additives. The effectiveness of MOS varies with the length of oligosaccharides, medium length MOSs such as mannotetraose and mannopentaose being the most efficient. This study aims at improving specificity of β-mannanase from Aspergillus niger toward the desirable product size through rational-based enzyme engineering. Tyr 42 and Tyr 132 were mutated to Gly to extend the substrate binding site, allowing higher molecular weight MOS to non-catalytically bind to the enzyme. Hydrolysis product content was analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection. Instead of mannobiose, the enzyme variants yielded mannotriose and mannotetraose as the major products, followed by mannobiose and mannopentaose. Overall, 42% improvement in production yield of highly active mannotetraose and mannopentaose was achieved. This validates the use of engineered β-mannanase to selectively produce larger MOS, making them promising candidates for large-scale MOS enzymatic production process.

甘露寡糖（MOS）是最常用的源自生物质的饲料添加剂之一。MOS的有效性随寡糖的长度而变化，其中中等长度的MOS（例如甘露糖和甘露戊糖）最为有效。本研究旨在提高黑曲霉β-甘露聚糖酶的特异性通过基于理性的酶工程实现理想的产品尺寸。将Tyr 42和Tyr 132突变为Gly以扩展底物结合位点，从而允许更高分子量的MOS非催化结合至酶。通过高效阴离子交换色谱和脉冲安培检测分析水解产物的含量。酶变体以甘露二糖和甘露糖二糖为主要产物，而不是甘露二糖，其次是甘露二糖和甘露戊糖。总体而言，高活性甘露糖和甘露戊糖的产量提高了42％。这验证了使用工程化的β-甘露聚糖酶选择性生产更大的MOS的潜力，使其成为大规模MOS酶促生产工艺的有希望的候选者。