固定化尿苷-胞苷激酶和聚磷酸激酶偶联催化制备5′-胞苷酸

Concomitant use of immobilized uridine-cytidine kinase and polyphosphate kinase for 5′-cytidine monophosphate production

摘要:

尿苷-胞苷激酶作为生物体核苷酸代谢补偿途径中的重要催化剂，可以催化胞苷的磷酸化反应合成5′-胞苷酸 (简称胞苷酸)，但需要NTP作为磷酸供体。为了提高胞苷酸的生产效率，文中首先使用大肠杆菌分别异源表达来源于嗜热栖热菌Thermus thermophiles HB8的尿苷-胞苷激酶和来源于类球红细菌Rhodobacter sphaeroides的聚磷酸激酶，其中尿苷-胞苷激酶用于催化胞苷和ATP形成胞苷酸，聚磷酸激酶则用于ATP的循环再生。然后，使用D403金属螯合树脂吸附Ni2+形成固定化载体，再利用固定化载体特异性吸附重组酶形成固定化酶。最后，单因素优化实验确定固定化酶的催化反应条件，在30 ℃、pH 8.0的条件下，以60 mmol/L胞苷和0.5 mmol/L ATP为底物，可实现5批次的高效连续催化反应，胞苷酸平均摩尔得率达到91.2%。上述制备方法反应成本低，产物得率高，酶利用率高，在工业生产中具有较好的应用潜力。

Abstract:

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5′-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni2+ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.