基于壳聚糖墨水3D打印固定化腺苷酸激酶及其ATP再生应用  

3D Bioprinting of Immobilized Adenylate Kinase Using Chitosanbased Bioink and Its Application in ATP Regeneration

作　　者：张哲扬 王健杰 刘长军 ZHANG Zheyang;WANG Jianjie;LIU Changjun(School of Life and Health Sciences,Hunan University of Science and Technology,Xiangtan 411201,Hunan,China;Key Laboratory of Genetic Improvement and Multiple Utilization of Economic Crops in Hunan Province,Hunan University of Science and Technology,Xiangtan 411201,Hunan,China)

机构地区：[1]湖南科技大学生命科学与健康学院,中国湖南湘潭411201 [2]湖南科技大学经济作物遗传改良与综合利用湖南省重点实验室,中国湖南湘潭411201

出　　处：《生命科学研究》2025年第1期21-31,共11页Life Science Research

基　　金：2022年度湖南省教育厅科学研究优秀青年项目(22B0482);湖南科技大学博士启动基金项目(E51993)。

摘　　要：腺苷酸激酶(adenylate kinase, ADK)可逆催化2分子腺苷二磷酸(adenosine diphosphate, ADP)转化为腺苷三磷酸(adenosine triphosphate, ATP)和腺苷一磷酸(adenosine monophosphate, AMP),实现ATP再生。由于游离酶的反应温度和pH适应范围窄、稳定性差且无法重复使用,导致游离酶的使用成本较高,这阻碍了其在工业规模生产中的应用。为了实现ADK的固定化,本研究首先构建了ADK的表达质粒pET-ADK,并经大肠杆菌BL21(DE3)诱导表达获得重组ADK;然后优化了壳聚糖的质量浓度以及固定化过程中酶液的pH、酶的质量浓度和固定化时间,并检测了3D打印固定化ADK的理化性质及ATP再生能力。结果表明:基于壳聚糖墨水3D打印固定化酶的最优条件是——壳聚糖的质量浓度为3%,酶液的pH为7.5,酶的质量浓度为0.10mg/m L,固定化时间为3 h;利用3D打印固定化的ADK,葡萄糖激酶催化葡萄糖生成D-葡萄糖-6-磷酸的产率提高了52.00%,而且重复使用8次后, 3D打印固定化的ADK仍能保留初次活性的74.83%。上述结果为可用于ATP再生的ADK固定化研究提供了重要参考。Adenylate kinase(ADK),which could be applied in adenosine triphosphate(ATP)regeneration,reversibly catalyzes the conversion of two adenosine diphosphate(ADP)molecules to ATP and adenosine monophosphate(AMP).As free enzymes have the narrow range of reaction temperature and pH adaptation,poor stability and non-reusability,they are expensive in application and not suitable for industrial-scale production.In order to achieve the immobilization of ADK,the expression plasmid pET-ADK was first constructed,and recombinant ADK was obtained by isopropylthio-β-D-galactoside(IPTG)induction of E.coli BL21(DE3).Then,the chitosan concentration for 3D bioprinting,the pH value of enzyme solution,enzyme concentration and immobilization time in the process of immobilization were optimized.Finally,the physicochemical properties and ATP regeneration ability of 3D-bioprinted immobilized ADK were examined.The results showed that the optimal conditions for ADK immobilization by 3D bioprinting using chitosan-based bioink were 3%of chitosan concentration,pH 7.5,0.10 mg/mL of enzyme concentration,and 3 h of immobilization time.By using 3D-bioprinted immobilized ADK hydrogels,the yield of D-glucose-6-phosphate increased by 52.00%when glucose was catalyzed with glucose kinase.In addition,after repeated use for 8 times,the immobilized ADK still retained 74.83%of its initial activity.These results provide the groundwork for studying ADK immobilization that can be applied to ATP regeneration.

关 键 词：酶固定化 3D生物打印 生物墨水 壳聚糖 腺苷酸激酶(ADK) ATP再生 

分 类 号：Q814[生物学—生物工程]