解纤维素热酸菌来源的耐热磷酸酶的酶学性质与应用

Characterization and application of thermostable sugar phosphatase from Acidothermus cellulolyticus

卤代酸脱卤酶(HAD)超家族的磷酸酶,其底物谱广泛,催化功能多样,因此鉴别和表征耐热的磷酸酶对于其在生物制品生产中的应用具有重要意义。对解纤维素热酸菌(Acidothermus cellulolyticus)来源的磷酸酶(AcPase)进行研究,将其基因经密码子偏好性优化后,在E.coli Rosetta(DE3)中进行异源表达,并由HisTrap树脂柱分离纯化重组蛋白,研究其酶学性质。结果发现,该酶属于Mg^(2+)依赖型的磷酸酶,最适温度为65℃,最适pH为6.0。AcPase在45~65℃的条件下稳定,在45℃的半衰期为17.4 h,展现出很好的热稳定性。AcPase在50℃下对D-葡萄糖-6-磷酸有较高的催化活性,比酶活为0.68 U/mg。由于AcPase对D-葡萄糖-6-磷酸具有较高的催化活性,笔者进而构建了一个基于该磷酸酶的纤维素磷解多酶催化系统,通过4个酶的体外多酶级联催化将纤维素转化成葡萄糖,底物转化率达到97.6%。该研究通过对一个偏好D-葡萄糖-6-磷酸的磷酸酶的表征,为纤维素完全转化提供了新的研究思路。

Phosphatase of the haloacid dehalogenase(HAD)-like hydrolase superfamily has a broad substrate spectrum and diverse catalytic functions.Identification and characterization of thermostable phosphatases provides great potential for exploring their applications in biochemical production.Herein,the phosphatase from Acidothermus cellulolyticus(AcPase)was investigated.The gene encoding AcPase was codon-optimized and synthesized,and was then transformed into E.coli Rosetta(DE3).The recombinant enzyme was subsequently expressed and purified by His Trap affinity chromatography.Studies of enzymatic properties revealed that AcPase was a Mg^(2+)-dependent HAD-like phosphatase which functioned optimally at 65℃and pH 6.0.AcPase was thermostable at 45-65℃,with a half-life of 17.4 h at 45℃.At 50℃,AcPase exhibited a relatively high specific activity for D-glucose-6-phosphate(0.68 U/mg).An in vitro synthetic enzymatic biosystem containing four enzymes including AcPase was therefore constructed to transform cellulose into glucose in one pot,achieving a yield of 97.6%.To sum up,based on the characterization of a phosphatase which prefers D-glucose-6-phosphatase,this study suggests a new strategy for the complete conversion of cellulose.