Sahewalla Surbhi, Sihag Sonam, Duhan Anil, Saharan Vinod, Pal Ajay
Department of Biochemistry, College of Basic Sciences and Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana, 125 004, India.
DRDO- Defence Research and Development Establishment, Gwalior, 474 002, India.
Protein J. 2025 Sep 15. doi: 10.1007/s10930-025-10291-8.
The urease enzyme has an inevitable application in cereal crops, particularly in response to foliar urea application. A holistic and novel approach was employed in the present work with the aim to purify and characterize the wheat leaf urease. This will help in exploring and enhancing its activity in assimilation of foliar urea application and a move towards sustainability. Wheat urease was purified to electrophoretic homogeneity with a 41.98 fold purification and 36.3% recovery. The molecular weight of the native enzyme was found to be ~ 290 kDa by Gel Filtration Chromatography (GFC), and a single band in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of ~ 103 kDa deduced its homotrimeric nature. The enzyme had a K of 1.0 mM, V of 63.25 units mL, turnover number of 30.26 min, and a specificity constant of 504.33 M sec. Further, the optimum pH was 7.5 with 40 °C optimum temperature. The E of the purified urease was 61.36 kJ mol, with the E as 104.3 kJ mol. The half-life and D-value decreased with an increase in temperature owing to the rapid loss of its catalytic activity. The z-value was calculated as 44.6 °C. The thermodynamic study revealed the interplay between ΔH, ΔG and ΔS during enzyme deactivation. Histidine was found to be present at the active site and Nickel enhanced the urease activity, whereas copper displayed an inhibitory effect. Hence, this study of wheat urease offers novel insights into an enzyme that has remained largely unexplored despite its inevitable importance in cereal crops. The measures for enhancing its activity in vivo can also be abstracted from this study.
脲酶在谷类作物中具有不可避免的应用，特别是在叶面喷施尿素方面。本研究采用了一种全面且新颖的方法，旨在纯化和表征小麦叶片脲酶。这将有助于探索和提高其在叶面尿素同化中的活性，并朝着可持续性发展迈进。小麦脲酶被纯化至电泳纯，纯化倍数为41.98倍，回收率为36.3%。通过凝胶过滤色谱（GFC）发现天然酶的分子量约为290 kDa，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中出现一条约103 kDa的单带，推断其为同三聚体性质。该酶的米氏常数为1.0 mM，最大反应速度为63.25单位/毫升，周转数为30.26分钟，特异性常数为504.33 M-1sec-1。此外，最适pH为7.5，最适温度为40°C。纯化脲酶的活化能为61.36 kJ/mol，失活能为104.3 kJ/mol。由于催化活性迅速丧失，半衰期和D值随温度升高而降低。z值计算为44.6°C。热力学研究揭示了酶失活过程中焓变、吉布斯自由能变和熵变之间的相互作用。发现活性位点存在组氨酸，镍可增强脲酶活性，但铜表现出抑制作用。因此，对小麦脲酶的这项研究为一种尽管在谷类作物中具有不可避免的重要性但在很大程度上仍未被探索的酶提供了新的见解。也可以从本研究中提取提高其体内活性的措施。
