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| Optimization of lipase activity assay system based on enzyme reaction kinetics theory |
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| DOI： |
| KeyWord:lipase enzyme activity assay enzyme reaction kinetics surfactant |
| FundProject:国家自然科学基金项目（21266029）；浙江省自
然科学基金项目（LY17B060011） |
| Author Name | Affiliation | | ZHANG Fan1,2, CHENG Lufeng1, CAO Hong2, HE Defei3, ZHENG Lanlan2, LI Chun4 | 1.College of Pharmacy, Xinjiang Medical University, Urumqi 830011, China
2.College of Biological
Chemical Sciences and Engineering, Jiaxing University, Jiaxing 314001, Zhejiang, China
3.Zhejiang Gress Biotechnology Co. ，Ltd. ， Jiaxing 314000, Zhejiang, China
4.Department of
Chemical Engineering, Tsinghua University, Beijing 100084, China |
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| Abstract: |
| In order to improve the sensitivity and accuracy of p-nitrophenol palmitate (p-NPP) method for the assay of lipase activity, the Lipase F-AP 15 from Rhizopus oryzae was taken as the research object, and the optimum reaction pH and reaction temperature were firstly optimized, then surfactant was introduced into the enzyme activity assay system as an activating factor, and the influence mechanism was explored. The assay conditions (substrate concentration, enzyme mass concentration, reaction time) were optimized according to the theory of enzyme reaction kinetics. The repeatability and accuracy of the optimized p-NPP method were examined. Finally, Lipase-PPL, another source of lipase, was used as a model to improve and optimize its enzyme activity assay system with the same idea and method to examine the applicability of the optimization method and idea. The results showed that with the introduction of surfactant PEG 8000, the α-helix structure covering the active site of lipase was rearranged and opened, and the lipase changed from closed conformation to and remained in the active conformation. The optimum conditions of the assay system were as follows: pH 8.0, reaction temperature 30 ℃, enzyme mass concentration 1.00 mg/mL, substrate concentration 6 mmol/L, and reaction time 5 min. Under the optimized conditions, the enzyme activity measured was 1.4 times that of the common p-NPP method (pH 8.0, reaction temperature 30 ℃, enzyme mass concentration 2.50 mg/mL, substrate concentration 10 mmol/L, and reaction time 8 min). The repeatability and accuracy experiments of the optimized p-NPP method showed that the relative standard deviation of the repeated assay results was 1.21%, and the recovery rate of standard addition was 95.60%, which was better than that of the common p-NPP method. The optimization method and idea was applicable to the optimization of lipase activity assay system from other sources. In conclusion, the introduction of surfactant in the lipase activity assay system of p-NPP method and the optimization of the assay conditions under the guidance of enzyme reaction kinetics theory can significantly improve the sensitivity of the method, effectively ensure the repeatability and accuracy of the assay results, shorten the assay time, and save the assay cost. |
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