Applied Biochemistry and Biotechnology

, Volume 183, Issue 3, pp 699–711 | Cite as

Modulating the pH Activity Profiles of Phenylalanine Ammonia Lyase from Anabaena variabilis by Modification of Center-Near Surface Residues

  • Fan Zhang
  • Nan Huang
  • Li Zhou
  • Wenjing Cui
  • Zhongmei Liu
  • Longbao Zhu
  • Yi Liu
  • Zhemin ZhouEmail author


Phenylalanine ammonia lyase from Anabaena variabilis (Av-PAL) is a candidate for the treatment of phenylketonuria (PKU). However, Av-PAL shows its optimal pH at 8.5 and maintains only 70% of its highest activity when pH decreases to 7.3–7.4 (the condition of human plasma). The objective of the study was to shift its optimal pH by mutating surface amino acid residues which interact with the general base Tyr78. Based on the crystal structure and the online program GETAREA, we selected five sites: Asn69, Glu72, Glu75, Asn89, and Val90. Removing negative charges or introducing positive charges near the general base Tyr78 by mutation, the pH optima were successfully shifted to more acidic range. Especially, the pH optima of E75A, E75L, and E75Q were shifted to 7.5 with 35, 30, and 24% higher specific activities than that of the wild, respectively. Half-lives of E75L and E75Q at 70 °C prolonged to 190 and 180 min from 130 min of the wild, respectively. In addition, the higher resistance to a low pH of 3.5 and protease made E75L a candidate for oral medicine of PKU. This work would improve the therapeutic prospect of Av-PAL and provide guidance in modulating optimal pH of enzymes.


Phenylalanine ammonia lyase pH shift Therapeutic application Mutation Surface residues Kinetic stability Resistance 



This work was mainly supported by Key Laboratory of Industrial Biotechnology, Ministry of Education, China.

Compliance with Ethical Standards

Funding Sources

This work was partly funded by the National Natural Science Foundation of China (31300087, 31400058, 31671797, and 21506172), the Natural Science Foundation of Jiangsu Province of China (BK20130131, BK20130139, and BK20140151), the National High Technology Research and Development Program of China (863 Program, 2014AA021304), the High Foreign Experts Project (GDW20123200114), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (111-2-06), the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” Industry Development Program and the Fundamental Research Funds for the Central Universities (JUSRP51411B, JUSRP51504, JUSRP51611A), and the Natural Science Foundation of Anhui Province University of China (KJ2016A801).

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12010_2017_2458_MOESM1_ESM.docx (164 kb)
ESM 1 (DOCX 164 kb)


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Fan Zhang
    • 1
  • Nan Huang
    • 1
  • Li Zhou
    • 1
  • Wenjing Cui
    • 1
  • Zhongmei Liu
    • 1
  • Longbao Zhu
    • 2
  • Yi Liu
    • 3
  • Zhemin Zhou
    • 1
    Email author
  1. 1.Key Laboratory of Industrial Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina
  2. 2.School of Biological and Chemical EngineeringAnhui Polytechnic UniversityWuhuChina
  3. 3.Key Laboratory of Food and Biotechnology, School of Food and BiotechnologyXihua UniversityChengduChina

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