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Expression and characterization of a thermostable l-aminoacylase in transgenic rice

  • Parawee Kanjanaphachoat
  • I-Wen Wang
  • Kun-Ting Hsieh
  • Ching-Shan Tseng
  • Liang-Jwu ChenEmail author
Original Article
  • 19 Downloads

Abstract

The gene encoding a thermostable l-aminoacylase (LAA) from Deinococcus radiodurans BCRC12827 was isolated and expressed in transgenic rice under the control of a rice actin gene promoter or a seed-specific promoter, Ose705. The recombinant LAA in the transgenic line Ose705:LAA was specifically detected in rice grains, but not in leaves, and its identity was confirmed by a LC/MS/MS assay. Furthermore, was efficiently purified via affinity chromatography using a nickel column. Enzymatic activity of this rice-produced LAA was determined by HPLC and a maximum activity at pH 8.0 and 45 °C in a phosphate buffer supplemented with the divalent metal ion Co2+ using NAc-l-HPA as a substrate was obtained, similar to its host counterpart. This rice-produced LAA maintained approximately 50% enzyme activity after 48 h of incubation under 45 °C and maintained approximately 90% activity compared to a freshly prepared sample after being stored in rice seeds for 4 years. The present study indicated that seed-specific protein production in transgenic rice is a good and safe source for mass production of LAA, and this system can be useful for the production of other biomedical proteins as well.

Keywords

l-Aminoacylase l-Homophenylalanine N-acetyl-l-homophenylalanine Transgenic rice Recombinant protein storage 

Abbreviations

ACE

Angiotensin-converting enzyme

ACEI

Angiotensin-converting enzyme inhibitor

BCIP

5-Bromo-4-chloro-3-indolyl-phosphate

LAA

l-Aminoacylase

l-HPA

l-Homophenylalanine

NAc-l-HPA

N-acetyl-l-homophenylalanine

NBT

Nitro blue tetrazolium

WT

Wild type

Notes

Acknowledgements

This work was financially supported through grants from the National Science Council (NSC 99-2324-B-005-008-MY2; NSC 101-2324-B-005-009) and the Council of Agriculture (98AS-1.1.1-FD-Z1) to Liang-Jwu Chen and in part by Advanced Plant Biotechnology Center from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) (Grant No. 107S0021G) in Taiwan. We thank Dr. Chien-Chen Lai for LC/MS/MS assay.

Funding

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interests.

Supplementary material

13562_2019_539_MOESM1_ESM.docx (463 kb)
Supplementary material 1 (DOCX 463 kb)

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Copyright information

© Society for Plant Biochemistry and Biotechnology 2019

Authors and Affiliations

  1. 1.Biotechnology Program, Faculty of ScienceMaejo UniversityChiang MaiThailand
  2. 2.Division of BiotechnologyTaiwan Agriculture Research InstituteTaichungTaiwan
  3. 3.Institute of Molecular BiologyNational Chung Hsing UniversityTaichungTaiwan
  4. 4.Agricultural Biotechnology CenterNational Chung Hsing UniversityTaichungTaiwan

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