Expression and characterization of a thermostable l-aminoacylase in transgenic rice
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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.
Keywordsl-Aminoacylase l-Homophenylalanine N-acetyl-l-homophenylalanine Transgenic rice Recombinant protein storage
Angiotensin-converting enzyme inhibitor
Nitro blue tetrazolium
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.
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.
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