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Screening of Fungi Isolates for C-4 Hydroxylation of R-2-Phenoxypropionic Acid Based on a Novel 96-Well Microplate Assay Method

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Abstract

R-2-(4-Hydroxyphenoxy)propionic acid (R-HPPA) is a pivotal intermediate for the synthesis of aryloxyphenoxypropionate (APP) herbicide. To rapidly screen microbial isolates with the capacity of hydroxylating R-2-phenoxypropionic acid to R-HPPA from various environmental samples, a convenient and safe 96-well microplate assay method with sodium nitrite (NaNO2) as chromogenic reagent was proposed and optimized. The optimum assay conditions were as follows: the detection wavelength was 420 nm, the concentration of NaNO2 solution was 6.0 g/L, color reaction temperature was 60 °C, the pH of the NaNO2 solution was 2.4, and the reaction time was 40 min. With the aid of this method, screening for microorganisms with C-4-specific hydroxylation activity of R-PPA was conducted. As a result, 23 strains among 3744 single colonies isolated from various samples exhibited the hydroxylation activity. Among these strains, the highest bioconversion rate was achieved by Penicillium oxalicum A5 and Aspergillus versicolor A12, respectively. After 72-h cultivation in shake flask, their conversion rates of R-HPPA from 10 g/L R-PPA reached 21.18% and 40.24%, respectively. The established method was effective in rapid screening of microbes capable of biosynthesizing R-HPPA through hydroxylation of R-PPA, and the obtained two fungi species could be potentially used for R-HPPA production.

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Funding

The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (No. 31500031) and the Natural Science Foundation of Zhejiang Province (No. LQ14B060004).

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Authors and Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Hai-Yan Zhou, Rui Jiang, Yi-Zuo Li, Wei Xu, Yuan-Shan Wang, Ya-Ping Xue & Yu-Guo Zheng

  2. Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People’s Republic of China

    Hai-Yan Zhou, Rui Jiang, Yi-Zuo Li, Wei Xu, Yuan-Shan Wang, Ya-Ping Xue & Yu-Guo Zheng

  3. The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People’s Republic of China

    Hai-Yan Zhou, Rui Jiang, Yi-Zuo Li, Wei Xu, Yuan-Shan Wang, Ya-Ping Xue & Yu-Guo Zheng

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  1. Hai-Yan Zhou
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Zhou, HY., Jiang, R., Li, YZ. et al. Screening of Fungi Isolates for C-4 Hydroxylation of R-2-Phenoxypropionic Acid Based on a Novel 96-Well Microplate Assay Method. Appl Biochem Biotechnol 192, 42–56 (2020). https://doi.org/10.1007/s12010-020-03303-z

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