Surface display and characterization of recombinant α-l-Rhamnosidase from Emiliania huxleyi on Pichia pastoris

Xi Xie; Ziwei Guo; Bihan Chen; Li Lin; Huifan Liu; Gengsheng Xiao; Qin Wang

doi:10.1016/j.bioorg.2025.108121

Surface display and characterization of recombinant α-l-Rhamnosidase from Emiliania huxleyi on Pichia pastoris

Bioorg Chem. 2025 Jan 4:155:108121. doi: 10.1016/j.bioorg.2025.108121. Online ahead of print.

Authors

Xi Xie¹, Ziwei Guo², Bihan Chen³, Li Lin⁴, Huifan Liu⁵, Gengsheng Xiao³, Qin Wang³

Affiliations

¹ Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; Key Laboratory of Green Processing and Intelligent Manufacturing of Lingnan Specialty Food, Ministry of Agriculture, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; Guangdong Provincial Department of Education of Medicinal and Edible Food Intensive Processing Engineering Technology Research Center, Guangzhou 510225, China. Electronic address: [email protected].
² Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; Key Laboratory of Green Processing and Intelligent Manufacturing of Lingnan Specialty Food, Ministry of Agriculture, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; Department of Wine, Food and Molecular Biosciences, Faculty of Agriculture and Life Sciences, Lincoln University, Christchurch 7647, New Zealand.
³ Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; Key Laboratory of Green Processing and Intelligent Manufacturing of Lingnan Specialty Food, Ministry of Agriculture, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China.
⁴ Guangdong Provincial Water Environment and Aquatic Products Security Engineering Technology Research Center, Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, College of Animal Science Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China.
⁵ Guangdong Provincial Key Laboratory of Lingnan Specialty Food Science and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; Key Laboratory of Green Processing and Intelligent Manufacturing of Lingnan Specialty Food, Ministry of Agriculture, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China; Guangdong Provincial Department of Education of Medicinal and Edible Food Intensive Processing Engineering Technology Research Center, Guangzhou 510225, China.

PMID: 39764918
DOI: 10.1016/j.bioorg.2025.108121

Abstract

An α-l-Rhamnosidase gene with an open reading frame of 3192 bp encoding a 1036-amino acid protein (EhRha) was cloned from Emiliania huxleyi for flavonoid hydrolysis on the cell surface of Pichia pastoris (P. pastoris) strain GS115 by fusing with the anchor protein (AGα1) from Saccharomyces cerevisiae. Fluorescence microscopy and flow cytometry assays revealed that EhRha was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity assay and substrate specificity analysis showed that the enzyme activity of displayed EhRha was 78 U/g (cell wet weight). EhRha demonstrated a preference for the α-1,6 linkage l-rhamnose in hesperidin and rutin as its optimal substrates, while showing low activity towards the α-1,2 linkage l-rhamnose in naringin. Furthermore, EhRha demonstrated optimal activity at pH 7.0 and 30 °C, maintaining stability within a pH range of 4.5-9.0 at temperatures below 50 °C, and remained functional at temperatures ranging from 15 °C-30 °C. The enzyme activity was significantly enhanced by the presence of 10 mM Mn²⁺ and Fe³⁺, whereas 10 mM Ca²⁺ and 1 mM Fe³⁺ had an inhibitory effect. These findings suggested that displayed EhRha holds promise for enhancing the bioavailability of health-beneficial polyphenols in low-temperature processing applications.

Keywords: Biochemical characterization; Emiliania huxleyi; Pichia pastoris; Surface display; α-l-Rhamnosidase.