CBM3-fused and signal peptide-truncated Rhodococcus hoagii cholesterol oxidase for stable cholesterol biosensing

Xiaozhen Huang

doi:10.1016/j.bej.2026.110182

CBM3-fused and signal peptide-truncated Rhodococcus hoagii cholesterol oxidase for stable cholesterol biosensing

2026-05-23

0

PRE

AI

X

Xiaozhen Huang

X

Xuan Li

Y

Yue Shao

Y

Yang, Furui

X

X. Rosalind Wang

C

Chunyu Song

Y

Yi Yu

M

Min Yang

B

Binglian Wang

Q

Qi Wang

L

Lihe Zhang

Y

Yaohong Ma

S

Sirong Zhu

W

Weili Gong*

DOI：10.1016/j.bej.2026.110182

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Abstract

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Aiming at the limited availability of experimentally characterised and engineered cholesterol oxidase (ChOx) suitable for oriented immobilization in cholesterol biosensor development, this study presents an unreported ChOx from Rhodococcus hoagii (Corynebacterium equii) with excellent pH and temperature stability, making it well-suited for sensor fabrication. To achieve oriented immobilization, the ChOx was further fused with carbohydrate-binding module 3 (CBM3) at either its N- or C-terminus, leveraging the affinity between CBM and cellulose-modified electrodes. Under ambient temperature, the C-terminally fused ChOx-CBM3 exhibited excellent catalytic activity within the linear detection range of 0-0.375 mM for cholesterol. Notably, it maintained a strong linear relationship (R-2 > 0.99) even after 15 days of storage at room temperature. Its stability was superior to that of the N-terminally fused CBM3-ChOx (R-2 > 0.99, 9 days) and the wild-type ChOx cross-linked with glutaraldehyde (R-2 > 0.99, 10 days). Mechanistic studies revealed that the signal peptide at the N-terminus of ChOx caused instability in CBM3-ChOx. Then N-terminal CBM3 fusion combined with signal peptide truncation (CBM3-ChOx-Q) achieved the best balance of stability, catalytic activity, and electrochemical sensing performance, maintaining stable biosensing performance for up to 20 days at room temperature, R-2 >= 0.99. This study expands the enzyme toolbox of ChOx for the development of improved cholesterol biosensors and provides a valuable case for developing other biosensors.

Keywords:

Fusion cholesterol oxidase

Carbohydrate-binding module (CBM3)

Electrochemical biosensor

Enzyme immobilization

CBM3-fused and signal peptide-truncated Rhodococcus hoagii cholesterol oxidase for stable cholesterol biosensing

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