The protein stability analyzer PSA-16 assists researchers from the Institute of Biology at Qilu University of Technology (Shandong Academy of Sciences) in publishing a paper titled "Structural guided high stable fusion urea oxidative engineering to self assembling with cellulose modified electrode for enhanced uric acid sensing" in the International Journal of Biological Macromolecules in April 2025.

Xingbao Wang, Weili Gong, Yunlong Xue, Yi Yu, Xiaozhen

Huang, Yue Shao, Xuan Li, Xin Wang, Chunyu Song, Binglian

Wang, Lihe Zhang, Yaohong Ma

PII: S0141-8130(25)04227-8

The thermal stability of proteins refers to the ability of protein polypeptide chains to deform under the influence of temperature, mainly reflected in the changes in the chemical properties and spatial conformation of polypeptide chains when temperature changes. The smaller the changes, the higher the thermal stability. The thermal stability of proteins is influenced by external factors such as temperature, pH value, and ionic strength, and is of great significance in fields such as biotechnology, drug development, and the food industry.

Protein denaturation temperature is an important concept for biologists to study the thermal stability of proteins. It refers to the structural changes that occur when proteins are heated, from a stable three-dimensional structure to a loose and disordered structure. The thermal stability of proteins is generally expressed using the melting temperature (Tm), also known as the denaturation temperature, which is the temperature at which the protein is unfolded by 50%.

01 Preface

The high stability and retained catalytic activity of immobilized uric acid oxidase (UOX) are key to achieving accurate uric acid (UA) sensors. This study developed an electrochemical UA biosensor based on self-assembled immobilized UOX, which promotes the detection of UA by fusing carbohydrate binding modules (CBM) onto cellulose modified electrodes. By utilizing a computational structure oriented enzyme engineering strategy, it has been demonstrated that the structural stability of N-terminal CBM3 fusion is superior to that of C-terminal fusion of CBM3 or CBM2. Further deletion of CBM3 N-terminal residue (P2VSG5) and linker residue (K158EPMSN163) that were predicted to be harmful to the stability of CBM3-UOX structure was carried out, and CBM3- Δ 10aa-UOX was constructed. Compared with the UOX biosensor crosslinked with glutaraldehyde (0-0.375 mM, 5 days), the reliability of the UA biosensor was significantly improved, showing a linear relationship in the UA range of 0 to 0.625 mM, R2 > 0.99， Sustainable for 20 days. In addition, co immobilization of wild-type UOX and CBM 3- Δ 10aa-UOX (0-0.875 mM,>34 days, R2 > 0.99）， We investigated the mechanism of current increase in the initial stage of UOX fusion and attributed it to the interactions between UOX subunits and peptide cleavage, further demonstrating that the enhanced stability of the CBM 3- Δ 10aa-UOX linking peptide can prolong the current increase process and improve the working life. This study emphasizes the effectiveness of self-assembly immobilization in advancing the field of biosensors and provides robust strategies for the evolution of similar polymeric biosensors.

02 Abstract

Uric acid (UA) is a key nitrogen-containing compound widely present in biological fluids such as serum and urine, and is the main end product of purine metabolism. The normal levels of UA in the serum of healthy individuals are approximately 0.21-0.43 mM in adult males and 0.15-0.36 mM in adult females, while in gout patients, the concentration of UA can be as high as 1.49-4.46 mM. Abnormal levels of UA in biological fluids can indicate diseases such as gout, metabolic syndrome, and kidney disease, making it a useful biomarker for detecting purine metabolism related diseases. Therefore, rapid and reliable detection of UA in biological fluids is often crucial for its diagnosis and treatment.

At present, various UA determination methods have been developed, including high-performance liquid chromatography (HPLC), spectrophotometry, ion chromatography, HPLC/isotope dilution mass spectrometry (ID-MS), and electrochemical biosensing methods. Among these methods, current type enzyme biosensors based on uric acid oxidase (UOX or uricase, EC 1.7.3.3) redox catalysis have received widespread research attention due to their simplicity, sensitivity, specificity, and rapid response. The UOX reaction equation is as follows: UA+2H2O+O2 Allantoin+H2O2+CO2. During the biosensing process, UOX fixed on the electrode reacts with UA, consuming O2 and producing H2O2. The reduced O2 or oxidized H2O2 is directly proportional to the concentration of UA, and the oxidation or reduction signal can be converted and amplified into a readable physical signal through a transducer and electronic amplifier. Therefore, the high stability and maintained catalytic activity of immobilized UOX are key to achieving precise sensing of UA.

In previously reported studies, different types of enzyme immobilization techniques have been utilized to construct current type UA biosensors. Physical adsorption and embedding methods are commonly used for UOX immobilization, but their adsorption process has weak binding force and is easily affected by environmental changes, thereby reducing the operational and storage stability of the corresponding biosensors. UOX embedding is usually achieved using polymers such as polyaniline (PANi) and polypyrrole. However, leakage of immobilized enzymes from these polymers may affect their stability. The covalent binding and cross-linking method using bifunctional reagents such as glutaraldehyde or carbodiimide to form stable covalent bonds between enzymes and carriers or bovine serum albumin (BSA) is also a common method for chemically immobilizing UOX, which can avoid enzyme desorption and leakage. However, the widespread enzyme inactivation and low repeatability caused by covalent binding limit its application in biosensors. Recently, affinity adsorption has been proven to be a promising enzyme immobilization strategy that can improve the performance of enzyme biosensors. In addition, the carbohydrate binding module (CBM2) from family 2 was fused with glucose oxidase through genetic engineering to enhance glucose sensing performance. However, the structure and catalytic mechanism of UOX are different from glucose oxidase, so it is still crucial to explore the construction of stable and active CBM fused UOX as a UA biosensing element.

UOX is a functional tetramer barrel structure, with its active site formed by interfacial residues from two monomers. Each monomer contains two identical structural domains with beta beta alpha beta antiparallel super secondary structures, known as tunnel folding domains. Some weak interactions (non covalent bonds, secondary bonds), including van der Waals forces, hydrogen bonds, salt bonds (dissociation bonds), and hydrophobic interactions, participate in stabilizing the quaternary structure. In addition, the complementary arrangement of polar and hydrophobic groups at the tight interface formed between subunits promotes the association of specific subunits. In the UOX catalytic process, a non cofactor approach was used to promote and control O2 chemistry, and different catalytic mechanisms similar to flavin dependent oxidase or flavin dependent monooxygenase were proposed, but their mechanisms of action have not yet been elucidated. Due to the multi subunit dependent catalytic activity and ambiguous catalytic mechanism of UOX, there have been few research reports on performance improvement of engineered UOX in the past few decades. Therefore, building a stable gene fusion UOX is a major challenge.

Tandem fusion is a popular and feasible strategy for constructing synthetic fusion proteins. When using this strategy to obtain an ideal fusion protein, two elements should be carefully considered: the optimal sequence of domain placement between fusion partners and the selection of appropriate linkers to connect protein domains together. Blind design of fusion proteins may lead to various adverse consequences, including domain misfolding, low protein yield, and reduced biological activity. Due to the significant development of computational programs specifically designed for protein structure prediction and rational protein design, such as AlphaFold2, PROCHECK, AutoDock Vina, and GROMACS, it is possible to better understand fusion protein structures and avoid many adverse outcomes.

03 Results

In order to provide a more comprehensive structural characterization of all UOXs,authorDifferential scanning fluorescence (DSF) [34] was used for thermal stability determination. As shown in Figure S7, the peak shapes of UOX-CBM3 and UOX-CBM2 are more asymmetric, especially UOX-CBM2, indicating that CBM and UOX fracture induce synergistic denaturation of multiple structural domains. In contrast, the DSF peak of CBM 3- Δ 10aa-UOX is the most symmetrical and narrow, confirming its structural stability, which is consistent with the results of molecular dynamics simulations and SDS-PAGE analysis.

04 Method

The thermal stability of the target protein was evaluated using the PSA-16 protein stability analyzer produced by Beijing Best Technology Co., Ltd. Firstly, dilute the sample to a concentration of 0.5 milligrams per milliliter. Then, place 20 microliters of the diluted sample into a quartz glass tube (product number LG-002, also from Beijing Best Technology Co., Ltd.).

Measure the intrinsic protein fluorescence intensity at 330 nm and 350 nm using linear temperature scanning method. The temperature range is 23 to 97 ℃, and the heating rate is 1 ℃ per minute. Calculate the midpoint temperature (Tm) of thermal denaturation based on the slope of the F350/F330 curve. Each sample was measured four times.

05 Results

In this study, the authors utilized a structure guided enzyme engineering strategy to construct a series of CBM fused uricase enzymes and explored the key factors affecting the stability of fused UOX as a biosensing element. The results showed that the continuous residues (P2, V3, S4, G5) at the N-terminus of CBM 3 and the anchoring region between domains 158 to 163 (K158EPMSN163) played a critical role in the structural stability of CBM fused UOX. After removing unstable residues, the reliability of UA biosensors was significantly improved. This study contributes to the rational construction of fused UOX with tunable and predictable characteristics, and provides an ideal candidate for manufacturing excellent UA biosensors.

In order to provide a more comprehensive structural characterization of all UOX,authorThe thermal stability was determined using differential scanning fluorescence (DSF) [34]. As shown in Figure S7, the peak shapes of UOX-CBM3 and UOX-CBM2 are more asymmetric, especially UOX-CBM2, indicating that multiple structural domains synergistically denature due to the fracture of CBM and UOX. In contrast, the differential scanning fluorescence (DSF) peak of CBM3- Δ 10aa UOX is the most symmetrical and narrowest, which confirms its structural stability, as shown by molecular dynamics (MD) simulation and SDS-PAGE analysis.