Improving the thermostability of Pseudoalteromonas Porphyrae κ-carrageenase by rational design and MD simulation

Yuyan Sang; Xiaoyi Huang; Hebin Li; Tao Hong; Mingjing Zheng; Zhipeng Li; Zedong Jiang; Hui Ni; Qingbiao Li; Yanbing Zhu

doi:10.1186/s13568-024-01661-z

Improving the thermostability of Pseudoalteromonas Porphyrae κ-carrageenase by rational design and MD simulation

AMB Express. 2024 Jan 20;14(1):8. doi: 10.1186/s13568-024-01661-z.

Authors

Yuyan Sang¹, Xiaoyi Huang¹, Hebin Li², Tao Hong^{1

3

4}, Mingjing Zheng^{1

3

4}, Zhipeng Li^{1

3

4}, Zedong Jiang^{1

3

4}, Hui Ni^{1

3

4}, Qingbiao Li^{1

3

4}, Yanbing Zhu^{5

6

7}

Affiliations

¹ College of Ocean Food and Biological Engineering, Jimei University, 361021, Xiamen, China.
² Department of Pharmacy, Xiamen Medical College, 361008, Xiamen, China.
³ Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, 361021, Xiamen, China.
⁴ Research Center of Food Biotechnology of Xiamen City, 361021, Xiamen, China.
⁵ College of Ocean Food and Biological Engineering, Jimei University, 361021, Xiamen, China. yanbingzhu@jmu.edu.cn.
⁶ Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, 361021, Xiamen, China. yanbingzhu@jmu.edu.cn.
⁷ Research Center of Food Biotechnology of Xiamen City, 361021, Xiamen, China. yanbingzhu@jmu.edu.cn.

Abstract

The industrial applications of the κ-carrageenases have been restricted by their poor thermostability. In this study, based on the folding free energy change (ΔΔG) and the flexibility analysis using molecular dynamics (MD) simulation for the alkaline κ-carrageenase KCgCD from Pseudoalteromonas porphyrae (WT), the mutant S190R was identified with improved thermostability. After incubation at 50 °C for 30 min, the residual activity of S190R was 63.7%, 25.7% higher than that of WT. The T_m values determined by differential scanning calorimetry were 66.2 °C and 64.4 °C for S190R and WT, respectively. The optimal temperature of S190R was 10 °C higher than that of WT. The κ-carrageenan hydrolysates produced by S190R showed higher xanthine oxidase inhibitory activity compared with the untreated κ-carrageenan. MD simulation analysis of S190R showed that the residues (V186-M194 and P196-G197) in F5 and the key residue R150 in F3 displayed the decreased flexibility, and residues of T169-N173 near the catalytic center displayed the increased flexibility. These changed flexibilities might be the reasons for the improved thermostability of mutant S190R. This study provides a useful rational design strategy of combination of ΔΔG calculation and MD simulation to improve the κ-carrageenase's thermostability for its better industrial applications.

Keywords: Folding free energy change; Molecular dynamic simulation; Mutant κ-carrageenase; Thermostability improvement.

Abstract

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