Enhancing the Thermostability of Alkaline Protease 2709 by Computation-Based Rational Design

Overview

Journal Molecules

Publisher MDPI

Specialty Biology

Date 2025 Mar 13

PMID 40076384

Authors

Yuan Yuan

Guowei Zhao

Jing Lu

Lei Wang

Yawei Shi

Jian Zhang

Affiliations

Soon will be listed here.

Abstract

The alkaline protease from strain 2709 (AprE 2709) is widely used in Chinese industries but faces stability challenges under high-temperature conditions. This study employed molecular modeling and mutagenesis to identify Asn residues at positions 61, 160, and 211 as key sites affecting the stability of AprE 2709. By leveraging the additive and cooperative effects of mutations, the mutant enzyme AprE 2709 (N61G/N160G/N211G) was engineered, exhibiting enhanced thermostability and catalytic activity. The mutant demonstrated a 2.89-fold increase in half-life at 60 °C and a 1.56-fold improvement in catalytic efficiency compared to the wild-type enzyme. Structural analysis revealed that the improved thermostability was due to altered electrostatic interactions and strengthened hydrophobic contacts. Targeting Asn residues prone to deamidation presents a promising strategy for improving protein heat tolerance. These findings not only enhance our understanding of enzyme stability but also lay a foundation for future research aimed at optimizing alkaline proteases for diverse industrial applications, particularly in high-temperature processes.

References

Zhou C, Zhang H, Fang H, Sun Y, Zhou H, Yang G . Transcriptome based functional identification and application of regulator AbrB on alkaline protease synthesis in Bacillus licheniformis 2709. Int J Biol Macromol. 2020; 166:1491-1498. DOI: 10.1016/j.ijbiomac.2020.11.028. View

Zhou C, Yang G, Zhang L, Zhang H, Zhou H, Lu F . Construction of an alkaline protease overproducer strain based on Bacillus licheniformis 2709 using an integrative approach. Int J Biol Macromol. 2021; 193(Pt B):1449-1456. DOI: 10.1016/j.ijbiomac.2021.10.208. View

Hains P, Truscott R . Age-dependent deamidation of lifelong proteins in the human lens. Invest Ophthalmol Vis Sci. 2010; 51(6):3107-14. PMC: 2891470. DOI: 10.1167/iovs.09-4308. View

Kusalik P, Svishchev I . The spatial structure in liquid water. Science. 1994; 265(5176):1219-21. DOI: 10.1126/science.265.5176.1219. View

Zhou C, Yang G, Meng P, Qin W, Li Y, Lin Z . Identification and engineering of the aprE regulatory region and relevant regulatory proteins in Bacillus licheniformis 2709. Enzyme Microb Technol. 2023; 172:110310. DOI: 10.1016/j.enzmictec.2023.110310. View

Liu B, Zhang J, Fang Z, Gu L, Liao X, Du G . Enhanced thermostability of keratinase by computational design and empirical mutation. J Ind Microbiol Biotechnol. 2013; 40(7):697-704. DOI: 10.1007/s10295-013-1268-4. View

Yi Z, Pei X, Wu Z . Introduction of glycine and proline residues onto protein surface increases the thermostability of endoglucanase CelA from Clostridium thermocellum. Bioresour Technol. 2010; 102(3):3636-8. DOI: 10.1016/j.biortech.2010.11.043. View

Aderinwale T, Bharadwaj V, Christoffer C, Terashi G, Zhang Z, Jahandideh R . Real-time structure search and structure classification for AlphaFold protein models. Commun Biol. 2022; 5(1):316. PMC: 8983703. DOI: 10.1038/s42003-022-03261-8. View

Bandi S, Singh S, Shah D, Upadhyay V, Mallela K . 2D NMR Analysis of the Effect of Asparagine Deamidation Versus Methionine Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Protein. Mol Pharm. 2019; 16(11):4621-4635. DOI: 10.1021/acs.molpharmaceut.9b00719. View

10.

Lee C, Yu K, Kim S, Han S . Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE. J Biosci Bioeng. 2010; 109(4):331-6. DOI: 10.1016/j.jbiosc.2009.10.014. View

11.

Victorino da Silva Amatto I, da Rosa-Garzon N, de Oliveira Simoes F, Santiago F, Pereira da Silva Leite N, Raspante Martins J . Enzyme engineering and its industrial applications. Biotechnol Appl Biochem. 2021; 69(2):389-409. DOI: 10.1002/bab.2117. View

12.

Shi Y, Rhodes N, Abdolvahabi A, Kohn T, Cook N, Marti A . Deamidation of asparagine to aspartate destabilizes Cu, Zn superoxide dismutase, accelerates fibrillization, and mirrors ALS-linked mutations. J Am Chem Soc. 2013; 135(42):15897-908. DOI: 10.1021/ja407801x. View

13.

Ullah S, Khan S, Khan A, Junaid M, Rafiq H, Htar T . Prospect of Anterior Gradient 2 homodimer inhibition via repurposing FDA-approved drugs using structure-based virtual screening. Mol Divers. 2021; 26(3):1399-1409. DOI: 10.1007/s11030-021-10263-x. View

14.

Kamble A, Singh R, Singh H . Structural and Functional Characterization of Obesumbacterium proteus Phytase: A Comprehensive In-Silico Study. Mol Biotechnol. 2024; 67(2):588-616. DOI: 10.1007/s12033-024-01069-x. View

15.

Zhou C, Liu H, Yuan F, Chai H, Wang H, Liu F . Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing. Int J Biol Macromol. 2018; 122:329-337. DOI: 10.1016/j.ijbiomac.2018.10.170. View

16.

Tang X, Shen W, Lakay F, Shao W, Wang Z, Prior B . Cloning and over-expression of an alkaline protease from Bacillus licheniformis. Biotechnol Lett. 2004; 26(12):975-9. DOI: 10.1023/b:bile.0000030042.91094.38. View

17.

Hiraga K, Nishikata Y, Namwong S, Tanasupawat S, Takada K, Oda K . Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5. Biosci Biotechnol Biochem. 2005; 69(1):38-44. DOI: 10.1271/bbb.69.38. View

18.

Zhu F, Li G, Wei P, Song C, Xu Q, Ma M . Rational engineering of a metalloprotease to enhance thermostability and activity. Enzyme Microb Technol. 2022; 162:110123. DOI: 10.1016/j.enzmictec.2022.110123. View

19.

Bhanuramanand K, Ahmad S, Rao N . Engineering deamidation-susceptible asparagines leads to improved stability to thermal cycling in a lipase. Protein Sci. 2014; 23(10):1479-90. PMC: 4287003. DOI: 10.1002/pro.2516. View

20.

Zhou C, Kong Y, Zhang N, Qin W, Li Y, Zhang H . Regulator DegU can remarkably influence alkaline protease AprE biosynthesis in Bacillus licheniformis 2709. Int J Biol Macromol. 2024; 266(Pt 1):130818. DOI: 10.1016/j.ijbiomac.2024.130818. View