As a green biocatalyst, transaminase with high thermostability can be better employed to synthesize many pharmaceutical intermediates in industry. To improve the thermostability of ()-selective amine transaminase from W3, related mutation sites were determined by multiple amino acid sequence alignment between wild-type ω-transaminase and four potential thermophilic ω-transaminases, followed by replacement of the related amino acid residues with proline by site-directed mutagenesis. Three stabilized mutants (D192P, T237P, and D192P/T237P) showing the highest stability were obtained and used for further analysis. Comparison with the wild-type enzyme revealed that the double mutant D192P/T237P exhibited the largest shift in thermostability, with a 2.5-fold improvement of at 40 °C, and a 6.3 °C increase in , and a 5 °C higher optimal catalytic temperature. Additionally, this mutant exhibited an increase in catalytic efficiency ( / ) relative to the wild-type enzyme. Modeling analysis indicated that the improved thermostability of the mutants could be associated with newly formed hydrophobic interactions and hydrogen bonds. This study shown that proline substitutions guided by sequence alignment to improve the thermostability of ()-selective amine transaminase was effective and this method can also be used to engineering other enzymes.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324462 | PMC |
| http://dx.doi.org/10.1007/s13205-020-02321-2 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Newcastle Disease Virus Displaying an Ectodomain of Middle East Respiratory Syndrome Coronavirus Spike Protein Elicited Robust Humoral and Cellular Immunity in Mice.
Vaccines (Basel)
December 2024
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand.
Background: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory illness in humans and currently lacks an approved vaccine. The Newcastle disease virus (NDV) vector is a well-established, safe, and effective platform for vaccine development. With recent advancements in stabilizing coronavirus spike proteins to enhance their antigenicity, this study aimed to determine whether modifications to the MERS-CoV spike protein could improve its presentation on NDV particles, allowing the resulting virus to be used as an inactivated vaccine.
View Article and Find Full Text PDFCRISPR-Cas12a is widely used for genome editing and biomarker detection since it can create targeted double-stranded DNA breaks and promote non-specific DNA cleavage after identifying specific DNA. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Cas12a variant (FnoCas12a ) by introducing double proline substitutions (K969P/D970P) in a conserved helix called the bridge helix (BH). In this work, we used cryogenic electron microscopy (cryoEM) to understand the molecular mechanisms of BH- mediated activation of Cas12a.
View Article and Find Full Text PDFDevelopment and Evaluation of a Newcastle Disease Virus-like Particle Vaccine Expressing SARS-CoV-2 Spike Protein with Protease-Resistant and Stability-Enhanced Modifications.
Viruses
December 2024
Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou 225012, China.
The ongoing global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates the continuous development of innovative vaccine strategies, especially in light of emerging viral variants that could undermine the effectiveness of existing vaccines. In this study, we developed a recombinant virus-like particle (VLP) vaccine based on the Newcastle Disease Virus (NDV) platform, displaying a stabilized prefusion form of the SARS-CoV-2 spike (S) protein. This engineered S protein includes two proline substitutions (K986P, V987P) and a mutation at the cleavage site (RRAR to QQAQ), aimed at enhancing both its stability and immunogenicity.
View Article and Find Full Text PDFEnhancement of the Thermostability of Esterase by Combinatorial Rational Design.
Molecules
December 2024
School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China.
The esterase EstSIT01 from can catalyze the asymmetric hydrolysis of -dimethyl ester to produce the crucial chiral intermediate (4, 5)-hemimethyl ester for -biotin synthesis. Despite its high yields and stereoselectivity, the low thermostability of EstSIT01 limits its practical application. Herein, two kinds of rational strategies were combined to enhance the thermostability of EstSIT01.
View Article and Find Full Text PDFIn vitro and in silico analyses of amino acid substitution effects at the conserved N-linked glycosylation site in hepatitis B virus surface protein on antigenicity, immunogenicity, HBV replication and secretion.
PLoS One
January 2025
Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand.
The "a" determinant, a highly conformational region within the hepatitis B virus large surface protein (LHBs), is crucial for antibody neutralization and diagnostic assays. Mutations in this area can lead to conformational changes, resulting in vaccination failure, diagnostic evasion, and disease progression. The "a" determinant of LHBs contains a conserved N-linked glycosylation site at N320, but the mechanisms of glycosylation in LHBs remain unclear.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!