DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability

Abstract

Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.

Characteristics of Thermo-stable Serine Peptidase Vpr from Endophytic Bacillus cereus strain InaCC-B1657

“…For instance, metal chelators and other inhibitors, in fact, can help modulate enzyme activity (Akbar and Sharma 2017; Zhao et al 2019). In addition, several factors also need to be considered to tackle the inhibitors problem, such as understanding their biochemical properties and implementation of gene manipulation methods such as DNA shu ing (Yao et al 2022) and similar protocols. The genes associated with brinolytic activity in Bacillus species are diverse and may encode various brinolytic enzymes and proteases.…”

Empowering Protein Engineering through Recombination of Beneficial Substitutions

“…The substantially increased thermostability and higher activity. Recombinant AprEFSM4 (shuffled gene from 4 aprE genes) [111] variant library. [49] Recombining ends of linearized plasmids after PCR (REPLACR-mutagenesis, Figure 4b) generated mutations in a single-step through in vivo recombination and can transform bacteria that express the Red/ET recombinant protein, offering an effective and robust in vivo mutagenesis protocol.…”

Molecular Modification Strategies of Nitrilase for Its Potential Application in Agriculture

“…In addition, they obtained another mutant (pHNIT45), which was highly active at a pH as low as 4.5 and was able to convert (R)-2-chloro-mandelonitrile to (R)-2-chloro-mandelic acid within 10 min, with an enantiomeric excess value of more than 99% . Zhuang et al used DNA recombination technology to improve the thermal stability and fibrinogen hydrolysis activity of the plasmin AprEFM4, indicating that DNA recombination can be used to improve the activity of the Bacillus fibrinolytic enzyme . De Santis et al used DNA recombination technology to perform molecular modification of nitrilase and obtained a variety of nitrilase mutants.…”

“…119 Zhuang et al used DNA recombination technology to improve the thermal stability and fibrinogen hydrolysis activity of the plasmin AprEFM4, indicating that DNA recombination can be used to improve the activity of the Bacillus fibrinolytic enzyme. 120 De Santis et al 111 used DNA recombination technology to perform molecular modification of nitrilase and obtained a variety of nitrilase mutants. Using two stereoselective nitrilases to catalyze 3hydroxyglutaronitrile, the ee values of the S-type and R-type products were greater than 95%, and the yield was 98%.…”