Acidithiobacillus caldus, a typical sulfur oxidizer, derives the majority of its energy from sulfur oxidation. And the essential enzyme for sulfide oxidation catalysis is sulfide quinone oxidoreductase (SQR), an ancient flavoprotein. Here, the catalytic mechanism of SQR generated from A. caldus was investigated (SQRAc). According to phylogenetic study, SQRAc (ACAty RS11315) is closely related to SQR (BAD99305) of Acidithiobacillus ferrooxidans NASF-1 and is classified as a type I Sqr enzyme. SQRAc heterologously produced in Escherichia coli exhibits the distinctive absorption peaks (375, 450 nm) of the flavoproteins family of proteins in its absorption spectrum. Utilizing site-directed mutagenesis, the function of conserved cysteines in the catalytic pathway was determined. Based on the sulfide quinone redox reactions in vitro of SQRAc and variations, Cys160 and Cys356 have been identified as enzyme-active residues. Mutation of another cysteine present in all type I SQRs (Cys128) decreased enzyme activity by 56%, indicating that this residue plays an important but non-essential role in enzyme function. In addition, the binding affinities of SQRAc, the visualization of its 3D structure, and the interaction between receptors and ligands were investigated. Finally, a suitable sulfide quinone redox catalytic mechanism for A. caldus was proposed.
For many crucial industrial applications, enzyme-catalyzed processes take place in harsh organic solvent environments. However, it remains a challenging problem to improve enzyme stability in organic solvents. This study utilized the MLDE (machine learning-assisted directed evolution) protocol to improve the methanol tolerance of Proteus mirabilis lipase (PML). The machine learning (ML) models were trained based on 266 combinatorial mutants. Using top 3 in 22 regression models based on evaluation of tenfold cross-validation, the fitness landscape of the 8000 full-space combinatorial mutants was predicted. All mutants in the restricted library showed higher methanol tolerance, among which the methanol tolerance of G202N/K208G/G266S (NGS) was up to 13-fold compared with the wild-type. Molecular dynamics (MD) simulation showed that reconstructing of critical hydrogen bond network in the mutant region of NGS provides a more stable local structure. This compact structure may improve the methanol tolerance by preventing organic solvent molecules into the activity site and resisting structural destruction. This work provides a successful case of evolution guided by ML for higher organic solvent tolerance of enzyme, and may also be a reference for broad enzyme modifications.
l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesize β-hydroxy-α-amino acids with unsatisfactory enzyme activity. Here, we expressed the l-threonine aldolase from Pseudomonas putida KT2440 (l-PpTA) in Escherichia coli BL21 (DE3) and improved the activity and thermostability by protein engineering. Five amino acid residues (Ser10, His89, Asp93, Arg177, and Arg321) located in the substrate-binding pocket were selected and for mutation. Eight mutants (D93A, D93G, D93M, D93F, D93S, D93Q, D93Y and D93H) with increased enzyme activity were identified and their k cat/K M values showed about 1–7-fold higher than wild-type. Among all the variants, D93H showed the highest catalytic efficiency with 2925 and 4515 s−1 mM−1 of k cat/K M values toward l-threonine and l-allo-threonine, respectively. In addition, circular dichroism spectrum exhibited that the melting temperature of D93H (54.2 °C) was 5 °C higher than wild-type (49.2 °C). Molecular dynamics simulations illustrated that the D93H variant shortens the distance between the imidazole group of H93 and the hydroxyl group of substrate, which facilitated the proton extraction and promote the enzymatic reaction. This work affords a candidate for the synthesis of β-hydroxy-α-amino acids with improved catalytic efficiency and thermostability and provides structural insights into the l-TA family by protein engineering.
N-acetylneuraminic acid (NeuAc) is an important nutrient that plays a key role in brain development in infants NeuAc is mainly produced by extraction from natural resources such as edible birds’s nests, crucian eggs, caviar and human breast milk. The extraction process is complicated, resulting in the disadvantages of low NeuAc content and low recovery rate. In this study, a crude enzyme immobilization-based cell-free system (CEICFS) was developed for efficient NeuAc biosynthesis. First, N-terminal coding sequences that improved the expression levels of N-acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid aldolase (NanA) were obtained by high-throughput screening. And these sequences resulted in up to 1.5-fold (1.2-fold) increase in AGE (NanA) enzyme levels. And then, a CEICFS for NeuAc biosynthesis was proposed by directly immobilizing crude enzyme containing AGE and NanA on amino resin. Subsequently, NeuAc production from GlcNAc using CEICFS in one reactor was carried out, resulting 68 g/L of NeuAc and the highest productivity of 6.8 g/L/h. Further, the enzyme activity was still higher than 75% after five repeated uses. The functional properties of CEICFS were studied and compared to those of the free enzyme, immobilization can extend the application of enzyme to some harsh environments, such as low temperature and acidic environment. Therefore, CEICFS with excellent heat resistance, storage stability and reusability exhibit great potential for industrial application.
Trehalose is a disaccharide with many applications in cosmetics, refrigeration, and food. Trehalose synthase is a significant enzyme in trehalose production. Escherichia coli is usually used to express this enzyme heterologously. Since E. coli is a pathogenic strain, we chose Corynebacterium glutamicum ATCC13032 as an engineering strain in this study for food safety reasons. Because of its poor permeability, we constructed two recombinant C. glutamicum strains using two anchor proteins, PorH, and short-length NCgl1337, to anchor trehalose synthase from Streptomyces coelicolor on the cell surface and synthesize trehalose directly from maltose. Studies on enzymatic properties indicated that NCgl1337S–ScTreSK246A had better enzyme activity and thermal stability than the free enzyme. After optimizing the whole-cell transformation, the optimal transformation condition was 35 °C, pH 7.0, and OD600 of 30. Under this condition, the conversion rate of 300 g/L maltose reached 69.5% in a 5 L fermentor. The relative conversion rate was still above 75% after repeated five times.