Lipases for targeted industrial applications, focusing on the development of biotechnologically significant aspects: A comprehensive review of recent trends in protein engineering.

Lipases are remarkable biocatalysts, adept at catalyzing the breakdown of diverse compounds into glycerol, fatty acids, and mono- and di-glycerides via hydrolysis. Beyond this, they facilitate esterification, transesterification, alcoholysis, acidolysis, and more, making them versatile in industrial applications. In industrial processes, lipases that exhibit high stability are favored as they can withstand harsh conditions. However, most native lipases are unable to endure adverse conditions, making them unsuitable for industrial use. Protein engineering proves to be a potent technology in the development of lipases that can function effectively under challenging conditions and fulfill criteria for various industrial processes. This review concentrated on new trends in protein engineering to enhance the diversity of lipase genes and employed in silico methods for predicting and comprehensively analyzing target mutations in lipases. Additionally, key molecular factors associated with industrial characteristics of lipases, including thermostability, solvent tolerance, catalytic activity, and substrate preference have been elucidated. The present review delved into how industrial traits can be enhanced through directed evolution (epPCR, gene shuffling), rational design (FRESCO, ASR), combined engineering strategies (i.e. CAST, ISM, and FRISM) as protein engineering methodologies in contexts of biodiesel production, food processing, and applications of detergent, pharmaceutics, and plastic degradation.

Marine Microalgae Schizochytrium sp. S31: Potential Source for New Antimicrobial and Antibiofilm Agent.

BACKGROUND: The rise of antibiotic-resistant bacteria necessitates the discovery of new, safe, and bioactive antimicrobial compounds. The antibacterial and antibiofilm activity of microalgae makes them a potential candidate for developing natural antibiotics to limit microbial infection in various fields. OBJECTIVE: This study aimed to analyze the antibacterial effect of the methanolic extract of Schizochytrium sp. S31 microalgae by broth microdilution and spot plate assays. METHODS: The antibacterial effects of Schizochytrium sp. S31 extract was studied on gramnegative pathogens, Pseudomonas aeruginosa, Escherichia coli 35218, Klebsiella pneumonia, which cause many different human infections, and the gram-positive pathogen Streptococcus mutans. At the same time, the antibiofilm activity of the Schizochytrium sp. S31 extract on Pseudomonas aeruginosa and Escherichia coli 35218 bacteria were investigated by crystal violet staining method. RESULTS: Schizochytrium sp. S31 extract at a 60% concentration for 8 hours displayed the highest antibacterial activity against P. aeruginosa, E. coli 35218, and K. pneumonia, with a decrease of 87%, 92%, and 98% in cell viability, respectively. The experiment with Streptococcus mutans revealed a remarkable antibacterial effect at a 60% extract concentration for 24 hours, leading to a notable 93% reduction in cell viability. Furthermore, the extract exhibited a dose-dependent inhibition of biofilm formation in P. aeruginosa and E. coli 35218. The concentration of 60% extract was identified as the most effective dosage in terms of inhibition. CONCLUSION: This research emphasizes the potential of Schizochytrium sp. S31 as a natural antibacterial and antibiofilm agent with promising applications in the pharmaceutical sectors. This is the first study to examine the antibacterial activity of Schizochytrium sp. S31 microalgae using broth microdilution, spot plate assays, and the antibiofilm activity by a crystal staining method. The findings of this study show that Schizochytrium sp. S31 has antibacterial and antibiofilm activities against critical bacterial pathogens.

Regiospecific Oxidation of Chlorobenzene to 4-Chlororesorcinol, Chlorohydroquinone, 3-Chlorocatechol and 4-Chlorocatechol by Engineered Toluene o-Xylene Monooxygenases.

Toluene o-xylene monooxygenase (ToMO) was found to oxidize chlorobenzene to form 2-chlorophenol (2-CP, 4%), 3-CP (12%), and 4-CP (84%) with a total product formation rate of 1.2 ± 0.17 nmol/min/mg protein. It was also discovered that ToMO forms 4-chlorocatechol (4-CC) from 3-CP and 4-CP with initial rates of 0.54 ± 0.10 and 0.40 ± 0.04 nmol/min/mg protein, respectively, and chlorohydroquinone (CHQ, 13%), 4-chlororesorcinol (4-CR, 3%), and 3-CC (84%) from 2-CP with an initial product formation rate of 1.1 ± 0.32 nmol/min/mg protein. To increase the oxidation rate and alter the oxidation regiospecificity of chloroaromatics, as well as to study the roles of active site residues L192 and A107 of the alpha hydroxylase fragment of ToMO (TouA), we used the saturation mutagenesis approach of protein engineering. Thirteen TouA variants were isolated, among which some of the best substitutions uncovered here have never been studied before. Specifically, TouA variant L192V was identified which had 1.8-, 1.4-, 2.4-, and 4.8-fold faster hydroxylation activity toward chlorobenzene, 2-CP, 3-CP, and 4-CP, respectively, compared to the native ToMO. The L192V variant also had the regiospecificity of chlorobenzene changed from 4% to 13% 2-CP and produced the novel product 3-CC (4%) from 3-CP. Most of the isolated variants were identified to change the regiospecificity of oxidation. For example, compared to the native ToMO, variants A107T, A107N, and A107M produced 6.3-, 7.0-, and 7.3-fold more 4-CR from 2-CP, respectively, and variants A107G and A107G/L192V produced 3-CC (33 and 39%, respectively) from 3-CP whereas native ToMO did not. IMPORTANCE Chlorobenzene is a commonly used toxic solvent and listed as a priority environmental pollutant by the US Environmental Protection Agency. Here, we report that Escherichia coli TG1 cells expressing toluene o-xylene monooxygenase (ToMO) can successfully oxidize chlorobenzene to form dihydroxy chloroaromatics, which are valuable industrial compounds. ToMO performs this at room temperature in water using only molecular oxygen and a cofactor supplied by the cells. Using protein engineering techniques, we also isolated ToMO variants with enhanced oxidation activity as well as fine-tuned regiospecificities which make direct microbial oxygenations even more attractive. The significance of this work lies in the ability to degrade environmental pollutants while at the same time producing valuable chemicals using environmentally benign biological methods rather than expensive, complex chemical processes.