ABSTRACT
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.
ABSTRACT
We report on the fabrication of AqpZ immobilized flat sheet membranes. The effects of interfacial polymerization conditions as well as proteoliposome concentration were evaluated. Commercial AqpZ were used as positive control for cloned AqpZ. Specific permeate flux of membranes at higher proteoliposome concentrations increased up to 25 times higher than thin film composite membranes; however; MgSO4 rejection is lowered almost to 1.5%. FTIR and SEM confirm immobilization of proteoliposomes. Thermal analysis showed that increasing proteoliposome concentration has no positive effect on the incorporation of proteoliposomes into polyamide structures. On the contrary, at lower proteoliposome concentrations, incorporation of proteoliposomes was found better. When combined membrane performances were compared in terms of specific permeate flux; MgSO4 and humic rejection and flux recovery after humic acid filtration, the performance of cloned AqpZ incorporated membranes (having 0.1â mg/mL proteoliposome concentration and polyamide formed with 2â min piperazine reaction time) improved 1.7 times regarding TFC membranes. According to the results, increasing proteoliposome concentration did not improve nanofiltration membrane performance. On the contrary, lower proteoliposome concentrations were found to be more effective in increasing membrane performance.
Subject(s)
Aquaporins , Membranes, Artificial , Filtration , ProteolipidsABSTRACT
The aim of this study was to isolate a novel esterase from a hypersaline lake by sequence-based metagenomics. The metagenomic DNA was isolated from the enriched hypersaline lake sediment. Degenerate primers targeting the conserved regions of lipolytic enzymes of halophilic microorganisms were used for polymerase chain reaction (PCR) and a whole gene was identified by genome walking. The gene was composed of 783 bp, which corresponds to 260 amino acids with a molecular weight of 28.2 kDa. The deduced amino acid sequence best matched with the esterase from Halomonas gudaonensis with an identity of 91%. Recombinantly expressed enzyme exhibited maximum activity towards pNP-hexanoate with a kcat value of 12.30 s-1. The optimum pH and temperature of the enzyme were found as 9 and 30 °C, respectively. The effects of NaCl, solvents, metal ions, detergents and enzyme inhibitors were also studied. In conclusion, a novel enzyme, named as hypersaline lake "Acigöl" esterase (hAGEst), was identified by sequence-based metagenomics. The high expression level, the ability to maintain activity at cold temperatures and tolerance to DMSO and metal ions are the most outstanding properties of the hAGEst.
