Title: Deletion and Randomization of Structurally Variable Regions in B. subtilis Lipase A (BSLA) Alter Its Stability and Hydrolytic Performance Against Long Chain Fatty Acid Esters Martinez R, Bernal C, Alvarez R, Concha C, Araya F, Cabrera R, Dhoke GV, Davari MD Ref: Int J Mol Sci, 21:, 2020 : PubMed
The continuous search for novel enzyme backbones and the engineering of already well studied enzymes for biotechnological applications has become an increasing challenge, especially by the increasing potential diversity space provided by directed enzyme evolution approaches and the demands of experimental data generated by rational design of enzymes. In this work, we propose a semi-rational mutational strategy focused on introducing diversity in structurally variable regions in enzymes. The identified sequences are subjected to a progressive deletion of two amino acids and the joining residues are subjected to saturation mutagenesis using NNK degenerate codons. This strategy offers a novel library diversity approach while simultaneously decreasing enzyme size in the variable regions. In this way, we intend to identify and reduce variable regions found in enzymes, probably resulting from neutral drift evolution, and simultaneously studying the functional effect of said regions. This strategy was applied to Bacillus. subtilis lipase A (BSLA), by selecting and deleting six variable enzyme regions (named regions 1 to 6) by the deletion of two amino acids and additionally randomizing the joining amino acid residues. After screening, no active variants were found in libraries 1% and 4%, 15% active variants were found in libraries 2% and 3%, and 25% for libraries 5 and 6 (n = 3000 per library, activity detected using tributyrin agar plates). Active variants were assessed for activity in microtiter plate assay (pNP-butyrate), thermal stability, substrate preference (pNP-butyrate, -palmitate), and compared to wildtype BSLA. From these analyses, variant P5F3 (F41L-DeltaW42-DeltaD43-K44P), from library 3 was identified, showing increased activity towards longer chain p-nitrophenyl fatty acid esters, when compared to BSLA. This study allowed to propose the targeted region 3 (positions 40-46) as a potential modulator for substrate specificity (fatty acid chain length) in BSLA, which can be further studied to increase its substrate spectrum and selectivity. Additionally, this variant showed a decreased thermal resistance but interestingly, higher isopropanol and Triton X-100 resistance. This deletion-randomization strategy could help to expand and explore sequence diversity, even in already well studied and characterized enzyme backbones such as BSLA. In addition, this strategy can contribute to investigate and identify important non-conserved regions in classic and novel enzymes, as well as generating novel biocatalysts with increased performance in specific processes, such as enzyme immobilization.
        
Title: Liver ethoxyresorufin-O-deethylase and brain acetylcholinesterase in two freshwater fish species of South America; the effects of seasonal variability on study design for biomonitoring Chiang G, Munkittrick KR, Urrutia R, Concha C, Rivas M, Diaz-Jaramillo M, Barra R Ref: Ecotoxicology & Environmental Safety, 86:147, 2012 : PubMed
Responses at low levels of biological organization to evaluate environmental changes and water quality have been used for many years. South America is no different, and recently biochemical endpoints in fish have been used to assess the impacts of industrial and sewage effluents on wild fish populations. For Chilean native freshwater fish, basic biological data is scarce and data on 7-ethoxyresorufin-O-deethylase (EROD) and Acetylcholinesterase (AChE) activity is practically absent. Moreover, extensive variation in these two biochemical endpoints exists among species and seasons. In this article we evaluate seasonal variation in liver EROD and brain AChE activities in Trichomycterus areolatus and Percilia gillissi, two widely distributed native freshwater fish species in central Chile. We observed a marked seasonality in hepatic EROD activity in both species, with maximums for P. gillissi during winter months and sex differences in February, July, August and December. T. areolatus showed no sex differences, and peaks in EROD activity in the middle of summer, winter and late spring. Species differences in EROD activity were observed with activity being 1-2 orders of magnitude higher in P. gillissi compared to T. areolatus. Scarce seasonal variation and no sex related differences in brain AChE for both species were observed. Multivariate analysis (PCA) indicated that physical water quality parameters had some degree of responsibility for the seasonal responses found. The seasonal variability data of these biochemical endpoints were used to optimize study design for future monitoring programs, planning timing of sampling, increasing statistical power by collecting specific sample sizes required.