The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.
Engineered bacteria with synthetic gene circuits are attractive tools to detect environmental contaminants. However, their applications in realistic settings are hindered by its relatively low sensitivity, long response time, and limited portability. Here, we present a synthetic bacterial consortium-based system for detecting organophosphorus pesticides (OPs). The system consists of two Escherichia coli strains with divided tasks, including one for hydrolyzing OPs to p-nitrophenol (PNP) and the other for converting the PNP signal into β-galactosidase production for colorimetric detection. Upon optimization, the system was able to detect ethyl-paraoxon at the concentration of 1 × 10 M within 3.5 h of induction at 28 °C, which is approximately 200-fold more sensitive than single-cell based whole-cell sensing. In addition, it was capable of detecting several OPs, commonly used in agriculture. Furthermore, the system showed promise for on-site detection through the demonstration of a paper-based setting and real apple and soil samples. This study provides a rapid, sensitive, and portable biosensing platform for contaminant detection and also demonstrates the utility of engineered microbial ecosystems for novel environmental applications.
Two-dimensional transient natural convective flow in a vertical tube of plain and uneven side-walls containing cobalt-kerosene nanofluids is analyzed using a nonhomogeneous dynamic model. The vertical right wall of the enclosure is maintained at a constant low temperature and the left wall is heated by a uniform thermal condition whereas the horizontal side-walls are insulated. The Brownian motion and thermophoretic phenomena of the nanoparticles are considered in the model. The governing nonlinear momentum, energy, and concentration equations are solved numerically using a Galerkin weighted residual finite element method. The thermal, flow and concentration fields are obtained to understand the flow dynamics of cobalt-kerosene nanofluid in two types of enclosures. The local and average Nusselt numbers are analyzed for plain and uneven side walls of the tube for different parameters of the problem. The simulated results are compared with the experimental as well as with the numerical data available in the literature for some special cases. The outcomes show that the tube of having uneven vertical side-walls give higher heat transfer for lower values of the thermal Rayleigh number; whereas for the higher values of the thermal Rayleigh number, the tube of plain vertical side-walls exhibit significantly higher heat transfer rate.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.