Engineering topics

Murphy, Natasha P.

Publications and source records attributed to Murphy, Natasha P..

4 records

DOE OSTI2024

Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline

Abstract As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.

36 MATERIALS SCIENCE↗

DOE OSTI2024

Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis

Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1–2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

09 BIOMASS FUELS↗

DOE OSTI2024

A lipase from Lacticaseibacillus rhamnosus IDCC 3201 with thermostability and pH resistance for use as a detergent additive

Lipases are important biocatalysts and ubiquitous in plants, animals, and microorganisms. The high growth rates of microorganisms with low production costs have enabled the wide application of microbial lipases in detergent, food, and cosmetic industries. Herein, a novel lipase from Lacticaseibacillus rhamnosus IDCC 3201 (Lac-Rh) was isolated and its activity analyzed under a range of reaction conditions to evaluate its potential industrial application. The isolated Lac-Rh showed a molecular weight of 24 kDa and a maximum activity of 3438.5 ± 1.8 U/mg protein at 60 °C and pH 8. Additionally, Lac-Rh retained activity in alkaline conditions and in 10% v/v concentrations of organic solvents, including glycerol and acetone. Interestingly, after pre-incubation in the presence of multiple commercial detergents, Lac-Rh maintained over 80% of its activity and the stains from cotton were successfully removed under a simulated laundry setting. Overall, the purified lipase from L. rhamnosus IDCC 3201 has potential for use as a detergent in industrial applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Production of chitosan-based composite film reinforced with lignin-rich lignocellulose nanofibers from rice husk

Lignocellulosic nanofibers (LCNFs), implying lignin-containing cellulose fibers, maintain the properties of both lignin and cellulose, which are hydrophobic and hydrophilic, respectively. The presence of hydrophobic lignin in LCNFs is expected to be an economical and attractive option that can improve the thermal and mechanical properties of polymers. Thus, this study was conducted to produce lignin-rich LCNFs from sugar-rich waste obtained from rice husks after acidic pretreatment. The LCNFs were produced from the lignin-rich solid fractions obtained after pretreatment and enzymatic hydrolysis, which were then incorporated as an additive into a chitosan-based film. The variations in lignin content in the range of approximately 50.6%-66.8% in differently obtained LCNFs gave significantly different optical strengths and mechanical properties. These controllable processes may allow for customized film formation. Additionally, the glucose-rich liquid fractions obtained after pretreatment and enzymatic hydrolysis were used as a substrate for ethanol fermentation to achieve total utilization of rice husk biomass waste. In conclusion, the lignin-rich biomass fraction holds promise as a suitable material for chitosan-LCNF film and has the potential to increase the economic feasibility of the biomaterial industry.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗