ABSTRACT
OBJECTIVES: This study was aimed at engineering charged residues on the surface of Thermomyces lanuginosus lipase (TLL) to obtain TLL variant with elevated performance for industrial applications. RESULTS: Site-directed mutagenesis of eight charged amino acids on the TLL surface were conducted and substitutions on the negatively charged residues D111, D158, D165, and E239 were identified with elevated specific activities and biodiesel yields. Synergistic effect was not discovered in the double mutants, D111E/D165E and D165E/E239R, when compared with the corresponding single mutants. One TLL mutant, D165E, was identified with increased specific activity (456.60 U/mg), catalytic efficiency (kcat/Km: 44.14 s-1 mM-1), the highest biodiesel conversion yield (93.56%), and comparable thermostability with that of the TLL. CONCLUSIONS: Our study highlighted the importance of surface charge engineering in improving TLL activity and biodiesel production, and the resulting TLL mutant, D165E, is a promising candidate for biodiesel industry.
Subject(s)
Eurotiales/enzymology , Lipase/metabolism , Mutagenesis, Site-Directed/methods , Amino Acid Substitution , Biocatalysis , Biofuels , Enzymes, Immobilized/genetics , Enzymes, Immobilized/metabolism , Eurotiales/genetics , Fungal Proteins/metabolism , Lipase/genetics , Protein EngineeringABSTRACT
The existing index systems on sustainable evaluation are mostly based on a multi index comprehensive evaluation method. The main disadvantage of this approach is that the selection and assignment of evaluation indexes are greatly influenced by subjective factors, which can result in poor comparability of results. By contrast, the Footprint Family (including ecological footprint, carbon footprint, and water footprint) is not affected by subjective factors. The Footprint Family also covers the basic tenets of sustainable development. This paper proposes use of a sustainable development evaluation index system based on the principle of the Footprint Family, and including the ecological pressure index (EPI), the ecological occupancy index (EOI), the ecological economic coordination index (EECI), the GHG (Greenhouse Gas) emission index (CEI), the water resources stress index (WSI), and the sustainable development index (SDI). Furthermore, a standard for grading the evaluated results based on global benchmarks is formulated. The results of an empirical study in China were the following. The development situation deteriorated from 1990 to 2015. The results showed that the SDI decreased from a medium level (grade 5) to a lower-medium level (grade 4). The results of this empirical study also showed that the method of evaluation can avoid the influence of subjective factors and can be used in the evaluation of sustainable development for various temporal and spatial conditions.
