Rigidifying Flexible Sites: A Promising Strategy to Improve Thermostability of Lysophospholipase From Pyrococcus abyssi.

High thermostability of the enzymes is one of the distinguishing characteristics that increase their industrial utility. In the current research work, rigidifying the flexible amino acid residues of a lysophospholipase (Pa-LPL) from Pyrococcus abyssi was used as a protein engineering approach to improve its thermostability. A truncated variant of Pa-LPL (t-LPL∆12) was constructed by trimming its 12 amino acid residues (50-61) through overlap extension PCR. The truncated enzyme worked optimally at 65°C and pH 6.5 with remarkable thermostability at 65°C-85°C. In comparison to wild-type Pa-LPL, 5.8 and 1.2-fold increase in half-life (t1/2) of t-LPL∆12 was observed at 65 (optimum temperature) and 95°C, respectively. The activity of t-LPL∆12 was stimulated by 1 mM Cu2+ followed by Ca2+, Ni2+, Co2+, and Mg2+. Both substrate docking and experimental results indicated that the truncated enzyme could hydrolyze a variety of p-nitrophenyl esters. Km, Vmax, and Kcat for enzymatic hydrolysis of p-nitrophenyl butyrate were calculated to be 1 ± 0.087 mM, 1456 ± 36.474 U/mg, and 1.397 × 1011 min-1, respectively. In short, broad substrate specificity and thermostability of t-LPL∆12 are some of the distinctive features that make it an ideal candidate for degumming of vegetable oils.

Engineering the cofactor binding site of 7α-hydroxysteroid dehydrogenase for improvement of catalytic activity, thermostability, and alteration of substrate preference.

Hydroxysteroid dehydrogenases (HSDHs) are crucial for bile acid metabolism and influence the size of the bile acid pool and gut microbiota composition. HSDHs with high activity, thermostability, and substrate selectivity are the basis for constructing engineered bacteria for disease treatment. In this study, we designed mutations at the cofactor binding site involving Thr15 and Arg16 residues of HSDH St-2-2. The T15A, R16A, and R16Q mutants exhibited 7.85-, 2.50-, and 4.35-fold higher catalytic activity than the wild type, respectively, while also displaying an altered substrate preference (from taurocholic acid (TCA) to taurochenodeoxycholic acid (TCDCA)). These mutants showed lower Km and higher kcat values, indicating stronger binding to the substrate and resulting in 3190-, 3123-, and 3093-fold higher kcat/Km values for TCDCA oxidation. Furthermore, the Tm values of the T15A, R16A, and R16Q mutants were found to increase by 4.3 °C, 6.0 °C, and 7.0 °C, respectively. Molecular structure analysis indicated that reshaped internal hydrogens and surface mutations could improve catalytic activity and thermostability, and altered interactions among the catalytic triad, cofactor binding sites, and substrates could change substrate preference. This work provides valuable insights into modifying substrate preference as well as enhancing the catalytic activity and thermostability of HSDHs by targeting the cofactor binding site.

Recent advances in simultaneous thermostability-activity improvement of industrial enzymes through structure modification.

Engineered thermostable microbial enzymes are widely employed to catalyze chemical reactions in numerous industrial sectors. Although high thermostability is a prerequisite of industrial applications, enzyme activity is usually sacrificed during thermostability improvement. Therefore, it is vital to select the common and compatible strategies between thermostability and activity improvement to reduce mutants̕ libraries and screening time. Three functional protein engineering approaches, including directed evolution, rational design, and semi-rational design, are employed to manipulate protein structure on a genetic basis. From a structural standpoint, integrative strategies such as increasing substrate affinity; introducing electrostatic interaction; removing steric hindrance; increasing flexibility of the active site; N- and C-terminal engineering; and increasing intramolecular and intermolecular hydrophobic interactions are well-known to improve simultaneous activity and thermostability. The current review aims to analyze relevant strategies to improve thermostability and activity simultaneously to circumvent the thermostability and activity trade-off of industrial enzymes.

Thermostability enhancement of l-glutamate oxidase from Streptomyces sp. NT1 by full consensus protein design.

Thermostable l-glutamate oxidases (LGOXs) are desirable for use in l-glutamate (L-Glu) assay kits, enzymatic synthesis of α-ketoglutarate and for biosensor development. However, protein engineering efforts to improve thermostability often lead to a decrease in enzymatic activity. In this report, we aimed to enhance the thermostability (melting temperature, Tm) of a mesophilic LGOX from Streptomyces sp. NT1 (LGOXNT1) without a reduction in activity by a sequence-based protein design approach, termed full consensus (Fc) protein design. Among the 690 amino acids of LGOXNT1, 104 amino acids were substituted by the Fc protein design. The mutant gene was artificially synthesized and expressed in Escherichia coli BL21(DE3) cells. The Tm of the purified, recombinant LGOX mutant (FcLGOX) was determined to be ∼72 °C, which is an increase on the Tm of 65 °C for LGOXNT1 and the highest among known LGOXs. Importantly, purified FcLGOX showed no loss of specific activity or substrate specificity after a 30-min incubation at 70 °C. Our findings provide a new approach to improve the thermostability of enzymes.