Role of Q177A and K173A/Q177A substitutions in thermostability and activity of the ELBn12 lipase

Abstract

Thermostable lipases have many applications in detergent industries and in organic synthesis. There are many ways to improve thermal stability of enzymes, for example, higher hydrophobicity, greater structural packing, higher content of the charged residues, and lower thermolabile ones. In this study, thermolabile Gln (sensitive to higher temperatures) was substituted with Ala in native ELBn12 and mutated K173A lipases to examine its effect on thermal stability and activity of the lipases. Single (Q177A) and double mutants (K173A/Q177A) were expressed in Escherichia coli pLysS. The Q177A variant increased both activity and thermostability of the lipase, whereas K173A/Q177A had a negative effect on the lipase activity and only had better thermal stability than the native at 50 °C. pH stability of the double mutant between 9.0 and 11 was also lower than the other variants. Stability analysis in the presence of chemicals showed that Q177A mutant had better activity with 50% (v/v) organic solvents. On the other hand, K173A lipase showed increased activity with 1% (w/v) nonionic surfactant, and finally K173A/Q177A had better stability with 10 mM metal ions compared to the native lipase.

Cyanobacteria as an eco‐friendly resource for biofuel production: A critical review

“…This genetic method is called thermorecovery, which could be used for the biofuel feedstock collection. Other thermostable lipases seem to be appropriate candidates for making engineered cyanobacteria with the capability to release FFA.…”

Structure-guided protein engineering increases enzymatic activities of the SGNH family esterases

“…Currently, many protein engineering methods have been developed to improve esterases (or lipases) properties, including substrate specificity [41,42], activity [43,44], thermostability [45], and enantioselectivity [46,47]. However, limited studies for enhancing alkali tolerance have been reported, especially based on structural information.…”

“…S5). According to previous studies, the thermal stability can be affected by (i) more abundant, high hydrophobicity, charged residues (such as Glu, Arg, and Lys) rather than uncharged polar amino acid (such as Ser, Thr, Asn, and Gln) in the amino acid sequences [41,48,49]; (ii) more α-helices in structures [50,51]; and (iii) some intermolecular forces, such as hydrogen bonds [52]. AlinE4 has 6 glutamine residues and 12 lysine residues, while CrmE10 has 12 glutamine residues and only 1 lysine residue, which might lead to the difference in thermal stability.…”

Structure guided protein engineering increases enzymatic activities of the SGNH family esterases

“…Currently, many protein engineering methods have been developed to improve esterases (or lipases) properties, including substrate speci city [41,42], activity [43,44], thermostability [45], and enantioselectivity [46,47]. However, limited studies for enhancing alkali-tolerance have been reported, especially based on structural information.…”

“…S5). According to previous studies, the thermal stability can be affected by (i) more abundant, high hydrophobicity, charged residues (such as Glu, Arg, and Lys) rather than uncharged polar amino acid (such as Ser, Thr, Asn, and Gln) in the amino acid sequences [41,48,49]; (ii) more α-helices in structures [50,51]; and (iii) some intermolecular forces, such as hydrogen bonds [52]. AlinE4 has six glutamine residues and twelve lysine residues, while CrmE10 has twelve glutamine residues and only one lysine residue, which might lead to the difference in thermal stability.…”