Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

Abstract

Rational design is widely employed in protein engineering to tailor wild-type enzymes for industrial applications. The typical target region for mutation is a functional region like the catalytic site to improve stability and activity. However, few have explored the role of other regions which, in principle, have no evident functionality such as the N-terminal region. In this study, stability prediction software was used to identify the critical point in the non-functional N-terminal region of L2 lipase and the effects of the substitution towards temperature stability and activity were determined. The results showed 3 mutant lipases: A8V, A8P and A8E with 29% better thermostability, 4 h increase in half-life and 6.6 °C higher thermal denaturation point, respectively. A8V showed 1.6-fold enhancement in activity compared to wild-type. To conclude, the improvement in temperature stability upon substitution showed that the N-terminal region plays a role in temperature stability and activity of L2 lipase.

Crucial Residues of C-Terminal Oligopeptide C60 to Improve the Yield of Prebiotic Xylooligosaccharides by Truncated Mutation

“…Generally, functional regions like the catalytic site are usually recognized as the typical target regions to modify wild-type enzymes. In addition to the catalytic domain, it has been well documented that N- and C-terminal regions are also crucial factors for enzyme properties [ 13 ]. For example, proline-rich sequence broadens the optimal pH and temperature ranges of xylanase from Geobacillus thermodenitrificans C5 [ 23 ], the chaperone-like ability of artemin is reduced by deletion of its extra C-terminal 39 residues [ 24 ], and the thermostability and optimal temperature of GH11 xylanase increased after C-terminal region deletion [ 25 ].…”

“…Generally, the function of linkers is determined by many factors such as its sequence length and critical residues types [ 13 , 20 ]. As shown in sequence alignment ( Figure 2 ), the first 15 residues of C60 showed high identity with the C-terminal sequences of xylanases with a single domain (MBQ5171507 and MBQ7162149).…”

“…Terminal regions or residues have previously shown their significant effects on the physical and catalytic properties of enzymes such as thermostability and catalytic efficiency [ 11 , 12 ]. These effects of terminal regions or residues are related to their sequence length, amino acid type, and structure flexibility [ 13 ]. In this study, in order to understand and investigate the key factors contributing to the enhanced function of C60, three truncated mutants with different lengths of C-terminal regions were constructed on the basis of sequence analysis, and their effects on enzymatic properties and XOS production were comparatively investigated with the wild type.…”

Discovery of the Key Mutation Site Influencing the Thermostability of Thermomyces lanuginosus Lipase by Rosetta Design Programs

“…TLL was characterized by higher yields than many other heat-resistant lipases: the enzyme activity of TLL after fermentation in a 150 mL test tube is closed to 1000 U/mL, while this value is only 400 U/mL in another thermal stable lipase WT-L2 from Bacillus sp. L2 [ 16 , 32 ]. In addition, when TLL was fermented in a 50 L fermenter in industry, its enzyme activity in the supernatant could reach 30,000 U/mL, which meets most of the conditions for the industrial production [ 33 ].…”

“…The crystal structure of TLL (1DT3) was used as the wild-type template for Gibbs free energy calculation [ 36 ]. The Rosetta ddg_monomer and Cartesian_ddG programs were used to calculate the Gibbs free energy changes (ΔΔG) between wild-type TLL and all of the possible single substitutions of the TLL (5111 in total) [ 15 , 16 ]. Default parameters were applied in both two calculations.…”