Improving the Thermostability of Rhizopus chinensis Lipase Through Site-Directed Mutagenesis Based on B-Factor Analysis

Abstract

In order to improve the thermostability of lipases derived from Rhizopus chinensis, we identified lipase (Lipr27RCL) mutagenesis sites that were associated with enhanced flexibility based upon B-factor analysis and multiple sequence alignment. We found that two mutated isoforms (Lipr27RCL-K64N and Lipr27RCL-K68T) exhibited enhanced thermostability and improved residual activity, with respective thermal activity retention values of 37.88% and 48.20% following a 2 h treatment at 50 • C relative to wild type Lipr27RCL. In addition, these Lipr27RCL-K64N and Lipr27RCL-K68T isoforms exhibited 2.4-and 3.0-fold increases in enzymatic half-life following a 90 min incubation at 60 • C. Together these results indicate that novel mutant lipases with enhanced thermostability useful for industrial applications can be predicted based upon B-factor analysis and constructed via site-directed mutagenesis.

Improving the Thermostability of Serine Protease PB92 from Bacillus alcalophilus via Site-Directed Mutagenesis Based on Semi-Rational Design

“…Firstly, with the rapid development of computational biology and structural biology, many parameters are provided for protein modification engineering, mainly including the temperature factor (B-factor), protein folding free energy, and other parameters to determine protein mutation sites and improve the interaction of related noncovalent forces to modify proteins, among which the B-factor is one of the most widely employed [ 22 ]. B-factor analysis of protease PB92 was the key step in the design of thermostable recombinant isoforms of this enzyme, given that B-factors reflect protein fluctuations and the rigidity of atoms in specific positions [ 25 ], and previous work has shown that a higher level of rigidity is known to improve thermostability [ 26 ]. As such, this study analyzed B-factor values to identify residues with pronounced degrees of flexibility for protease PB92 and aligned this sequence with those of other thermolysin sequences using the PDB, and then it subsequently engineered recombinant mutants by mutating these flexible residues.…”

Expression and immobilization of trypsin‐like domain of serine protease from Pseudomonas aeruginosa for improved stability and catalytic activity

“…42 The thermostability and catalytic efficiency of many industrial enzymes are improved experimentally to enhance their industrial applications. [43][44][45][46][47][48] Hermanová et al have reported that thermal stability and solvent tolerance of graphene oxide immobilized oxidoreductase increases markedly compared to free soluble enzyme. 49 The lower K m value and improved thermostability of immobilized protease make it a robust catalyst and potential future candidate for industrial applications.…”

“…From the industrial perspective, catalytic efficiency and improved thermostability are desirable features that help to support improved reaction time and enzyme cost 42 . The thermostability and catalytic efficiency of many industrial enzymes are improved experimentally to enhance their industrial applications 43–48 . Hermanová et al have reported that thermal stability and solvent tolerance of graphene oxide immobilized oxidoreductase increases markedly compared to free soluble enzyme 49 .…”

Conceptual Study of Transesterification of Vegetable Oils in the Continuous-Stirred-Tank Reactor at Unsteady-State and Isothermal Conditions

“…These types of enzymes can be obtained by isolating from bacterial species growing in a harsh environment [32]. Otherwise, the thermal properties of the enzymes could be also improved via directed mutagenesis or directed evolution [33,34]. The enzyme usually lose their shape and activity with a prolonged use.…”